TW202002999A - Use of an essentially pure mesenchymal stem cell population of the amniotic membrane of umbilical cord for generating a mammalian stem cell carrying a transgene - Google Patents

Use of an essentially pure mesenchymal stem cell population of the amniotic membrane of umbilical cord for generating a mammalian stem cell carrying a transgene Download PDF

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TW202002999A
TW202002999A TW108112053A TW108112053A TW202002999A TW 202002999 A TW202002999 A TW 202002999A TW 108112053 A TW108112053 A TW 108112053A TW 108112053 A TW108112053 A TW 108112053A TW 202002999 A TW202002999 A TW 202002999A
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東森 樊
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新加坡商細胞研究私人有限公司
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    • C12N2510/00—Genetically modified cells

Abstract

The present invention relates to a method of generating a transgenic mesenchymal stem cell population carrying a transgene, a transgenic mesenchymal stem cell population obtained by or obtainable by the method and a pharmaceutical composition and uses of the transgenic mesenchymal stem cell population in particular for gene therapy. The invention is also directed to methods of treating a disease or disorder comprising administering the transgenic mesenchymal stem cell population or a pharmaceutical composition containing a transgenic mesenchymal stem cell population of the invention to a subject in need thereof.

Description

臍帶羊膜的基本上純的間質幹細胞群用於產生攜帶轉殖基因的哺乳動物幹細胞的用途Use of substantially pure mesenchymal stem cell population of umbilical cord amniotic membrane for generating mammalian stem cells carrying transgene

相關申請案。本申請案請求於2018年4月6日申請之美國臨時申請案號為62/654,126的優先權權益，其內容係在此透過引用之方式為了所有目的而整體併入本文。Related applications. This application claims the priority interest of US Provisional Application No. 62/654,126 filed on April 6, 2018, the contents of which are hereby incorporated by reference in its entirety for all purposes.

本發明涉及一種產生攜帶轉殖基因的轉殖基因間質幹細胞群之方法，透過該方法產生的轉殖基因間質幹細胞群以及醫藥組合物以及該轉殖基因間質幹細胞群之用途。本發明還涉及治療疾病或病症之方法，包括將該轉殖基因間質幹細胞群或含有本發明之轉殖基因間質幹細胞群的醫藥組合物給予有需要之個體。The invention relates to a method for generating a colony of mesenchymal stem cells carrying a colony gene, a colony of mesenchymal stem cells produced by the method and a pharmaceutical composition, and uses of the colony of mesenchymal stem cells of the colony. The present invention also relates to a method for treating a disease or condition, which comprises administering the colony of mesenchymal stem cells or the pharmaceutical composition containing the colony of mesenchymal stem cells of the present invention to an individual in need.

A型血友病治療的基礎為因子VIII (FVIII)蛋白質替代物，其將止血能力恢復到足以在日常生活活動期間實現正常血液凝固的程度。無血漿重組FVIII產品現應為首選的治療方法(Grillberger等人，2009年；Franchini與Lippi，2010年)。相較於按需求的治療，使用無血漿重組FVIII蛋白產品進行定期預防可大幅降低急性出血發作的頻率、慢性肌肉骨骼殘疾，並改善與健康相關的生活品質(Manco-Johnson等人，2007年；Plug等人，2008年；Walsh與Valentino，2009年；Collins等人，2010年；Gringeri等人，2011年；Manco-Johnson等人，2013年)。然而，許多患者根本沒有接受最佳甚至任何FVIII替代治療，這主要是因為根據體重、臨床表現型以及個體FVIII藥物代謝動力學，預估一名年幼兒童所需的成本為138,000美元至300,000美元這麼高(Bernstein，2007年；Manco-Johnson等人，2007年；Roosendaal與Lafeber，2007年)。在目前全球約有140,000名患有A型血友病的人口，其中75%的人接受很少或沒有接受FVIII替代品治療(Mannucci，2011年；世界血友病聯盟，2013年)。即使FVIII產品在高收入國家能被負擔得起，定期預防也會導致頻繁的突破性出血(Collins等人，2009年；Walsh與Valentino，2009年)，而需要頻繁的靜脈注射也限制了此種治療，特別是針對兒童而言，有效的早期干預對兒童尤其重要(Ljung，2007年；Santagostino與Mancuso，2008年)。Hemophilia A treatment is based on factor VIII (FVIII) protein substitutes, which restore hemostatic capacity to a level sufficient to achieve normal blood clotting during daily activities. Plasma-free recombinant FVIII products should now be the treatment of choice (Grillberger et al., 2009; Franchini and Lippi, 2010). Compared with on-demand treatment, regular prevention with plasma-free recombinant FVIII protein products can significantly reduce the frequency of acute bleeding episodes, chronic musculoskeletal disabilities, and improve health-related quality of life (Manco-Johnson et al., 2007; Plug et al., 2008; Walsh and Valentino, 2009; Collins et al., 2010; Gringeri et al., 2011; Manco-Johnson et al., 2013). However, many patients simply do not receive optimal or even any FVIII replacement therapy, mainly because the estimated cost of a young child is between US$138,000 and US$300,000 based on body weight, clinical phenotype, and individual FVIII pharmacokinetics So high (Bernstein, 2007; Manco-Johnson et al., 2007; Roosendaal and Lafeber, 2007). There are currently approximately 140,000 people with hemophilia A in the world, 75% of whom receive little or no FVIII replacement treatment (Mannucci, 2011; World Hemophilia Alliance, 2013). Even if FVIII products are affordable in high-income countries, regular prevention can lead to frequent breakthrough bleeding (Collins et al., 2009; Walsh and Valentino, 2009), and the need for frequent intravenous injections limits this Treatment, especially for children, effective early intervention is particularly important for children (Ljung, 2007; Santagostino and Mancuso, 2008).

現可獲得的高品質FVIII替代產品對於世界上超過一半的A型血友病患者而言成本太高，此一事實激發了開發替代療法的嘗試。使用病毒載體的體內基因治療對單基因FVIII缺乏具有吸引力。儘管在B型血友病基因治療中尚未得到相同的成功案例(Nathwani等人，2014年)，但AAV載體中FVIII轉殖基因表現及包裝的改善似乎很有希望(High等人，2014年)，像是對AAV載體免疫反應的最小化方法 (Masat等人，2013年)。The fact that currently available high-quality FVIII replacement products are too costly for more than half of patients with hemophilia A in the world, this fact has inspired attempts to develop alternative therapies. In vivo gene therapy using viral vectors is unattractive to single gene FVIII. Although the same success stories have not been obtained in gene therapy for hemophilia B (Nathwani et al., 2014), the improvement of the expression and packaging of FVIII transgenes in AAV vectors seems promising (High et al., 2014) , Such as the method of minimizing the immune response to AAV vectors (Masat et al., 2013).

另一種策略是離體將FVIII轉殖基因以非病毒方式遞送到自體細胞中。在離體轉染的自體真皮纖維母細胞的臨床試驗中證實了概念，該質體遞送了從纖維接合素啟動子表現的B結構域缺失的FVIII轉殖基因(Roth等人，2001年)。對6名患有嚴重A型血友病的患者中的4名植入分泌FVIII的自體纖維母細胞在長達10個月內得到了可測量到的，雖然不太多的治療功效。從那時起，已經出現了幾種具有高準確度修飾基因組的可編程核酸酶，且可以透過非病毒載體遞送。其中，鋅指核酸酶(zinc finger nuclease，ZFN)技術目前在可能的臨床應用方面最為先進。在12名慢性HIV感染患者的自體T細胞中鋅指核酸酶(ZFN)調節的CCR5 不活化的臨床第1期試驗報告沒有可歸因於使用鋅指核酸酶(ZFN)的不良事件(Tebas等人，2014年)。儘管如此，人們對潛在的致癌性併發症有了更高的認識，因為γ反轉錄病毒載體調節的轉殖基因整合的臨床試驗受到治療誘導的白血病的損害(Hacein-Bey-Abina等人，2003年；Hacein-Bey-Abina等人，2008年；Howe等人，2008年；Stein等人，2010年；Avedillo Diez等人，2011年；Gaspar等人，2011年)。發生這些危及生命的併發症使得所有基因組修飾技術的生物安全變得非常重要。Another strategy is to deliver FVIII transgenes to autologous cells ex vivo. The concept was confirmed in a clinical trial of autologous dermal fibroblasts transfected ex vivo, the plastid delivered the FVIII transgenic gene deleted from the B domain expressed by the fibronectin promoter (Roth et al., 2001) . Implantation of autologous fibroblasts secreting FVIII in 4 out of 6 patients with severe hemophilia A had measurable results for up to 10 months, although not much therapeutic efficacy. Since then, several programmable nucleases with highly accurate modified genomes have appeared and can be delivered through non-viral vectors. Among them, zinc finger nuclease (Zinc finger nuclease, ZFN) technology is currently the most advanced in possible clinical applications. The clinical phase 1 trial of zinc finger nuclease (ZFN)-mediated CCR5 inactivation in autologous T cells of 12 patients with chronic HIV infection reported no adverse events attributable to the use of zinc finger nuclease (ZFN) (Tebas Et al., 2014). Nonetheless, people have a higher understanding of potential carcinogenic complications, because clinical trials of the integration of transgenes regulated by gamma retroviral vectors are impaired by treatment-induced leukemia (Hacein-Bey-Abina et al., 2003 Year; Hacein-Bey-Abina et al., 2008; Howe et al., 2008; Stein et al., 2010; Avedillo Diez et al., 2011; Gaspar et al., 2011). The occurrence of these life-threatening complications makes the biological safety of all genome modification techniques very important.

儘管已採用各種方法，但尚未全面評估鋅指核酸酶(ZFN)處理的細胞中偏離目標基因組修飾的程度及性質。已經透過篩選生物資訊學預測的偏離目標位點(Hockemeyer等人，2009年)，偏向基因庫的體外切割(Pattanayak等人，2011年)或對整合酶缺陷型慢病毒載體的整合位點進行定序(Gabriel等人，2011年)來評估基因組修飾的特異性。由不同方法產生的大部分非重疊偏離目標位點的問題(Gabriel等人，2011年；Pattanayak等人，2011年)僅透過機器學習分類器(Sander等人，2013年)部分解決，且仍然存在偏離目標位點識別中的非平凡之方法依賴性差異。如果如所建議的那樣，鋅指核酸酶(ZFNs)在整個基因組中的結合能量非常低(Hendel等人，2014年)，電腦預測以及實際偏離目標位點之間的相關性差並不令人驚訝。此外，很明顯地，沒有一種用於詢問基因組的技術足以全面揭示偏離目標修飾(Hendel等人，2014年；Pattanayak等人，2014年)，目前也沒有用於評估離體生物安全性的共同標準。此外，許多因素(例如疾病背景、細胞類型、靶位點選擇、核酸酶與供體DNA設計、鋅指核酸酶(ZFN)濃度以及核酸酶處理條件)的相互作用導致意外的臨床效果，這些因素可能對不同的治療環境具有特異性。有鑑於這種複雜性，仍需要攜帶一轉殖基因的哺乳動物細胞，該轉殖基因例如為編碼因子VIII多胜肽的轉殖基因或其缺陷表現或產生的任何其他多胜肽與可安全使用的疾病或病症相關，例如，在基因治療中，其中轉殖基因已經穩定地與位點特異性地整合。這種攜帶轉殖基因的哺乳動物細胞被描述於國際專利申請WO 2017/105350以及Sivalingam, J.等人，「Multidimensional genome-wide analyses show accurate FVIII integration by ZFN in primary human cells」，Molecular Therapy, 2016年，第24卷第3期，第607-619頁。然而，儘管在WO 2017/105350以及Sivalingam, J.等人，Molecular Therapy，2016年，第24卷第3期，第607-619頁中描述的臍帶襯裡幹細胞取得了進展，期望具有攜帶這種轉殖基因的另一種哺乳動物細胞，具體而言是作為高度同源的哺乳動物幹細胞群的種類，因此可用於臨床試驗。Although various methods have been used, the extent and nature of modification of the target genome in cells treated with zinc finger nuclease (ZFN) have not been fully evaluated. It has been screened by bioinformatics to predict deviation from the target site (Hockemeyer et al., 2009), bias in vitro cleavage of the gene pool (Pattanayak et al., 2011), or to determine the integration site of integrase-deficient lentiviral vectors Sequence (Gabriel et al., 2011) to assess the specificity of genome modification. Most of the non-overlapping off-target problems generated by different methods (Gabriel et al., 2011; Pattanayak et al., 2011) are only partially solved by machine learning classifiers (Sander et al., 2013), and still exist Non-trivial method-dependent differences in target site identification. If, as suggested, the binding energy of zinc finger nucleases (ZFNs) in the entire genome is very low (Hendel et al., 2014), the poor correlation between computer predictions and actual deviations from the target site is not surprising . In addition, it is clear that no technique for interrogating the genome is sufficient to fully reveal off-target modifications (Hendel et al., 2014; Pattanayak et al., 2014), and there is currently no common standard for assessing the safety of ex vivo organisms . In addition, the interaction of many factors (such as disease background, cell type, target site selection, nuclease and donor DNA design, zinc finger nuclease (ZFN) concentration, and nuclease treatment conditions) have led to unexpected clinical effects. These factors May be specific to different treatment environments. In view of this complexity, there is still a need for mammalian cells carrying a transgene, such as the transgene encoding factor VIII polypeptide or its defective expression or any other polypeptide produced and safe The disease or condition used is related, for example, in gene therapy, where the transgene has been stably integrated with the site-specific. This mammalian cell carrying a transgenic gene is described in international patent application WO 2017/105350 and Sivalingam, J. et al., "Multidimensional genome-wide analyses show accurate FVIII integration by ZFN in primary human cells", Molecular Therapy, 2016 Year, Volume 24, Number 3, pages 607-619. However, despite the progress made in umbilical cord lining stem cells described in WO 2017/105350 and Sivalingam, J. et al., Molecular Therapy, 2016, Volume 24, Number 3, pages 607-619, it is expected that Another type of mammalian cell that reproduces genes is specifically a species of highly homologous mammalian stem cell population, and therefore can be used in clinical trials.

因此，本發明之一目的為提供滿足這種需要的這種哺乳動物幹細胞群。因此，本發明之另一目的為提供產生這種哺乳動物幹細胞群之方法。Therefore, one object of the present invention is to provide such a mammalian stem cell population that meets this need. Therefore, another object of the present invention is to provide a method for producing such a mammalian stem cell population.

該目的透過具有申請專利範圍獨立項的特徵之方法、細胞，以及醫藥組合物來實現。This objective is achieved by methods, cells, and pharmaceutical compositions that have features that are independent of the scope of the patent application.

於第一方面，本發明提供一種產生一轉殖基因間質幹細胞群之方法，該方法包括透過酶調節的整合將一轉殖基因***間質幹細胞群的基因組中，其中該間質幹細胞群為臍帶羊膜的分離的間質幹細胞群，其中至少約90%或更多的幹細胞群細胞表現以下標記中的每一種：CD73、CD90以及CD105。用於將該轉殖基因***該間質幹細胞群的基因組中的酶調節的整合可以透過選自由一重組酶、一類轉錄活化劑效應子核酸酶，以及一核酸酶所組成之群組的酶進行。In a first aspect, the present invention provides a method of generating a population of transgenic mesenchymal stem cells, the method comprising inserting a transgenic gene into the genome of a population of mesenchymal stem cells through enzyme-mediated integration, wherein the population of mesenchymal stem cells is An isolated mesenchymal stem cell population of umbilical cord amniotic membrane, wherein at least about 90% or more of the stem cell population cells exhibit each of the following markers: CD73, CD90, and CD105. The enzyme-regulated integration for inserting the transferred gene into the genome of the mesenchymal stem cell population can be performed by an enzyme selected from the group consisting of a recombinase, a type of transcriptional activator effector nuclease, and a nuclease .

在該第一方面之具體實施例中，至少約91%或更多、約92%或更多、約93%或更多、約94%或更多、約95%或更多、約96%或更多、約97%或更多、約98%或更多、約99%或更多的分離的間質幹細胞群的細胞表現每一個CD73、CD90以及CD105。此外，在該第一方面的這些具體實施例中，至少約91%或更多、約92%或更多、約93%或更多、約94%或更多、約95%或更多、約96%或更多、約97%或更多、約98%或更多、約99%或更多的分離的間質幹細胞群的細胞較佳缺乏標記CD34、CD45以及HLA-DR的表現。In specific embodiments of the first aspect, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% The cells of the isolated mesenchymal stem cell population of or more, about 97% or more, about 98% or more, about 99% or more express each CD73, CD90 and CD105. In addition, in these specific embodiments of the first aspect, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, Cells of about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cell population preferably lack the performance of the markers CD34, CD45 and HLA-DR.

於第二方面，本發明提供了透過本發明(第一方面)之方法獲得的轉殖基因間質幹細胞群。因此，本發明之轉殖基因間質幹細胞群為來自臍帶羊膜的或衍生自臍帶羊膜的轉殖基因間質幹細胞群，其中至少約81%或更多，或90%或更多的間質幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105。此外，該轉殖基因間質幹細胞群的至少約81%或更多，或約90%或更多的細胞可能缺乏以下標記的表現：CD34、CD45以及HLA-DR。在該轉殖基因間質幹細胞群的具體實施例中，至少約81%或更多、約82%或更多、至少83%或更多、至少84%或更多、至少約85%或更多、約86%或更多、約87%或更多、約88%或更多、約89%或更多、約90%或更多、約91%或更多、約92%或更多、約93%或更多、約94%或更多、約95%或更多、約96%或更多、約97%或更多、約98%或更多，約99%或更多的該轉殖基因間質幹細胞群的細胞表現CD73、CD90以及CD105的每一種，且缺乏表現CD34、CD45以及HLA-DR的每一種。In a second aspect, the present invention provides a population of transgenic mesenchymal stem cells obtained by the method of the present invention (first aspect). Therefore, the colony of mesenchymal stem cells of the present invention is a colony of mesenchymal stem cells derived from or derived from umbilical cord amniotic membrane, wherein at least about 81% or more, or 90% or more mesenchymal stem cells The cells of the cluster exhibited each of the following markers: CD73, CD90, and CD105. In addition, at least about 81% or more, or about 90% or more, cells of the transgenic mesenchymal stem cell population may lack the expression of the following markers: CD34, CD45, and HLA-DR. In a specific embodiment of the transgenic gene mesenchymal stem cell population, at least about 81% or more, about 82% or more, at least 83% or more, at least 84% or more, at least about 85% or more More, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more , About 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more The cells of this colony of mesenchymal stem cells expressed each of CD73, CD90, and CD105, and lacked each of CD34, CD45, and HLA-DR.

於第三方面，本發明提供一種含有本發明(第二方面)之轉殖基因間質幹細胞群的醫藥組合物。In a third aspect, the present invention provides a pharmaceutical composition containing the transgenic mesenchymal stem cell population of the present invention (second aspect).

於第四方面，本發明提供一種治療患有一疾病的一患者之方法，該方法包括向該患者施用本發明之轉殖基因間質幹細胞群(透過本發明第一方面之方法或第二方面之轉殖基因間質幹細胞群所獲得)。In a fourth aspect, the present invention provides a method of treating a patient suffering from a disease, the method comprising administering to the patient the population of transgenic mesenchymal stem cells of the present invention (through the method of the first aspect of the invention or the second aspect (Transgenic gene mesenchymal stem cell population obtained).

於第五方面，本發明提供一種本發明(第二方面)之轉殖基因間質幹細胞群用於治療一疾病之用途。In a fifth aspect, the present invention provides a use of the transgenic gene mesenchymal stem cell population of the present invention (second aspect) for treating a disease.

於第六方面，本發明提供該第二方面之轉殖基因間質幹細胞群用於基因治療之用途。In a sixth aspect, the present invention provides the use of the transgenic gene mesenchymal stem cell population of the second aspect for gene therapy.

於第七方面，本發明提供一臍帶羊膜的間質幹細胞群之用途，其中至少約90%或更多的間質幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105，用於產生該第二方面之轉殖基因間質幹細胞群。於第七方面的這種用途之具體實施例中，至少約90%或更多的間質幹細胞群的細胞缺乏下列標記的表現：CD34、CD45以及HLA-DR。在該用途的其他具體實施例中，使用至少約91%或更多、約92%或更多、約93%或更多、約94%或更多、約95%或更多、約96%或更多、約97%或更多、約98%或更多，約99%或更多的分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，且缺乏表現CD34、CD45以及HLA-DR的每一種。In a seventh aspect, the present invention provides the use of an umbilical cord amniotic mesenchymal stem cell population, wherein at least about 90% or more of the cells of the mesenchymal stem cell population exhibit each of the following markers: CD73, CD90, and CD105 for The population of transgenic mesenchymal stem cells of the second aspect is generated. In a specific embodiment of this use of the seventh aspect, at least about 90% or more of the cells of the mesenchymal stem cell population lack the expression of the following markers: CD34, CD45, and HLA-DR. In other specific embodiments of this use, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% are used Or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population expressing each of CD73, CD90 and CD105, and lack of expressing CD34, CD45 And every kind of HLA-DR.

如下所述，本發明尤其基於一種從臍帶的羊膜分離及/或提供間質幹細胞群之方法，該方法包括在包含DMEM (Dulbecco’s modified eagle medium)、F12 (Ham's F12培養基)、M171 (培養基171)以及FBS (胎牛血清)的培養基中培養臍帶組織。本案發明人驚訝地發現，使用這種培養基提供了(用於分離)來自臍帶之羊膜的間質幹細胞群，其中超過90%，或甚至99%或更多的細胞對於三種間質幹細胞標記CD73、CD90呈現陽性，而同時這些幹細胞缺乏CD34、CD45與HLA-DR的表現(參閱實驗部分)，表示該細胞群的99%或甚至更多的細胞表現幹細胞標記CD73、CD90以及CD105，而不表現標記CD34、CD45與HLA-DR。這種非常均勻且明確界定的細胞群是臨床試驗與基於細胞之治療的理想候選者，因為例如它們完全符合通常所接受的用於細胞治療的人類間質幹細胞之標準，例如透過Dominici等人，「Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement」，Cytotherapy (2006年)第8卷，第4期，第315-317頁；Sensebe等人，「Production of mesenchymal stromal/stem cells according to good manufacturing practices：a，review」，Stem Cell Research & Therapy，2013年，第4卷：第66頁；Vonk等人，Stem Cell Research & Therapy (2015年)第6卷：第94頁，或Kundrotas Acta Medica Lituanica，2012年，第19卷，第2期，第75-79頁。此外，使用量子細胞擴增系統等生物反應器，每次運行可以獲得大量的間質幹細胞，例如300至700百萬個間質幹細胞(亦參閱實驗部分)。因此，本發明允許提供治療應用所需的幹細胞量，例如它們在基因治療中的用途，例如，以成本有效的方式治療患有A型血友病的患者。因此，本發明之轉殖基因間質幹細胞群為基因治療的理想平台。另外，用於製備本發明中使用的培養基的所有組成分都是以GMP品質所購得的。因此，本發明還開啟了從臍帶羊膜中GMP產生這種高度同源的間質幹細胞群的途徑，因此也開闢了相應的高度同源的轉殖基因間質幹細胞群的GMP產生途徑。此外，本發明還具有避免例如安全風險以及與使用腺相關病毒載體的基因治療相關的放大問題等問題的附加優點。As described below, the present invention is particularly based on a method for separating and/or providing a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, the method including the inclusion of DMEM (Dulbecco's modified eagle medium), F12 (Ham's F12 medium), M171 (medium 171) And FBS (fetal bovine serum) culture medium. The inventor of the present invention was surprised to find that the use of this culture medium provides (for separation) a population of mesenchymal stem cells derived from amniotic membrane of the umbilical cord, of which more than 90%, or even 99% or more, cells are labeled for the three mesenchymal stem cells CD73, CD90 was positive, and at the same time these stem cells lacked the expression of CD34, CD45 and HLA-DR (see the experimental section), indicating that 99% or even more cells of this cell population expressed stem cell markers CD73, CD90 and CD105, but not markers CD34, CD45 and HLA-DR. Such very uniform and well-defined cell populations are ideal candidates for clinical trials and cell-based therapies because, for example, they fully meet the generally accepted standards for human mesenchymal stem cells for cell therapy, such as through Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Volume 8, Issue 4, pages 315-317; Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review”, Stem Cell Research & Therapy, 2013, Volume 4: Page 66; Vonk et al., Stem Cell Research & Therapy (2015) Volume 6: Page 94, Or Kundrotas Acta Medica Lituanica, 2012, Volume 19, Number 2, pages 75-79. In addition, using bioreactors such as quantum cell expansion systems, a large number of mesenchymal stem cells can be obtained per operation, such as 300 to 700 million mesenchymal stem cells (see also the experimental section). Therefore, the present invention allows to provide the amount of stem cells required for therapeutic applications, such as their use in gene therapy, for example, to treat patients with hemophilia A in a cost-effective manner. Therefore, the transgenic mesenchymal stem cell population of the present invention is an ideal platform for gene therapy. In addition, all components used to prepare the medium used in the present invention were purchased in GMP quality. Therefore, the present invention also opens a way to produce such highly homogenous mesenchymal stem cell population from umbilical cord amniotic membrane by GMP, and thus also opens up a corresponding highly homologous production route of GM mesenchymal stem cell population. In addition, the present invention has the additional advantage of avoiding problems such as safety risks and amplification problems associated with gene therapy using adeno-associated viral vectors.

預防性地給予許多蛋白質，例如無血清重組FVIII蛋白，而非僅為停止出血事件是對A型血友病患者的最佳治療方法。由於安全的FVIII蛋白質產品成本非常高，世界上超過一半的A型血友病患者接受的治療效果不理想，甚至根本沒有治療。這導致數千名患者終生患有慢性殘疾。自體細胞療法為一種潛在的解決方案。使用鋅指核酸酶(ZFNs)，一種FVIII轉殖基因可以離體整合到體細胞的基因組中。因此，這些細胞獲得FVIII分泌，然後在體內植入後可作為FVIII恆定且持久的來源。鋅指核酸酶(ZFNs)基因組修飾的安全性是臨床開發中需要解決的關鍵問題。意外的偏離目標基因組改變已知有致癌後果。迄今為止，已經使用僅關注基因組DNA的單一技術評估鋅指核酸酶(ZFN)的偏離目標效應。Preventive administration of many proteins, such as serum-free recombinant FVIII protein, rather than just stopping bleeding events, is the best treatment for patients with hemophilia A. Because the cost of safe FVIII protein products is very high, more than half of the patients with hemophilia A in the world receive unsatisfactory treatment effects, or even no treatment at all. This has led to thousands of patients with chronic disabilities throughout their lives. Autologous cell therapy is a potential solution. Using zinc finger nucleases (ZFNs), a FVIII transgene can be integrated ex vivo into the genome of somatic cells. Therefore, these cells obtain FVIII secretion, which can then be used as a constant and durable source of FVIII after implantation in vivo. The safety of genome modification of zinc finger nucleases (ZFNs) is a key issue to be solved in clinical development. Unexpected deviations from the target genome are known to have carcinogenic consequences. To date, a single technique focusing only on genomic DNA has been used to assess the off-target effects of zinc finger nucleases (ZFN).

於此，AAVS1 鋅指核酸酶(ZFN)用於將FVIII轉殖基因整合到臍帶襯裡的上皮細胞(cord lining epithelial cells，CLECs)以及如本文所述之間質臍帶襯裡幹細胞中。組合幾個胺基酸取代以驚人地增強鋅指核酸酶(ZFN)準確度及活性(即專性異二聚化以及增強的DNA裂解活性)，且短暫的次低溫能夠提高轉殖基因整合的效率。使用基因捕獲策略以及嘌呤黴素抗性的篩選來促進FVIII轉殖基因的正確目標整合，FVIII轉殖基因為一種B結構域截短的人-豬雜交體，其分泌比人類FVIII更高的FVIII活性。在AAVS1鋅指核酸酶(ZFN)處理的優化條件下，完整的FVIII轉殖基因整合在臍帶襯裡的上皮細胞(CLECs)中，其分泌了大量的FVIII。在臍帶襯裡的上皮細胞(CLECs)中整合FVIII轉殖基因的相同條件下，也可以修飾這些成體細胞以整合並分泌FVIII。AAVS1鋅指核酸酶(ZFN)還可用於整合任何轉殖基因，包括，但不限於，FVIII，在臍帶羊膜的分離的間質幹細胞群中，其中至少約90%或更多的幹細胞群細胞表現以下標記中的每一種：CD73、CD90以及CD105。Here, AAVS1 zinc finger nuclease (ZFN) is used to integrate the FVIII transgene into cord lining epithelial cells (CLECs) and interstitial umbilical cord lining stem cells as described herein. Combining several amino acid substitutions to surprisingly enhance the accuracy and activity of zinc finger nuclease (ZFN) (ie, obligate heterodimerization and enhanced DNA cleavage activity), and a brief sub-low temperature can improve the integration of transgenic genes effectiveness. Use gene capture strategies and puromycin resistance screening to promote the correct target integration of the FVIII transgene, a human-porcine hybrid with a truncated B domain that secretes higher FVIII than human FVIII active. Under the optimized conditions of AAVS1 zinc finger nuclease (ZFN) treatment, the complete FVIII transfer gene was integrated into umbilical cord-lined epithelial cells (CLECs), which secreted a large amount of FVIII. These adult cells can also be modified to integrate and secrete FVIII under the same conditions as the integration of FVIII transgenes in umbilical cord-lined epithelial cells (CLECs). AAVS1 zinc finger nuclease (ZFN) can also be used to integrate any transgenic gene, including, but not limited to, FVIII, in an isolated mesenchymal stem cell population of umbilical cord amniotic membrane, where at least about 90% or more of the stem cell populations perform Each of the following marks: CD73, CD90, and CD105.

因此，本發明提供了具有設計特徵的AAVS1鋅指核酸酶(ZFN)以及FVIII供體DNA，該設計特徵有利於準確並有效的正確目標整合，其修飾如本文所述之間質幹細胞以分泌FVIII而不會引起可預示致癌風險的顯著偏離目標改變。這些結果顯示AAVS1 鋅指核酸酶(ZFN)調節的FVIII轉殖基因整合用於A型血友病的自體細胞治療不太可能引起不良併發症，因此是一種非常有前景的方法，可以進一步開發用於臨床治療。Therefore, the present invention provides an AAVS1 zinc finger nuclease (ZFN) and FVIII donor DNA with design features that facilitate accurate and effective correct target integration, which are modified as described herein to mesenchymal stem cells to secrete FVIII It will not cause a significant off-target change that predicts the risk of cancer. These results indicate that the integration of the FVIII transgene regulated by AAVS1 zinc finger nuclease (ZFN) for autologous cell therapy of hemophilia A is unlikely to cause adverse complications, so it is a very promising method that can be further developed Used for clinical treatment.

此外，如上所述，使用包含轉殖基因之同源且定義明確的間質幹細胞群是臨床試驗以及基於細胞的療法的理想候選者，因為它們例如完全符合人類間質幹細胞普遍接受的標準，用於，例如，由以下所定義的細胞治療，Dominici等人，「Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement」，Cytotherapy (2006年)第8卷第4期，第315-317頁，Sensebe等人， 「Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review」， Stem Cell Research & Therapy 2013年, 4:66)，Vonk等人，tem Cell Research & Therapy (2015年) 6:94，或Kundrotas Acta Medica Lituanica. 2012年，第19卷第2期，第75-79頁。In addition, as mentioned above, the use of homogenous and well-defined mesenchymal stem cell populations containing transgenes is an ideal candidate for clinical trials and cell-based therapies, because they fully meet, for example, the generally accepted standards for human mesenchymal stem cells. In, for example, cell therapy as defined by Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Vol. 8, No. 4, No. 315 -317 pages, Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review", Stem Cell Research & Therapy 2013, 4:66), Vonk et al., tem Cell Research & Therapy ( 2015) 6:94, or Kundrotas Acta Medica Lituanica. 2012, Volume 19, Number 2, pages 75-79.

如上所述，於第一方面，本發明涉及產生轉殖基因間質幹細胞群之方法，該方法包括透過酶調節的整合將轉殖基因***間質幹細胞群的基因組中，例如， 透過選自由一重組酶、一類轉錄活化劑效應子核酸酶，以及一核酸酶所組成之群組的酶進行整合，其中該間質幹細胞群為臍帶羊膜的分離的間質幹細胞群，其中至少約90%或更多的幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105。此處應當注意的是，任何臍帶羊膜的分離的間質幹細胞群，其中至少約90%或更多的幹細胞群的細胞表現以下標記中的每一種CD73、CD90以及CD105均可用於本發明。As described above, in the first aspect, the present invention relates to a method for generating a population of transgenic mesenchymal stem cells, which method includes inserting the transgene into the genome of the population of mesenchymal stem cells through enzyme-mediated integration, for example, by selecting from Recombinase, a type of transcriptional activator effector nuclease, and a group of enzymes consisting of a nuclease are integrated, wherein the mesenchymal stem cell population is an isolated mesenchymal stem cell population of umbilical cord amniotic membrane, of which at least about 90% or more The cells of the large stem cell population exhibited each of the following markers: CD73, CD90, and CD105. It should be noted here that any isolated mesenchymal stem cell population of umbilical cord amniotic membrane in which at least about 90% or more of the stem cell population's cells exhibit each of the following markers CD73, CD90, and CD105 can be used in the present invention.

在這種情況下，亦當指出的是，只要臍帶組織含有羊膜(這也稱為「臍帶襯裡」)，如本文所述之間質幹細胞群可以從任何臍帶組織中分離並培養(即衍生)。因此，除了羊膜之外，該臍帶組織可以包含臍帶的任何其他組織/組成分。例如，如在美國專利申請2006/0078993或國際專利申請WO2006/019357的圖16所示，該臍帶的羊膜為臍帶的最外部分，覆蓋著臍帶。此外，臍帶含有一條靜脈(攜帶含氧、營養豐富的血液給胎兒)以及二條動脈(攜帶去氧、營養物消耗的血液遠離胎兒)。為了保護與機械支持這三種血管嵌入華通氏膠(Wharton's Jelly)中，這種凝膠狀物質主要由黏多醣製成。In this case, it should also be pointed out that as long as the umbilical cord tissue contains amniotic membrane (this is also called "umbilical cord lining"), as described herein, the mesenchymal stem cell population can be isolated and cultured (ie derived) from any umbilical cord tissue . Therefore, in addition to the amniotic membrane, the umbilical cord tissue may contain any other tissue/component of the umbilical cord. For example, as shown in FIG. 16 of US Patent Application 2006/0078993 or International Patent Application WO2006/019357, the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord, covering the umbilical cord. In addition, the umbilical cord contains a vein (carrying oxygenated, nutrient-rich blood to the fetus) and two arteries (carrying deoxygenated, nutrient-depleted blood away from the fetus). In order to protect and mechanically support the three blood vessels embedded in Wharton's Jelly, this gel-like substance is mainly made of mucopolysaccharides.

間質幹細胞的分離可透過組織外植體(例如，包含臍帶的全部(完整)部分的外植體)開始，如果需要更大量的間質幹細胞，例如用於臨床試驗，則可隨後培養(培養作用)分離的間質幹細胞。或者，也可以首先將羊膜與臍帶的其它成分分離，並透過在本發明之培養基中培養羊膜來將間質臍帶襯裡幹細胞與羊膜分離。這種培養也可以透過組織外植體進行，任選地接著培養分離的間質幹細胞。在本文中，「組織外植體」或「組織外植體方法」等詞以其在本領域中的常規含義使用，係指一種方法，其中一旦組織被收穫，或一片組織被放置在含有培養(生長)培養基的細胞培養皿中，隨著時間的推移，幹細胞從組織中遷移出來到培養皿表面。然後，這些原代幹細胞可以進一步擴大，並透過微繁殖(培養)轉移到新鮮的培養皿中。Isolation of mesenchymal stem cells can be initiated by tissue explants (eg, explants that contain the entire (complete) part of the umbilical cord), and if larger amounts of mesenchymal stem cells are needed, such as for clinical trials, they can be subsequently cultured (cultivated Role) isolated mesenchymal stem cells. Alternatively, the amniotic membrane can be first separated from other components of the umbilical cord, and the interstitial umbilical cord lining stem cells can be separated from the amniotic membrane by culturing the amniotic membrane in the medium of the present invention. This culture can also be performed through tissue explants, optionally followed by culturing the isolated mesenchymal stem cells. In this article, the terms "tissue explant" or "tissue explant method" are used in their conventional meaning in the art and refer to a method in which once a tissue is harvested, or a piece of tissue is placed in a culture containing (Growth) In the cell culture dish of culture medium, stem cells migrate out of the tissue to the surface of the culture dish over time. Then, these primary stem cells can be further expanded and transferred to fresh petri dishes through micropropagation (cultivation).

如上所述，用於本發明之間質幹細胞群是從臍帶的羊膜中分離的間質幹細胞群(其在本文中也可互換，稱為「臍帶襯裡幹細胞」)。於一特定具體實施例中，該臍帶襯裡幹細胞為一間質幹細胞，其描述於國際專利申請WO 2006/019357或相應公開的美國專利申請US 2006/078993或授權的美國專利9,085,755中。因此，可以如WO 2006/019357、美國專利申請2006/078993或美國專利號9,085,755中所述分離臍帶羊膜的這種間質幹細胞。As described above, the mesenchymal stem cell population used in the present invention is a population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord (which is also interchangeable herein, referred to as "umbilical cord lining stem cells"). In a specific embodiment, the umbilical cord lining stem cell is a mesenchymal stem cell, which is described in the international patent application WO 2006/019357 or the corresponding published US patent application US 2006/078993 or issued US patent 9,085,755. Therefore, such mesenchymal stem cells of umbilical cord amniotic membrane can be isolated as described in WO 2006/019357, US Patent Application 2006/078993, or US Patent No. 9,085,755.

一旦被分離，可將該臍帶羊膜的間質幹細胞轉移到培養容器中進行培養。培養也可以從冷凍原代細胞，即從主細胞庫開始。針對培養，可將任何適量的細胞接種在培養容器，如細胞培養盤中。該間質細胞可以，為此目的，被懸浮在合適的培養基(最方便地，如本文所述之PPT-6培養基中)，用於在濃度為，例如，約0.5×106 個細胞/ml至約5.0×106 個細胞/ml進行培養。於一具體實施例中，該懸浮細胞以約1.0×106 個細胞/ml的濃度進行培養。培養可以透過在簡單的培養瓶中進行，以及例如，在一多層系統如CellStacks (康寧公司，康寧市，紐約州，美國)或Cellfactory (Nunc公司，Thermo Fisher Scientific公司的一部分，沃爾瑟姆，麻州，美國)，細胞可以被堆放在培育器中。Once separated, the mesenchymal stem cells of the umbilical cord amniotic membrane can be transferred to a culture container for cultivation. Cultivation can also start from freezing primary cells, ie from the main cell bank. For culture, any suitable amount of cells can be seeded in a culture container, such as a cell culture dish. The interstitial cells can, for this purpose, be suspended in a suitable medium (most conveniently, as described in PPT-6 medium as described herein) for use at a concentration of, for example, about 0.5×10 6 cells/ml Up to about 5.0×10 6 cells/ml were cultured. In a specific embodiment, the suspended cells are cultured at a concentration of about 1.0×10 6 cells/ml. Cultivation can be carried out in simple culture flasks, and for example, in a multi-layer system such as CellStacks (Corning Corporation, Corning City, New York, USA) or Cellfactory (Nunc Corporation, part of Thermo Fisher Scientific, Waltham) , Massachusetts, USA), cells can be stacked in an incubator.

另外，培養也可以在封閉的自含式系統，例如，生物反應器中進行。生物反應器的不同設計是本領域技術人員已知的，例如，平行平板，中空纖維，或微流體生物反應器。參閱例如，Sensebe等人「Production of mesenchymal stromal/stem cells according to good manufacturing practices：a review」，同上。商業中空纖維生物反應器的一個說明性的實例為Quantum® Cell Expansion System (Terumo BCT公司)，其，例如，已被用於擴增骨髓間質幹細胞以進行臨床試驗(參閱，Hanley等人，Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System， Cytotherapy ， 2014年八月；16(8年): 1048–1058)。可用於本發明之間質幹細胞群培養的市售生物反應器的另一個例子為，GE Heathcare公司提供的Xuri Cell Expansion System。如果要在GMP條件下生產用於治療應用的工作細胞庫並且需要大量細胞，則在自動化系統，如Quantum® 細胞擴增系統，中培養間質幹細胞群是特別有益的。在示例性具體實施例中，將間質幹細胞培養至間質幹細胞達到約70至約80%匯合。In addition, cultivation can also be performed in a closed self-contained system, for example, a bioreactor. Different designs of bioreactors are known to those skilled in the art, for example, parallel flat plates, hollow fibers, or microfluidic bioreactors. See, for example, Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", ibid. An illustrative example of a commercial hollow fiber bioreactor is the Quantum ® Cell Expansion System (Terumo BCT), which, for example, has been used to expand bone marrow mesenchymal stem cells for clinical trials (see, Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System , Cytotherapy , August 2014; 16 (8 years): 1048–1058). Another example of a commercially available bioreactor that can be used for the cultivation of mesenchymal stem cell populations of the present invention is the Xuri Cell Expansion System provided by GE Heathcare. If a working cell bank for therapeutic applications is to be produced under GMP conditions and a large number of cells are required, it is particularly beneficial to cultivate the mesenchymal stem cell population in an automated system, such as the Quantum ® cell expansion system. In an exemplary embodiment, the mesenchymal stem cells are cultured until the mesenchymal stem cells reach about 70 to about 80% confluence.

間質臍帶襯裡幹細胞群的培養可以在培養哺乳動物細胞的標準條件下進行。例如，人類臍帶組織與間質臍帶襯裡幹細胞通常分別在37ºC、5%CO2 的空氣氛圍中培養。在這種情況下，注意到在本發明中，該間質細胞可以源自任何哺乳動物物種，例如小鼠、大鼠、天竺鼠、兔、山羊、馬、狗、貓、綿羊、猴或人類，於一個具體實施例中具有人源的間質幹細胞為較佳的。The cultivation of the interstitial umbilical cord lining stem cell population can be carried out under standard conditions for the cultivation of mammalian cells. For example, human umbilical cord tissue and interstitial umbilical cord lining stem cells are usually cultured in an air atmosphere of 37ºC and 5% CO 2 respectively. In this case, it is noted that in the present invention, the mesenchymal cells may be derived from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey, or human, In a specific embodiment, human-derived mesenchymal stem cells are preferred.

一旦從培養物中獲得所需/合適數量的間質臍帶襯裡幹細胞，透過從用於培養的培養容器中除去間質幹細胞來收穫間質幹細胞。間質幹細胞的收穫通常透過酶處理再次進行，包括細胞的胰蛋白酶化。隨後收集分離的間質幹細胞，並將其直接使用或保存備用。通常，保存是透過冷凍保存進行的。「冷凍保存」乙詞在本文中以其常規含義使用，以描述間質幹細胞透過冷卻至低於零度以下的溫度，例如(通常)-80ºC或-196ºC (液態氮的沸點)來保存。可以如本領域技術人員已知的那樣進行冷凍保存，並且可以包括使用冷凍保護劑，例如二甲基亞碸(DMSO)或甘油，其減慢臍帶細胞中冰晶的形成。Once the desired/appropriate number of interstitial umbilical cord lining stem cells is obtained from the culture, the mesenchymal stem cells are harvested by removing the mesenchymal stem cells from the culture vessel used for culture. Mesenchymal stem cells are usually harvested again by enzyme treatment, including trypsinization of the cells. The isolated mesenchymal stem cells are then collected and used directly or saved for later use. Generally, preservation is carried out by cryopreservation. The term "cryopreservation" is used herein in its conventional meaning to describe mesenchymal stem cells stored by cooling to a temperature below zero degrees, such as (typically) -80ºC or -196ºC (boiling point of liquid nitrogen). Cryopreservation may be performed as known to those skilled in the art, and may include the use of cryoprotectants, such as dimethyl sulfoxide (DMSO) or glycerin, which slows the formation of ice crystals in umbilical cord cells.

透過本文所述之分離方法獲得的間質臍帶襯裡幹細胞的分離群高度確定且均質。在該方法的典型具體實施例中，至少約90%或更多的幹細胞群細胞表現下列標記中的每一個： CD73、CD90以及CD105。在較佳具體實施例中，至少約91%或更多、約92%或更多、約93%或更多、約94%或更多、約95%或更多、約96%或更多、約97%或更多、約98%或更多、約99%或更多的分離的間質幹細胞群的細胞為CD73、CD90以及CD105陽性(CD73+、CD90+以及CD105+)，這代表該百分比的分離細胞群表現CD73、CD90以及CD105的每一個(參閱本申請案實驗的部分)。另外，至少約90%或更多、約91%或更多、約92%或更多、約93%或更多、約94%或更多、約95%或更多、約96%或更多、約97%或更多、約98%或更多、約99%或更多的分離的間質幹細胞可能缺乏表現以下標記：CD34、CD45，以及HLA-DR，表示該百分比的分離細胞群缺乏表現(不表現)每種「陰性標記」 CD34、CD45，以及HLA-DR。在特定的具體實施例中，分離的間質幹細胞群中約97%或更多、約98%或更多、或約99%或更多表現CD73、CD90以及CD105，同時缺乏CD34、CD45以及HLA-DR的表現。The isolated population of interstitial umbilical cord lining stem cells obtained by the separation method described herein is highly defined and homogeneous. In a typical embodiment of the method, at least about 90% or more of the stem cell population cells exhibit each of the following markers: CD73, CD90, and CD105. In a preferred embodiment, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more , About 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cell population are CD73, CD90 and CD105 positive (CD73+, CD90+ and CD105+), which represents the percentage of The isolated cell population exhibited each of CD73, CD90, and CD105 (see the experiment section of this application). In addition, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more More, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack the following markers: CD34, CD45, and HLA-DR, indicating the percentage of isolated cell population Lack of performance (not performance) for each "negative marker" CD34, CD45, and HLA-DR. In specific embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cell populations exhibit CD73, CD90, and CD105, while lacking CD34, CD45, and HLA -DR performance.

於2018年10月5日提出的同在申請中的美國申請號15/725,913中首次報導了源自臍帶羊膜的這種高度同質的間質幹細胞群，該美國專利申請主張於2017年10月5日提出之美國臨時申請號62/404,582的優先權，其內容透過引用整體併入本文)以及於2018年10月5日提出的同在申請中的PCT申請PCT/SG2017/050500，其主張於2017年10月5日提出的美國臨時申請號62/404,582的優先權，並符合用於細胞治療的間質幹細胞的標準(亦參閱實驗部分，以及例如，Sensebe等人「Production of mesenchymal stromal/stem cells according to good manufacturing practices：a review」，上文)。在本文中注意到，如果需要的話，可以透過本發明之分離方法獲得這種間質幹細胞群，但也可以透過不同之方法，如細胞分選法，獲得。此處還注意的是，為了產生相應的轉殖基因幹細胞群，可以使用源自臍帶羊膜的任何此類同質間質幹細胞群，例如，從組織外植體(原代培養物)直接獲得的細胞群，或透過這種原代培養物的繼代培養獲得的細胞群。This highly homogenous mesenchymal stem cell group derived from umbilical cord amniotic membrane was first reported in the co-pending US application No. 15/725,913 filed on October 5, 2018. This US patent application claims on October 5, 2017 Priority of US Provisional Application No. 62/404,582 filed on the Japanese date, the content of which is incorporated by reference in its entirety) and the PCT application PCT/SG2017/050500 filed on October 5, 2018 in the same application, which claims in 2017 The priority of US Provisional Application No. 62/404,582, which was filed on October 5, 2014, and meets the standards for mesenchymal stem cells for cell therapy (see also the experimental section, and for example, Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", above). It is noted in this article that this mesenchymal stem cell population can be obtained by the separation method of the present invention if necessary, but can also be obtained by different methods, such as cell sorting. It is also noted here that in order to generate a corresponding colony of stem cells for transplantation, any such homogeneous mesenchymal stem cell population derived from umbilical cord amniotic membrane can be used, for example, cells obtained directly from tissue explants (primary culture) Population, or a population of cells obtained by subculture of this primary culture.

此外，用於本發明方法之間質幹細胞群可以源自任何哺乳動物物種，例如小鼠、大鼠、天竺鼠、兔、山羊、狗、貓、綿羊、猴、猿、馬、獼猴或人類，於一個具體實施例中具有人源的間質幹細胞為較佳的。In addition, the mesenchymal stem cell population used in the method of the present invention can be derived from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, dog, cat, sheep, monkey, ape, horse, macaque, or human, In a specific embodiment, human-derived mesenchymal stem cells are preferred.

標記CD73為技術人員已知的。於此方面，CD73係指分化簇73，也稱為5'-核苷酸酶(5'-nucleotidase，5'-NT)或ecto-5'-核苷酸酶。人類CD73蛋白的序列可能具有如SEQ ID NO. 33所示之序列。The label CD73 is known to the skilled person. In this regard, CD73 refers to differentiation cluster 73, also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase. The sequence of human CD73 protein may have the sequence shown in SEQ ID NO. 33.

標記CD90為技術人員已知的。於此方面，CD90係指分化簇90，也稱為胸腺細胞分化抗原1 (Thymocyte differentiation antigen 1，Thy-1)。人類CD90蛋白的序列可能具有如SEQ ID NO: 34所示之序列。The label CD90 is known to the skilled person. In this regard, CD90 refers to differentiation cluster 90, also known as Thymocyte differentiation antigen 1 (Thy-1). The sequence of human CD90 protein may have the sequence shown in SEQ ID NO: 34.

標記CD105為技術人員已知的。CD105也稱為内皮糖蛋白(Endoglin，ENG)。人類CD105蛋白的序列可能具有如SEQ ID NO: 35所示之序列。The label CD105 is known to the skilled person. CD105 is also called endoglin (Endoglin, ENG). The sequence of human CD105 protein may have the sequence shown in SEQ ID NO: 35.

如上所述，用於產生攜帶轉殖基因的間質幹細胞群的間質幹細胞群可能缺乏以下標記的表現：CD34、CD45以及HLA-DR。在該上下文中，注意到標記CD34、CD45以及HLA-DR為本領域技術人員已知的。人類CD34蛋白可能具有如SEQ ID NO. 36所示之序列。人類CD45蛋白可能具有如SEQ ID NO: 37所示之序列。人類HLA-DR蛋白可能具有如SEQ ID NO: 38所示之序列。As mentioned above, the mesenchymal stem cell population used to generate the mesenchymal stem cell population carrying the transgene may lack the expression of the following markers: CD34, CD45, and HLA-DR. In this context, note that the markers CD34, CD45 and HLA-DR are known to those skilled in the art. Human CD34 protein may have the sequence shown in SEQ ID NO. 36. Human CD45 protein may have the sequence shown in SEQ ID NO: 37. The human HLA-DR protein may have the sequence shown in SEQ ID NO: 38.

於本發明之具體實施例中，至少約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多的用於產生本發明之攜帶幹細胞群的轉殖基因的分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種。另外，至少約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多的用於產生本發明之攜帶幹細胞群的轉殖基因的分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，且缺乏表現CD34、CD45以及HLA-DR的每一種。In a specific embodiment of the present invention, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more More, about 97% or more, about 98% or more, about 99% or more of the cells used to generate the isolated mesenchymal stem cell population carrying stem cell population transgenes of the present invention express CD73, CD90 and Each of the CD105. In addition, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more More, about 98% or more, about 99% or more of the cells used to generate the isolated mesenchymal stem cell population carrying stem cell population transgenes of the present invention express each of CD73, CD90 and CD105, and Lack of expression of each of CD34, CD45 and HLA-DR.

現轉向本發明之所用的培養基，該培養基可包括分離或培養間質臍帶襯裡幹細胞DMEM，終濃度為約55-65% (v/v)，F12最終濃度為約5至15% (v/v)，M171的終濃度為約15至30% (v/v)，FBS的終濃度為約1至8% (v/v)。如本文所用，「% (v/v)」的值係指個體組成分相對於培養基的最終體積之體積。這表示如果DMEM例如存在於培養基中，最終濃度為約55至65% (v/v)，則1升培養基含有約550至650 ml DMEM。Turning now to the culture medium used in the present invention, the culture medium may include isolated or cultured interstitial umbilical cord lining stem cells DMEM at a final concentration of about 55-65% (v/v), and a final concentration of F12 of about 5 to 15% (v/v) ), the final concentration of M171 is about 15 to 30% (v/v), and the final concentration of FBS is about 1 to 8% (v/v). As used herein, the value of "% (v/v)" refers to the volume of the individual components relative to the final volume of the culture medium. This means that if DMEM is present in the medium, for example, and the final concentration is about 55 to 65% (v/v), then 1 liter of medium contains about 550 to 650 ml of DMEM.

於其他具體實施例中，培養基可包含終濃度為約57.5至62.5% (v/v)的DMEM，終濃度為約7.5至12.5% (v/v)的F12，終濃度為約17.5至25.0% (v/v)的M171，以及終濃度為約1.75至3.5% (v/v)的FBS。於進一步的具體實施例中，該培養基可包含終濃度為約61.8% (v/v)的DMEM，終濃度為約11.8% (v/v)的F12，終濃度為約23.6% (v/v)的M171，以及最終濃度為約2.5% (v/v)的FBS。In other specific embodiments, the culture medium may include DMEM with a final concentration of about 57.5 to 62.5% (v/v), F12 with a final concentration of about 7.5 to 12.5% (v/v), and a final concentration of about 17.5 to 25.0% (v/v) M171, and FBS with a final concentration of about 1.75 to 3.5% (v/v). In a further specific embodiment, the medium may comprise DMEM with a final concentration of about 61.8% (v/v), F12 with a final concentration of about 11.8% (v/v), and a final concentration of about 23.6% (v/v) ) And MBS with a final concentration of about 2.5% (v/v).

除了上述組成分外，該培養基還可包含有利於培養間質臍帶襯裡幹細胞的補充物。本發明之培養基可包括例如表皮生長因子(Epidermal Growth Factor，EGF)。如果存在，表皮生長因子(EGF)可以約1 ng/ml至約20 ng/ml的終濃度存在於培養基中。於某些這樣的具體實施例中，該培養基可包含終濃度為約10 ng/ml的表皮生長因子(EGF)。In addition to the above components, the medium may also contain supplements that are beneficial for culturing interstitial umbilical cord lining stem cells. The medium of the present invention may include, for example, epidermal growth factor (EGF). If present, epidermal growth factor (EGF) can be present in the culture medium at a final concentration of about 1 ng/ml to about 20 ng/ml. In some such specific embodiments, the medium may contain epidermal growth factor (EGF) at a final concentration of about 10 ng/ml.

本發明之培養基還可包含胰島素。如果存在，胰島素可以約1 μg/ml至10 μg/ml的終濃度存在。於某些這樣的具體實施例中，該培養基可包含終濃度為約5 μg/ml的胰島素。The medium of the present invention may also contain insulin. If present, insulin can be present at a final concentration of about 1 μg/ml to 10 μg/ml. In some such specific embodiments, the medium may contain insulin at a final concentration of about 5 μg/ml.

該培養基可進一步包含下列補充物中的至少一種：腺嘌呤、氫皮質酮，以及3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(3,3′,5-Triiodo-L-thyronine sodium salt，T3)。於此類具體實施例中，該培養基可包含全部三種腺嘌呤、氫皮質酮，以及3,3’,5-三碘-L-甲腺胺酸鈉鹽(T3)。於這些具體實施例中，該培養基可包含終濃度為約0.05至約0.1 μg/ml的腺嘌呤，最終濃度為約1至約10 μg/ml的氫皮質酮，及/或終濃度為約0.5至約5 ng/ml的3,3',5-三碘-L-甲狀腺胺酸鈉鹽(T3)。The medium may further contain at least one of the following supplements: adenine, hydrocorticosterone, and 3,3',5-triiodo-L-thyroxine sodium salt (3,3',5-Triiodo-L- thyronine sodium salt, T3). In such specific embodiments, the culture medium may contain all three adenine, hydrocorticosterone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these specific embodiments, the medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg/ml, hydrocorticosterone at a final concentration of about 1 to about 10 μg/ml, and/or a final concentration of about 0.5 3,3',5-triiodo-L-thyroxine sodium salt (T3) to about 5 ng/ml.

在一具體實施例中，該間質幹細胞在PTT-6培養基中培養，以獲得本文描述及使用之高度純化的間質幹細胞群。於本文中，應注意透過混合獲得如本文所述之PTT-6培養基以獲得最終體積為500 ml的培養基： i. 250 ml的DMEM ii. 118 ml的M171 iii. 118 ml的DMEM/F12 iv. 12.5 ml的胎牛血清(FBS)以達到一2.5% (v/v)的終濃度 v. EGF的終濃度為10 ng/ml vi. 胰島素的終濃度為5 μg/ml vii. 0.175 ml 胰島素(終濃度為5 μg/ml)。In a specific embodiment, the mesenchymal stem cells are cultured in PTT-6 medium to obtain a highly purified mesenchymal stem cell population described and used herein. In this article, it should be noted that the PTT-6 medium as described herein is obtained by mixing to obtain a medium with a final volume of 500 ml: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM/F12 iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v/v) v. The final concentration of EGF is 10 ng/ml vi. The final concentration of insulin is 5 μg/ml vii. 0.175 ml insulin (final concentration 5 μg/ml).

「DMEM」係指Dulbecco改良的eagle培養基，其係在1969年被開發，且為基礎培養基(BME)的改良(參閱圖1，顯示了Lonza公司的DMEM數據表)。最初的DMEM配方含有1000 mg/L的葡萄糖，並首次報導用於培養胚胎小鼠細胞。然後，DMEM自此成為可從各種來源，例如，Thermo Fisher Scientific公司(目錄型號11965-084)、Sigma Aldrich公司(目錄型號D5546)或Lonza公司至僅有少數供應商商購獲得的標準培養基。因此，任何可商購的DMEM都可用於本發明。於較佳的具體實施例中，本文使用的DMEM可從Lonza公司的目錄型號12-604F獲得之DMEM培養基。該培養基為補充有4.5 g/L葡萄糖與L-麩醯胺酸的DMEM)。於另一較佳的具體實施例中，本文使用的DMEM為Sigma Aldrich公司目錄型號D5546之DMEM培養基，其含有1000 mg/L葡萄糖與碳酸氫鈉，但不含L-麩醯胺酸。"DMEM" refers to Dulbecco's modified eagle medium, which was developed in 1969 and is an improvement of the basic medium (BME) (see Figure 1, which shows Lonza's DMEM data sheet). The original DMEM formula contained 1000 mg/L glucose and was reported for the first time to culture embryonic mouse cells. DMEM has since become a standard medium commercially available from various sources, for example, Thermo Fisher Scientific (catalogue model 11965-084), Sigma Aldrich (catalogue model D5546) or Lonza Corporation to only a few suppliers. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein can be obtained from Lonza's catalog model 12-604F. The medium is DMEM supplemented with 4.5 g/L glucose and L-glutamic acid. In another preferred embodiment, the DMEM used herein is Sigma Aldrich catalog model D5546 DMEM medium, which contains 1000 mg/L glucose and sodium bicarbonate, but does not contain L-glutamic acid.

「F12」培養基係指Ham's F12培養基。這種培養基也是一種標準的細胞培養基，其最初被設計用來培養各種哺乳動物與雜交瘤細胞的營養混合物，當與血清及激素與運鐵蛋白組合使用時(參閱圖2，顯示了來自Lonza公司的Ham's F12培養基之數據表)。任何可商購的Ham's F12培養基(例如，來自Thermo Fisher Scientific公司(目錄型號11765-054)、Sigma Aldrich公司(目錄型號N4888)或Lonza公司，僅用於少數幾個供應商)可用於本發明。於較佳的具體實施例中，使用來自Lonza公司的Ham's F12培養基。"F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, which was originally designed to cultivate a nutrient mixture of various mammalian and hybridoma cells when used in combination with serum and hormones and transferrin (see Figure 2 which shows the company from Lonza Data table of Ham's F12 medium). Any commercially available Ham's F12 medium (for example, from Thermo Fisher Scientific (catalogue model 11765-054), Sigma Aldrich (catalogue model N4888), or Lonza Corporation, only for a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza Corporation is used.

「DMEM/F12」或「DMEM：F12」係指DMEM與Ham's F12培養基的1:1混合物(參閱圖3，顯示來自Lonza公司的DMEM：F12 (1：1)培養基的數據表)。另外，DMEM/F12 (1：1)培養基是用於支持許多不同哺乳動物細胞生長的廣泛使用的基礎培養基，並且可從各種供應商，如Thermo Fisher Scientific公司(目錄型號11330057年)、Sigma Aldrich公司(目錄型號D6421)或Lonza公司商購。任何可商購的DMEM：F12培養基均可用於本發明。於較佳的具體實施例中，本文使用的DMEM：F12培養基為可從Lonza公司的目錄型號12-719F獲得的DMEM/F12 (1：1)培養基(其為具有L-麩醯胺酸、15 mM HEPES，以及3.151 g/L葡萄糖)。"DMEM/F12" or "DMEM: F12" refers to a 1:1 mixture of DMEM and Ham's F12 medium (see Fig. 3, which shows a data table of DMEM: F12 (1:1) medium from Lonza). In addition, DMEM/F12 (1:1) medium is a widely used basic medium for supporting the growth of many different mammalian cells, and can be obtained from various suppliers, such as Thermo Fisher Scientific (catalogue model 11330057), Sigma Aldrich (Catalogue Model D6421) or commercially available from Lonza. Any commercially available DMEM: F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM/F12 (1:1) medium (which has L-glutamic acid, 15 mM HEPES, and 3.151 g/L glucose).

「M171」係指培養基171，其係以作為用於培養正常人類***上皮細胞生長的基礎培養基而被開發(參閱圖4，顯示來自Life Technologies公司的M171培養基的數據表)。這種基礎培養基也被廣泛使用，例如可以從Thermo Fisher Scientific公司或Life Technologies公司(目錄型號M171500)等供應商處購得。任何市售的M171培養基均可用於本發明。在較佳的具體實施例中，本文使用的M171培養基為可獲自Life Technologies公司的目錄型號為M171500的M171培養基。"M171" refers to medium 171, which was developed as a basal medium for growing normal human breast epithelial cells (see FIG. 4, which shows a data table of M171 medium from Life Technologies). This basic medium is also widely used, for example, it can be purchased from suppliers such as Thermo Fisher Scientific or Life Technologies (catalog model M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is M171 medium available from Life Technologies as catalog model number M171500.

「FBS」係指胎牛血清(也稱為「胎牛血清」)，即在血液自然凝固之後保留的血液部分，然後離心以除去任何剩餘的紅血球細胞。胎牛血清為用於真核細胞體外細胞培養的最廣泛使用的血清補充物，因為其具有非常低的抗體含量並含有更多的生長因子，允許在許多不同的細胞培養應用中具有多功能性。FBS較佳從國際血清工業協會(International Serum Industry Association, ISIA)的成員獲得，其主要焦點為血清與動物衍生產品的安全及安全使用，透過適當的原產地可追溯性、標籤真實性，以及適當的標準化及監督。作為ISIA成員的FBS供應商包括Abattoir Basics公司、Animal Technologies公司、Biomin Biotechnologia LTDA公司、GE Healthcare公司、Thermo Fisher Scientific公司的Gibco，以及Life Science Production公司等。在目前較佳的具體實施例中，FBS從GE Healthcare公司獲得，目錄型號為A15-151。"FBS" means fetal bovine serum (also known as "fetal bovine serum"), which is the portion of blood that remains after the blood has naturally coagulated, and then centrifuged to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells because it has a very low antibody content and contains more growth factors, allowing versatility in many different cell culture applications . FBS is preferably obtained from members of the International Serum Industry Association (ISIA). Its main focus is the safe and safe use of serum and animal-derived products, through appropriate traceability of origin, authenticity of labels, and appropriate Standardization and supervision. FBS suppliers that are members of ISIA include Abattoir Basics, Animal Technologies, Biomin Biotechnologia LTDA, GE Healthcare, Gibco of Thermo Fisher Scientific, and Life Science Production. In the presently preferred embodiment, FBS is obtained from GE Healthcare, catalog number A15-151.

如上所述，製備用於分離本發明中使用之間質幹細胞群的培養基之方法包括混合以獲得最終體積為500ml的培養基： i. 250 ml的DMEM ii. 118 ml的M171 iii. 118 ml的DMEM/F12 iv. 12.5 ml的胎牛血清(FBS)以達到一2.5% (v/v)的終濃度。As described above, the method of preparing the medium for separating the mesenchymal stem cell population used in the present invention includes mixing to obtain a medium with a final volume of 500 ml: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM/F12 iv. 12.5 ml of fetal bovine serum (FBS) to achieve a final concentration of 2.5% (v/v).

如上所述，DMEM/F12培養基為DMEM與Ham's F12培養基的1：1混合物。因此，118 ml的DMEM/F12培養基含有59 ml的DMEM與59 ml的F12。據此，當使用這種培養基的製備方法時，符合500 ml總體積的終濃度(v/v)如下： - DMEM：250 ml+ 59 ml = 309 ml，相當於309/500 = 61.8% (v/v) - M171：118 ml，相當於118/500 = 23.6% (v/v) - F12：59 ml，相當於59/500 = 11.8% (v/v)。As mentioned above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM/F12 medium contains 59 ml of DMEM and 59 ml of F12. According to this, when using this medium preparation method, the final concentration (v/v) corresponding to the total volume of 500 ml is as follows: -DMEM: 250 ml + 59 ml = 309 ml, equivalent to 309/500 = 61.8% (v/v) -M171: 118 ml, equivalent to 118/500 = 23.6% (v/v) -F12: 59 ml, equivalent to 59/500 = 11.8% (v/v).

這種培養基的製備方法之具體實施例還包括添加 v. 1 ml的EGF原液(5 μg/ml)以達到10 ng/ml的最終EGF濃度，及 vi. 0.175 ml的胰島素原液(14.28 mg/ml)以達到5 μg/ml的最終胰島素濃度。Specific examples of the preparation method of this medium also include addition v. 1 ml of EGF stock solution (5 μg/ml) to reach a final EGF concentration of 10 ng/ml, and vi. 0.175 ml of insulin stock solution (14.28 mg/ml) to reach a final insulin concentration of 5 μg/ml.

這裡要注意的是，在這些具體實施例中，這些組成分i至vi的上述體積，導致最終體積為499.675 ml的培養基。如果不向培養基中加入其他組成分，剩餘的0.325 ml (加起來至500 ml的體積)可為，例如，組成分i至iv中的任何一種，這表示DMEM、M171、DMEM/F12或FBS任一。或者，EGF或胰島素的原液之濃度當然可以被調整，以使得培養基的總體積為500 ml。另外，還注意到組成分i.至iv.不一定必須按照它們被列出的順序添加，但是當然也可以使用任何順序來混合這些組成分以達到本發明之培養基。這代表，例如，可以將M171與DMEM/F12混合在一起，然後與DMEM及FBS混合，以達到如本文所述之終濃度，即一終濃度為約55至65% (v/v)的DMEM、一終濃度為約5-15% (v/v)的F12、一終濃度為約15-30% (v/v)的M171，以及一終濃度為約1-8% (v/v)的FBS。It should be noted here that in these specific examples, the aforementioned volumes of these components i to vi result in a final volume of 499.675 ml of culture medium. If no other components are added to the medium, the remaining 0.325 ml (totaling the volume to 500 ml) can be, for example, any of components i to iv, which means that DMEM, M171, DMEM/F12 or FBS One. Alternatively, the concentration of the stock solution of EGF or insulin can of course be adjusted so that the total volume of the culture medium is 500 ml. In addition, it is also noted that the components i. to iv. do not necessarily have to be added in the order in which they are listed, but of course it is also possible to mix these components in any order to achieve the medium of the present invention. This means that, for example, M171 and DMEM/F12 can be mixed together and then mixed with DMEM and FBS to achieve the final concentration as described herein, that is, a final concentration of about 55 to 65% (v/v) DMEM , A final concentration of about 5-15% (v/v) F12, a final concentration of about 15-30% (v/v) M171, and a final concentration of about 1-8% (v/v) Of FBS.

在其他具體實施例中，該方法進一步包括向DMEM中加入0.325 ml體積的一種或多種以下補充物：腺嘌呤、氫皮質酮、3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(T3)，從而到達總體積為500 ml的培養基。在這個具體實施例中，這些補充物在DMEM中的終濃度可以如下： 約0.05至0.1 μg/ml的腺嘌呤，例如約0.025 μg/ml腺嘌呤， 約1至10 μg/ml的氫皮質酮， 約0.5至5 ng/ml的3,3',5-三碘-L-甲狀腺胺酸鈉鹽(T3)，例如1.36 ng/ml的3,3',5-三碘-L-甲狀腺胺酸鈉鹽(T3)。In other specific embodiments, the method further includes adding to the DMEM a volume of one or more of the following supplements in a volume of 0.325 ml: adenine, hydrocorticosterone, 3,3',5-triiodo-L-thyroxine sodium salt (T3) to reach a total volume of 500 ml of culture medium. In this particular embodiment, the final concentration of these supplements in DMEM can be as follows: About 0.05 to 0.1 μg/ml adenine, for example about 0.025 μg/ml adenine, About 1 to 10 μg/ml hydrocorticosterone, About 0.5 to 5 ng/ml of 3,3',5-triiodo-L-thyroxine sodium salt (T3), for example 1.36 ng/ml of 3,3',5-triiodo-L-thyroxine Sodium salt (T3).

根據上述公開內容，本發明還涉及透過如本文所述之培養基的製備方法可獲得或獲得的細胞培養基。According to the above disclosure, the present invention also relates to a cell culture medium obtainable or obtained by a method for preparing a culture medium as described herein.

另外，本發明還涉及一種從臍帶羊膜分離間質幹細胞之方法，其中該方法包括在以本文所述之方法製備的培養基中培養羊膜組織。In addition, the present invention also relates to a method of separating mesenchymal stem cells from umbilical cord amniotic membrane, wherein the method includes culturing amnion tissue in a culture medium prepared by the method described herein.

因此，本發明還涉及一種細胞培養基(的用途)，包含 - 終濃度為約55至65% (v/v)的DMEM， - 終濃度為約5至15% (v/v)的F12， - 終濃度約為15至30% (v/v)的M171，以及 - 終濃度為約1至8% (v/v)的FBS。Therefore, the present invention also relates to a cell culture medium (use), comprising -DMEM with a final concentration of about 55 to 65% (v/v), -F12 with a final concentration of about 5 to 15% (v/v), -M171 with a final concentration of approximately 15 to 30% (v/v), and -FBS with a final concentration of about 1 to 8% (v/v).

於本文所述之培養基的某些具體實施例中，該培養基包含終濃度為約57.5至62.5% (v/v)的DMEM、終濃度為約7.5至12.5% (v/v)的F12、終濃度為約17.5至25.0% (v/v)的M171，以及終濃度為約1.75至3.5% (v/v)的FBS。於其他具體實施例中，該培養基可以包含終濃度為約61.8% (v/v)的DMEM、終濃度為約11.8% (v/v)的F12、終濃度為約23.6% (v/v)的M171，以及終濃度約為2.5% (v/v)的FBS。In some specific embodiments of the medium described herein, the medium includes DMEM with a final concentration of about 57.5 to 62.5% (v/v), F12 with a final concentration of about 7.5 to 12.5% (v/v), final The concentration of M171 is about 17.5 to 25.0% (v/v), and the final concentration of FBS is about 1.75 to 3.5% (v/v). In other specific embodiments, the medium may comprise DMEM with a final concentration of about 61.8% (v/v), F12 with a final concentration of about 11.8% (v/v), and a final concentration of about 23.6% (v/v) M171, and FBS with a final concentration of about 2.5% (v/v).

此外，該培養基可進一步包含終濃度為約1 ng/ml至約20 ng/ml的表皮生長因子(Epidermal Growth Factor，EGF)。於某些具體實施例中，該培養基包含終濃度為約10 ng/ml的EGf. 本文所述之培養基可進一步包含終濃度為約1 μg/ml至10 μg/ml的胰島素。於這樣的具體實施例中，該培養基可以包含終濃度為約5 μg/ml的胰島素。In addition, the medium may further include epidermal growth factor (EGF) at a final concentration of about 1 ng/ml to about 20 ng/ml. In certain embodiments, the medium contains EGf at a final concentration of about 10 ng/ml. The medium described herein may further contain insulin at a final concentration of about 1 μg/ml to 10 μg/ml. In such a specific embodiment, the medium may contain insulin at a final concentration of about 5 μg/ml.

本發明之細胞培養基可進一步包含以下補充物中的至少一種：腺嘌呤、氫皮質酮，以及3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(T3)。於某些具體實施例中，該培養基包含腺嘌呤、氫皮質酮，以及3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(T3)中的全部三種。如果存在的話，培養基可包含終濃度為約0.01至約0.1 μg/ml腺嘌呤或約0.05至約0.1 μg/ml腺嘌呤的腺嘌呤，終濃度為約0.1至約10 μg/ml氫皮質酮或約1至約10 μg/ml氫皮質酮的氫皮質酮，及/或終濃度為約0.5至約5 ng/ml的3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(T3)。The cell culture medium of the present invention may further contain at least one of the following supplements: adenine, hydrocorticosterone, and 3,3',5-triiodo-L-thyroxine sodium salt (T3). In certain embodiments, the medium contains adenine, hydrocorticosterone, and all three of 3,3',5-triiodo-L-thyroxine sodium salt (T3). If present, the culture medium may contain adenine at a final concentration of about 0.01 to about 0.1 μg/ml adenine or about 0.05 to about 0.1 μg/ml adenine at a final concentration of about 0.1 to about 10 μg/ml hydrocortisone or Hydrocorticosterone from about 1 to about 10 μg/ml hydrocorticosterone, and/or 3,3',5-triiodo-L-thyroxine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng/ml ).

於該細胞培養基的具體實施例中，500 ml的本發明之細胞培養基包含： i. 250 ml的DMEM ii. 118 ml的M171 iii. 118 ml的DMEM/F12 iv. 12.5 ml的胎牛血清(FBS)(終濃度為2.5%)。In a specific embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention includes: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM/F12 iv. 12.5 ml of fetal bovine serum (FBS) (final concentration 2.5%).

於進一步的具體實施例中，該細胞培養基可以進一步包含 v. 終濃度為10 ng/ml的EGF；以及 vi. 終濃度為5 μg/ml的胰島素。In a further specific embodiment, the cell culture medium may further comprise v. EGF with a final concentration of 10 ng/ml; and vi. Insulin at a final concentration of 5 μg/ml.

胰島素與EGF都可以使用選擇的原液加入到該培養基中，使得該培養基的總體積不超過500 ml。Both insulin and EGF can be added to the medium using the selected stock solution so that the total volume of the medium does not exceed 500 ml.

在特定的實例中，本發明之培養基的組成分i.至vi.為圖5中所示之組成分，這表示它們是從使用圖5中所示的目錄型號之各個製造商所獲得的。混合在圖5中所示之組成分i.至vi.所得之培養基在本文中亦稱為「PTT-6」。在本文中再次指出，組成分i.至vi.以及任何其他成分例如任何其他商業供應商的抗生素可用於製備本發明之培養基。In specific examples, the components i. to vi. of the medium of the present invention are the components shown in FIG. 5, which means that they were obtained from various manufacturers using the catalog models shown in FIG. The medium obtained by mixing the components i. to vi. shown in FIG. 5 is also referred to herein as "PTT-6". It is pointed out again herein that components i. to vi. and any other ingredients such as antibiotics from any other commercial suppliers can be used to prepare the medium of the present invention.

此外，本發明之細胞培養基可包含一終濃度為約0.01至約0.1 μg/ml腺嘌呤或約0.05至約0.1 μg/ml的腺嘌呤，終濃度為約0.1至10 μg/ml的氫皮質酮，約0.5至約10 μg/ml或約1至約10 μg/ml的氫皮質酮，及/或終濃度為約0.1至約5 ng/ml或約0.5至約5 ng/ml的3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(T3)。In addition, the cell culture medium of the present invention may contain a final concentration of about 0.01 to about 0.1 μg/ml adenine or about 0.05 to about 0.1 μg/ml adenine, and a final concentration of about 0.1 to 10 μg/ml hydrocortisone , About 0.5 to about 10 μg/ml or about 1 to about 10 μg/ml hydrocorticosterone, and/or 3,3 with a final concentration of about 0.1 to about 5 ng/ml or about 0.5 to about 5 ng/ml ',5-Triiodo-L-thyroxine sodium salt (T3).

如上所述，在本發明之一方面，一轉殖基因整合到本文所述之羊膜的間質幹細胞群中。在本文中，「轉殖基因」或「轉殖基因的」等詞在本文中根據其在本領域中的常規含義用於描述編碼核酸的片段，例如包含已從一種細胞類型(或生物體)分離並被引入不同的細胞類型(或生物體)的基因序列的DNA。如本文所用的該非天然核酸(DNA)區段將保留在轉殖基因細胞(或生物體)中產生所需RNA分子或所需多胜肽(蛋白質)的能力。該轉殖基因還可改變所獲得的轉殖基因細胞的遺傳密碼的正常功能。通常，將核酸分子(DNA序列)摻入本文所述之間質幹細胞群的種系中。該轉殖基因可以來自與接收間質幹細胞群相同的物種。例如，該轉殖基因可為一人類基因，例如，一編碼胰島素的基因或一編碼因子VIII的基因，且該受體細胞可為如本文所定義的臍帶羊膜的人類間質幹細胞群。透過將這樣的轉殖基因整合到人類間質幹細胞群中，所得到的轉殖基因人類間質幹細胞群將能夠產生相應的編碼蛋白，例如，親本間質幹細胞群根本不會產生，或僅產生在生理上無關緊要的量的胰島素或因子VIII。As described above, in one aspect of the present invention, a transgene is integrated into the amniotic mesenchymal stem cell population described herein. As used herein, the terms "transgenic gene" or "transgenic gene" are used herein to describe fragments encoding nucleic acids, according to their conventional meaning in the art, for example, including a cell type (or organism) that has been derived from DNA isolated and introduced into gene sequences of different cell types (or organisms). The unnatural nucleic acid (DNA) segment as used herein will retain the ability to produce the desired RNA molecule or the desired multiple peptide (protein) in the transgenic cell (or organism). The transgenic gene can also change the normal function of the genetic code of the transgenic cell obtained. Generally, nucleic acid molecules (DNA sequences) are incorporated into the germline of the mesenchymal stem cell population described herein. The transferred gene may be from the same species as the recipient mesenchymal stem cell population. For example, the transgene may be a human gene, for example, a gene encoding insulin or a gene encoding factor VIII, and the recipient cell may be a human mesenchymal stem cell population of umbilical cord amniotic membrane as defined herein. By integrating such a transgenic gene into a human mesenchymal stem cell population, the resulting human mesenchymal stem cell population will be able to produce the corresponding encoded protein, for example, the parental mesenchymal stem cell population will not produce at all, or only produce A physiologically insignificant amount of insulin or factor VIII.

轉殖基因可以例如透過任何合適的酶將其整合到本發明之幹細胞群的基因組中。用於將轉殖基因整合到哺乳動物(包括人類細胞)基因組中的合適的酶為本領域技術人員已知的。參閱，例如，O'Gorman等人，Recombinase-mediated gene activation and site-specific integration in mammalian cells Science 1991年，第251卷第4999期，第1351-1355頁；Anastassiadis等人，Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice Disease Models & Mechanisms 2, 508-515 (2009年)；Xu等人，Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome BMC Biotechnology 2013年，13:87；Karow與Calos (2011年) The therapeutic potential of phiC31 integrase as a gene therapy system, Expert Opinion on Biological Therapy, 11:10, 1287-1296；Reis等人，CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology, NEB expressions Issue I, 2014年(可從以下網頁獲取 https://www.neb.com/-/media/nebus/files/brochures/expressions_issuei_2014.pdf)。The transgene can be integrated into the genome of the stem cell population of the invention, for example, by any suitable enzyme. Suitable enzymes for integrating the transferred gene into the genome of mammals (including human cells) are known to those skilled in the art. See, for example, O'Gorman et al., Recombinase-mediated gene activation and site-specific integration in mammalian cells Science 1991, Vol. 251, No. 4999, pages 1351-1355; Anastassiadis et al., Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice Disease Models & Mechanisms 2, 508-515 (2009); Xu et al., Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome BMC Biotechnology 2013, 13:87; Karow and Calos (2011) The therapeutic potential of phiC31 integrase as a gene therapy system, Expert Opinion on Biological Therapy, 11:10, 1287-1296; Reis et al., CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology, NEB expressions Issue I, 2014 (available from the following web page https://www.neb.com/-/media/nebus/files/ brochures/expressions_issuei_2014.pdf).

與上述一致，用於將轉殖基因***間質幹細胞群的基因組中的酶調節的整合可以例如透過一酶進行，該酶選自一重組酶、一類轉錄活化劑效應子核酸酶，以及一核酸酶。Consistent with the above, the enzyme-regulated integration for inserting the transgene into the genome of the mesenchymal stem cell population can be performed, for example, by an enzyme selected from a recombinase, a type of transcriptional activator effector nuclease, and a nucleic acid Enzyme.

可用於本發明之重組酶的說明性實例包括酪胺酸重組酶(tyrosine recombinases，YR)以及絲胺酸重組酶(serine recombinases，SR)及其混合物。合適的酪胺酸重組酶的說明性實例包括，但不限於，Cre (導致Cre重組酶調節的整合)、Dre (導致Dre重組酶調節的整合)，以及Flp (導致Flp重組酶調節的整合)。絲胺酸重組酶的合適實例包括，但不限於，重組酶phiC31 (導致phiC31整合酶調節的整合)、Bxb1，以及R4。合適的核酸酶的說明性實例包括，但不限於，鋅指核酸酶(ZFN)(鋅指核酸酶調節的整合)或Cas9 (CRISPR/Cas9調節的整合)。於此方面亦參閱實驗部分，其詳述了透過鋅指核酸酶(ZFN)以及CRISPR/Cas9系統整合轉殖基因。Illustrative examples of recombinases that can be used in the present invention include tyrosine recombinases (YR) and serine recombinases (SR) and mixtures thereof. Illustrative examples of suitable tyrosine recombinases include, but are not limited to, Cre (causing integration of Cre recombinase regulation), Dre (causing integration of Dre recombinase regulation), and Flp (causing integration of Flp recombinase regulation) . Suitable examples of serine recombinase include, but are not limited to, the recombinase phiC31 (resulting in phiC31 integrase-regulated integration), Bxb1, and R4. Illustrative examples of suitable nucleases include, but are not limited to, zinc finger nuclease (ZFN) (zinc finger nuclease regulated integration) or Cas9 (CRISPR/Cas9 regulated integration). See also the experimental section in this regard, which details the integration of transgenic genes through zinc finger nuclease (ZFN) and the CRISPR/Cas9 system.

更詳細地解釋CRISPR/Cas9調節的整合，來自化膿性鏈球菌(Streptococcus pyogenes )的聚集的常規間隙短回文重複(Clustered Regularly Interspaced Short Palindromic Repeat，CRISPR)以及CRISPR相關蛋白9 (CRISPR-associated protein 9，Cas9)核酸酶，統稱為CRISPR-Cas9，已經徹底改變了基因組編輯與轉殖基因整合到人類基因組。該CRISPR-Cas9系統依賴於在特定CRISPR-RNA (crRNA)-反式活化crRNA (trans-activating crRNAs，tracrRNA)識別的目標基因組位點處的DSB誘導，統稱為嚮導RNA (guide RNA，gRNA)。CRISPR-Cas9 gRNA通常識別並結合20個核苷酸的長目標序列，通常具有稱為結合位點上游的原型間隔區相鄰基序(protospacer adjacent motif，PAM)的5'-NGG-3'三核苷酸序列。設計針對給定基因組區域的gRNA的簡易性、簡單性以及相對低的成本使得該基因組工程策略成為基因治療的較佳選擇。參閱本文中的Swystun與Lillicrap Gene Therapy for Coagulation Disorders Circulation Research 2016年；118：1443-1452。目前在中國及美國也正在進行臨床試驗，利用CRISPR-Cas9方法破壞PD-1及/或T細胞受體編碼基因，以測試更有效的CAR-T細胞療法(Cornu，T. I.，C. Mussolino等人(2017年)。"Refining strategies to translate genome editing to the clinic." Nature Medicine 23(4): 415-423)Explain in more detail the integration of CRISPR/Cas9 regulation, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) from Streptococcus pyogenes and CRISPR-associated protein 9 , Cas9) nucleases, collectively known as CRISPR-Cas9, has completely changed the genome editing and transgene integration into the human genome. The CRISPR-Cas9 system relies on DSB induction at target genomic loci recognized by specific CRISPR-RNA (crRNA)-trans-activating crRNAs (tracrRNAs), collectively referred to as guide RNA (gRNA). CRISPR-Cas9 gRNA usually recognizes and binds to a 20-nucleotide long target sequence, usually with a 5'-NGG-3' triple called a protospacer adjacent motif (PAM) upstream of the binding site Nucleotide sequence. The simplicity, simplicity, and relatively low cost of designing gRNAs for a given genomic region make this genome engineering strategy a better choice for gene therapy. See Swystun and Lillicrap Gene Therapy for Coagulation Disorders Circulation Research 2016; 118:1443-1452 in this article. Clinical trials are currently underway in China and the United States, using the CRISPR-Cas9 method to destroy the gene encoding PD-1 and/or T cell receptors to test more effective CAR-T cell therapy (Cornu, TI, C. Mussolino et al. (2017). "Refining strategies to translate genome editing to the clinic." Nature Medicine 23(4): 415-423)

當將它們用於轉殖基因整合時，可以考慮CRISPR-Cas9方法的偏離目標效應。gRNA序列識別可能在目標位點容許多達6-7個錯配的基因組序列的潛力可能導致CRISPR-Cas9系統的潛在偏離目標效應在進行基因組工程時產生。使用生物資訊學工具，如Cas-OFFinder (http://www.rgenome.net/cas-offinder/)、CROP-IT (http://cheetah.bioch.virginia.edu/AdliLab/CROP-IT/homepage .html)或CHOPCHOP (http://chopchop.cbu.uib.no/index.php)可用於識別給定的gRNA序列可識別的潛在偏離目標位點。隨後，可以透過例如Cel-I/Surveyor Mismatch核酸酶測定或目標擴增子深度定序等工具評估這些潛在的偏離目標位點，以研究DSB誘導在這些潛在的偏離目標位點上產生的INDELS (***/缺失)的存在。提高針對HDR與定點整合的基因組目標特異性的策略包括使用透過使突變不活化至配對的核酸酶之一而產生的Cas9切口酶。透過同源定向修復(homology directed repair，HDR)更有效地修復Cas9切口酶(dCas9)調節的ssDNA斷裂，其改善目標基因整合。其他策略包括使用更高效且特異性的Cas9直系同源物，可以識別更長的PAM (Ran, F. A., L. Cong等人(2015年)。「In vivo genome editing using Staphylococcus aureus Cas9.」 Nature 520(7546)：186-191或Muller, M., C. M. Lee 等人(2016年) 「Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.」 Molecular Therapy: The Journal of the American Society of Gene Therapy 24(3): 636-644)或Cas9同源物如Cpf1具有更強的DNA結合特異性，如Kleinstiver, B. P., S. Q. Tsai等人所述(2016年) 「Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells.」 Nature Biotechnology 34(8): 869-874。When they are used for transgene integration, the off-target effects of the CRISPR-Cas9 method can be considered. The potential of gRNA sequence recognition may allow up to 6-7 mismatched genomic sequences at the target site, which may lead to the potential off-target effects of the CRISPR-Cas9 system during genome engineering. Use bioinformatics tools such as Cas-OFFinder (http://www.rgenome.net/cas-offinder/), CROP-IT (http://cheetah.bioch.virginia.edu/AdliLab/CROP-IT/homepage .html) or CHOPCHOP (http://chopchop.cbu.uib.no/index.php) can be used to identify potentially off-target sites that can be recognized by a given gRNA sequence. Subsequently, tools such as Cel-I/Surveyor Mismatch nuclease assay or target amplicon depth sequencing can be used to evaluate these potential off-target sites to investigate the DSB-induced INDELS generated at these potentially off-target sites ( Insertion/deletion). Strategies to increase the specificity of genomic targets for HDR and site-directed integration include the use of Cas9 nickases produced by inactivating mutations to one of the paired nucleases. By homology directed repair (HDR), ssDNA breaks regulated by Cas9 nickase (dCas9) are more effectively repaired, which improves target gene integration. Other strategies include the use of more efficient and specific Cas9 orthologs that can recognize longer PAM (Ran, FA, L. Cong et al. (2015). "In vivo genome editing using Staphylococcus aureus Cas9." Nature 520 (7546): 186-191 or Muller, M., CM Lee et al. (2016) "Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome." Molecular Therapy: The Journal of the American Society of Gene Therapy 24 (3): 636-644) or Cas9 homologs such as Cpf1 have stronger DNA binding specificity, as described by Kleinstiver, BP, SQ Tsai et al. (2016) "Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology 34(8): 869-874.

「類轉錄活化劑效應子核酸酶」(Transcription activator-like effector nucleases，TALEN)乙詞在本文中以其在本領域中使用的常規含義來表示可以被工程化以切割特定DNA序列的限制酶(參閱，例如，Joung與Sander TALENs：a widely applicable technology for targeted genome editing, Nat Rev Mol Cell Biol 2013年元月; 14(1): 49–55)。它們透過將效應子DNA結合結構域與DNA切割結構域(切割DNA鏈的核酸酶)融合而製成。可以設計類轉錄活化劑效應子(Transcription activator-like effectors，TALEs)以實際上與任何所需的DNA序列結合，因此當與核酸酶組合時，可以在特定位置切割DNA。類轉錄活化劑效應子核酸酶(TALENs)類似於鋅指核酸酶(ZNF)，且通常包含與可客製化的DNA結合結構域融合的非特異性FokI核酸酶結構域。該Fok核酸酶可為野生型FokI切割結構域或具有突變的FokI切割結構域變體，其設計用於改善切割特異性，如Doyon Y等人，Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat Methods 8: 74-79 (2011年)中所述。DNA結合結構域由高度保守的重複序列組成，這些重複序列來源於類轉錄活化劑效應子(TALEs)，它是由黃單胞菌屬細菌分泌的蛋白質，可改變宿主植物細胞中基因的轉錄(再次參閱，Joung與Sander，2013年，同上)。合適的類轉錄活化劑效應子核酸酶的說明性實例也描述於Ousterout與Gersbach，The Development of TALE Nucleases for Biotechnology，Methods Mol Biol 2016年；1338：27-42。The term "Transcription activator-like effector nucleases" (TALEN) is used herein to refer to the restriction enzymes that can be engineered to cleave specific DNA sequences in its conventional meaning in the art ( See, for example, Joung and Sander TALENs: a widely applicable technology for targeted genome editing, Nat Rev Mol Cell Biol 2013 January; 14(1): 49–55). They are made by fusing effector DNA binding domains with DNA cleavage domains (nucleases that cleave DNA strands). Transcription activator-like effectors (TALEs) can be designed to bind virtually any desired DNA sequence, so that when combined with a nuclease, DNA can be cleaved at specific locations. Transcription activator-like effector nucleases (TALENs) are similar to zinc finger nucleases (ZNF), and usually contain a non-specific FokI nuclease domain fused to a customizable DNA binding domain. The Fok nuclease can be a wild-type FokI cleavage domain or a mutant FokI cleavage domain variant designed to improve cleavage specificity, such as Doyon Y et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures . Nat Methods 8: 74-79 (2011). The DNA binding domain is composed of highly conserved repeating sequences derived from transcriptional activator-like effectors (TALEs), which are proteins secreted by bacteria of the genus Xanthomonas and can alter the transcription of genes in host plant cells ( (See again, Joung and Sander, 2013, ibid.). Illustrative examples of suitable transcription activator-like effector nucleases are also described in Ousterout and Gersbach, The Development of TALE Nucleases for Biotechnology, Methods Mol Biol 2016; 1338: 27-42.

在這種情況下，應注意轉殖基因的整合可在公開的條件下進行，這些條件為本領域普通技術人員已知的，或者可以憑經驗確定。酶(其可為一重組酶、一類轉錄活化劑效應子核酸酶或一核酸酶)可以透過DNA表現匣容易地遞送或透過常規轉染方法直接作為mRNA遞送。此外，轉殖基因整合的基因組位點可為已知位點，基因組安全港(genomic safe harbors，GSHs)為較佳的整合位點-GSHs為基因組中能夠容納新遺傳物質整合的位點。確保新***的遺傳元件的方式：(i) 可預測的作用，以及(ii) 不引起宿主基因組的改變對宿主細胞或生物體構成風險。因此，基因組安全港(GSHs)為轉殖基因***的理想位點，其用途可以增強人類基因治療的基礎研究以及治療應用中的功能遺傳學研究(參閱Papapetrou與Schambach的綜述，Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy, Mol Ther. 2016年4月；24(4): 678-684)。In this case, it should be noted that the integration of the transgene can be performed under published conditions, which are known to those of ordinary skill in the art, or can be determined empirically. The enzyme (which can be a recombinase, a type of transcriptional activator effector nuclease or a nuclease) can be easily delivered through the DNA expression cassette or directly as mRNA through conventional transfection methods. In addition, the genomic locus of integration of the transgenic gene may be a known locus, and genomic safe harbors (GSHs) are better integration loci-GSHs are sites in the genome that can accommodate the integration of new genetic material. Ways to ensure that newly inserted genetic elements: (i) predictable effects, and (ii) do not cause changes in the host genome to pose a risk to the host cell or organism. Therefore, genomic safe harbors (GSHs) are ideal sites for the insertion of transgenic genes, and their use can enhance basic research in human gene therapy and functional genetic studies in therapeutic applications (see review by Papapetrou and Schambach, Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy, Mol Ther. April 2016; 24(4): 678-684).

如上所述，於本發明之具體實施例中，該重組酶可選自酪胺酸重組酶(YR)以及絲胺酸重組酶(SR)及其混合物。酪胺酸重組酶可為，例如，Cre、Dre或Flp，且該絲胺酸重組酶可為重組酶phiC31 (phiC31整合酶調節的整合)、Bxb1或R4重組酶調節的整合。As described above, in specific embodiments of the present invention, the recombinase may be selected from tyrosine recombinase (YR) and serine recombinase (SR) and mixtures thereof. The tyrosine recombinase may be, for example, Cre, Dre, or Flp, and the serine recombinase may be a recombinase phiC31 (phiC31 integrase-regulated integration), Bxb1 or R4 recombinase-regulated integration.

亦如上所述，該核酸酶可為鋅指核酸酶(ZFN)。於一示例性具體實施例中，轉殖基因可透過本發明之基於鋅指的整合方法整合到人類臍帶襯裡幹細胞的AAVS1基因座(存在於人類染色體19q13.3-qter上)。AAVS1基因座為本領域技術人員已知的，並且例如由Kotin、Linden以及Berns在EMBO Journal第11卷第13期第5071-5078頁，1992年所述。在WO2017/105350以及Sivalingam, J.等人，Molecular Therapy，2016年，第24卷第3期，第607-619頁中描述了將目標轉殖基因整合到臍帶羊膜的上皮幹細胞(也稱為臍帶襯裡上皮幹細胞)的AAVS1基因座中。As also mentioned above, the nuclease may be zinc finger nuclease (ZFN). In an exemplary embodiment, the transgene can be integrated into the AAVS1 locus of human umbilical cord lining stem cells (present on human chromosome 19q13.3-qter) by the zinc finger-based integration method of the present invention. The AAVS1 locus is known to those skilled in the art and is described, for example, by Kotin, Linden, and Berns in EMBO Journal Volume 11, Issue 13, pages 5071-5078, 1992. In WO2017/105350 and Sivalingam, J. et al., Molecular Therapy, 2016, Volume 24, Number 3, pages 607-619, the epithelial stem cells (also called umbilical cord) that integrate the target transgene into the umbilical cord amniotic membrane are described Lining epithelial stem cells) in the AAVS1 locus.

或者，可以透過本發明之CRISPR/Cas9調節的整合方法將轉殖基因整合到AAVS1基因座中。可以使用各自的市售套組實現基因靶向AAVS1基因座。一實施例為OriGene Technologies公司， Rockville，馬里蘭州，美國)的商業套組，其具有用於AAVS1靶向的一體化CRISPR載體(pCas-Guide-AAVS1)，具有AAVS1同源臂的AAVS1供體載體，其中轉殖基因為可將目標選殖到AAVS1-int5 PCR引子對中，以確認整合到AAVS1區域。合適的商業套組的另一實例為「Genome-CRISPR™ Human AAVS1 Safe Harbor Gene Knock-in Kit」型號SH004，可從GeneCopoeia公司，Rockville，馬里蘭州獲得。該套組包含選殖載體-Puro (來自GeneCopoeia公司，含有AAVS1 CRISPR-Cas9選殖株，具有AAVS1 sgRNA、AAVS1 MCS供體選殖載體-Puro，具有AAVS1同源臂以及選殖位點，用於選殖目標轉殖基因以及綠色螢光蛋白(GFP)報告基因以追蹤具有AAVS1整合的細胞。該套組還配有AAVS1 5'和3'驗證PCR引子，用於檢測整合。另一種合適的套組為AAVS1 Safe Harbor Targeting All-purpose HR Donor Vector 2.0 Promoterless (AAVS1-SA-puro-MCS)套組(GE620A-KIT)，可從System Biosciences (SBI)公司，Palo Alto，加州，美國獲得。該套組具有額外的特徵，例如具有剪接受體序列的無啟動子嘌呤黴素選擇匣，其僅在整合到AAVS1基因座中並且在被內源性AAVS1啟動子驅動時產生嘌呤黴素抗性。因此，此類套組為本領域普通技術人員提供了易於使用的工具，以將任何所需的轉殖基因整合到本發明中使用的間質幹細胞群的AAVS1基因座中，進而產生本發明之轉殖基因間質幹細胞群。Alternatively, the CRISPR/Cas9 regulated integration method of the present invention can be used to integrate the transgene into the AAVS1 locus. The respective commercially available kits can be used to achieve gene targeting at the AAVS1 locus. An example is the commercial kit of OriGene Technologies, Rockville, Maryland, USA, which has an integrated CRISPR vector (pCas-Guide-AAVS1) for AAVS1 targeting and an AAVS1 donor vector with AAVS1 homology arms , Where the transgenic gene is to clone the target into the AAVS1-int5 PCR primer pair to confirm integration into the AAVS1 region. Another example of a suitable commercial kit is "Genome-CRISPR™ Human AAVS1 Safe Harbor Gene Knock-in Kit" model SH004, available from GeneCopoeia, Rockville, Maryland. The kit contains the selection vector-Puro (from GeneCopoeia, containing AAVS1 CRISPR-Cas9 selection strain, with AAVS1 sgRNA, AAVS1 MCS donor selection vector-Puro, with AAVS1 homology arm and selection site for Select the target transgene and green fluorescent protein (GFP) reporter gene to track cells with AAVS1 integration. The kit is also equipped with AAVS1 5'and 3'verification PCR primers to detect integration. Another suitable kit The group is AAVS1 Safe Harbor Targeting All-purpose HR Donor Vector 2.0 Promoterless (AAVS1-SA-puro-MCS) kit (GE620A-KIT), available from System Biosciences (SBI), Palo Alto, California, USA. The group has additional features, such as a promoter-free puromycin selection cassette with a splice acceptor sequence, which only develops puromycin resistance when integrated into the AAVS1 locus and when driven by the endogenous AAVS1 promoter. Such kits provide those of ordinary skill in the art with easy-to-use tools to integrate any desired transgenes into the AAVS1 locus of the mesenchymal stem cell population used in the present invention, thereby generating the transformation of the present invention Colony of mesenchymal stem cells.

在該上下文中注意到，以轉殖基因轉染/電穿孔的間質幹細胞群保持其幹性以及幹細胞特徵，但可顯示表現間質幹細胞標記如CD73、CD90以及CD105的細胞百分比降低。時間也可以顯示表現陰性標記如CD34、CD45或HLA-DR的細胞百分比的增加。參閱，Yap等人，Transfected human mesenchymal stem cells do not lose their surface markers and differentiation properties, Malaysian J Pathol 2009年；31(2): 113-120；比照也是Madeira等人發表的，Nonviral Gene Delivery to Mesenchymal Stem Cells UsingCationic Liposomes for Gene and Cell Therapy, Journal of Biomedicine and Biotechnology. 第2010卷，文章識別號735349，12頁doi：10.1155/2010/735349。因此，不希望受理論束縛，本發明之轉殖基因間質幹細胞群，其為透過將轉殖基因整合/***本文所述之間質幹細胞群的基因組中並從臍帶的羊膜中分離而產生的，可為轉殖基因幹細胞群，其中至少約81%或更多，約82%或更多，至少83%或更多，至少84%或更多，至少約85%或約86%或更多，約87%或更多，約88%或更多，約89%或更多，約90%或更多，約91%或更多，約92%或更多，約93%或更多、約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多轉殖基因間質幹細胞群的細胞表現以下標記中的每一種： CD73、CD90以及CD105。同樣不希望受理論束縛，本發明之轉殖基因幹細胞群可為群體，其中至少約81%或更多，約82%或更多，至少83%或更多，至少84%或更多，至少約85%，或約86%或更多，約87%或更多，約88%或更多，約89%或更多，約90%或更多，約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多缺乏表現CD34、CD45以及HLA-DR的每一種。這種轉殖基因間質幹細胞群的一個較佳實例為轉殖基因間質幹細胞群，其中分離的間質幹細胞群的約97%或更多，約98%或更多約99%或更多的細胞表現CD73、CD90以及CD105的每一種，且缺乏表現CD34、CD45以及HLA-DR的每一種。It is noted in this context that the population of mesenchymal stem cells transfected/electroporated with the transgene maintains their stemness and stem cell characteristics, but may show a decrease in the percentage of cells expressing mesenchymal stem cell markers such as CD73, CD90 and CD105. Time can also show an increase in the percentage of cells that exhibit negative markers such as CD34, CD45 or HLA-DR. See, Yap et al., Transfected human mesenchymal stem cells do not lose their surface markers and differentiation properties, Malaysian J Pathol 2009; 31(2): 113-120; cf. also published by Madeira et al., Nonviral Gene Delivery to Mesenchymal Stem Cells UsingCationic Liposomes for Gene and Cell Therapy, Journal of Biomedicine and Biotechnology. Volume 2010, article identification number 735349, 12 pages doi: 10.1515/2010/735349. Therefore, without wishing to be bound by theory, the transgenic mesenchymal stem cell population of the present invention is produced by integrating/inserting the transgenic gene into the genome of the interstitial stem cell population described herein and separating it from the amniotic membrane of the umbilical cord , May be a population of transgenic stem cells, of which at least about 81% or more, about 82% or more, at least 83% or more, at least 84% or more, at least about 85% or about 86% or more , About 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, About 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more cells transgenic for the mesenchymal stem cell population Represents each of the following marks: CD73, CD90, and CD105. Without wishing to be bound by theory, the population of transgenic stem cells of the present invention may be a population, wherein at least about 81% or more, about 82% or more, at least 83% or more, at least 84% or more, at least About 85%, or about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% Or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or More lack of expression of each of CD34, CD45 and HLA-DR. A preferred example of such a transgenic gene mesenchymal stem cell population is a transgenic gene mesenchymal stem cell population, wherein about 97% or more of the isolated mesenchymal stem cell population, about 98% or more about 99% or more Of cells expressed each of CD73, CD90 and CD105, and lacked each of CD34, CD45 and HLA-DR.

於本發明之另一說明性具體實施例中，該轉殖基因可以整合，例如，透過phiC31整合酶調節的轉殖基因整合到DLC1的內含子7中，如Sivalingam, J.等人針對臍帶襯裡上皮幹細胞的描述，「Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity」 Gene Therapy (2014年) 21, 402–412.。In another illustrative embodiment of the present invention, the transgene can be integrated, for example, the transgene regulated by phiC31 integrase is integrated into intron 7 of DLC1, such as Sivalingam, J. et al. Description of lining epithelial stem cells, "Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity" Gene Therapy (2014) 21, 402–412.

根據以上於本發明方法之一具體實施例中，該方法包括透過突變的鋅指核酸酶***轉殖基因。於本發明方法之另一具體實施例中，該方法包括透過phiC31整合酶***轉殖基因。According to the above in one specific embodiment of the method of the present invention, the method includes inserting a transgenic gene through a mutated zinc finger nuclease. In another specific embodiment of the method of the present invention, the method includes inserting a transgene through phiC31 integrase.

於本文中，「phiC31整合酶」乙詞以其在本領域中的標準含義被使用，係指在噬菌體phiC31的基因組內編碼的位點特異性重組酶，其已成為用於精確整合轉殖基因的有用且可商購的工具。例如Kervala與Cavos所述，「Site-specific chromosomal integration mediated by phiC31 integrase.」 Methods Mol Biol. 2008年；435:165-73。In this article, the term "phiC31 integrase" is used in its standard meaning in the art, and refers to the site-specific recombinase encoded in the genome of the bacteriophage phiC31, which has become used for the precise integration of transgenes Of useful and commercially available tools. For example, Kervala and Cavos said, "Site-specific chromosomal integration mediated by phiC31 integrase." Methods Mol Biol. 2008; 435:165-73.

「鋅指核酸酶」乙詞 (當作為核酸酶或TALEN時)也以其本領域的標準含義被使用，例如本文引用的Guo等人，2010年以及本文引用的Doyon等人，2011年所述。簡而言之，鋅指核酸酶(ZFN)是透過將鋅指DNA結合結構域與DNA切割結構域融合而產生的人工限制酶。可以設計鋅指結構域以將特定的所需DNA序列作為目標，這使得鋅指核酸酶能夠將複雜基因組內的獨特序列作為目標。透過利用內源DNA修復機制，這些試劑可用於精確改變高等生物體的基因組。鋅指核酸酶為一種位點特異性內切核酸酶，設計用於在特定位置結合並切割DNA。其有兩個蛋白質結構域。第一個結構域為DNA結合域，其由真核轉錄因子所組成，含有鋅指蛋白。第二個結構域為核酸酶結構域，其通常由FokI限制酶組成並負責DNA的催化切割。限制酶FokI (在本文中也稱為Fok1)天然存在於海床黃桿菌(Flavobacterium okeanokoites )中，且為細菌型IIS限制性內切核酸酶，其由N端DNA結合結構域以及C端非特異性DNA切割結構域所組成。根據本發明，任何鋅指核酸酶均可用於本發明。FokI (切割)結構域可與任何合適的鋅指蛋白組合使用。鋅指核酸酶可為同源二聚體或異源二聚體。FokI限制酶可包含SEQ ID NO. 6的序列或僅包含該序列的催化結構域的序列。該催化結構域在以下的表1中以粗體字母描述於SEQ ID NO. 6中。該催化結構域包含196個胺基酸。The term "zinc finger nuclease" (when used as a nuclease or TALEN) is also used in its standard meaning in the art, such as Guo et al. cited in 2010, and Doyon et al. cited in 2011, as described in 2011 . In short, zinc finger nuclease (ZFN) is an artificial restriction enzyme produced by fusing a zinc finger DNA binding domain with a DNA cleavage domain. Zinc finger domains can be designed to target specific desired DNA sequences, which enables zinc finger nucleases to target unique sequences within complex genomes. By using endogenous DNA repair mechanisms, these reagents can be used to precisely alter the genomes of higher organisms. Zinc finger nuclease is a site-specific endonuclease designed to bind and cleave DNA at specific locations. It has two protein domains. The first domain is the DNA binding domain, which is composed of eukaryotic transcription factors and contains zinc finger proteins. The second domain is a nuclease domain, which usually consists of a FokI restriction enzyme and is responsible for the catalytic cleavage of DNA. The restriction enzyme FokI (also referred to herein as Fok1) is naturally present in Flavobacterium okeanokoites and is a bacterial type IIS restriction endonuclease, which is non-specific by the N-terminal DNA binding domain and the C-terminal Sexual DNA cleavage domain. According to the present invention, any zinc finger nuclease can be used in the present invention. The FokI (cleavage) domain can be used in combination with any suitable zinc finger protein. Zinc finger nucleases can be homodimers or heterodimers. The FokI restriction enzyme may comprise the sequence of SEQ ID NO. 6 or only the sequence of the catalytic domain of the sequence. The catalytic domain is described in SEQ ID NO. 6 in bold letters in Table 1 below. The catalytic domain contains 196 amino acids.

在說明性具體實施例中，該鋅指核酸酶在FokI 切割結構域FokI 中包含至少一個突變，以提供具有增強的切割活性的變體。例如，如本文所述之突變/取代可對應於SEQ ID NO. 6的野生型Fok I 序列的位置。In an illustrative embodiment, the zinc finger nuclease contains at least one mutation in the FokI cleavage domain FokI to provide variants with enhanced cleavage activity. For example, the mutation/substitution as described herein may correspond to the position of the wild-type Fok I sequence of SEQ ID NO.

在說明性具體實施例中，該突變的鋅指核酸酶可以在鋅指核酸酶的一個或兩個單體中包含E490K及/或I538K取代。另外或可替代地，突變的鋅指核酸酶可以在鋅指核酸酶的一個或兩個單體中包含Q468E以及I499L取代。另外或可替代地，突變的鋅指核酸酶可以在左側單體中包含Q468E以及I499L取代，並且在鋅指核酸酶的右側單體中包含E490K以及I538K取代。因此，突變的鋅指核酸酶可包含SEQ ID NOs. 7及/或8的序列。In an illustrative embodiment, the mutated zinc finger nuclease may include E490K and/or I538K substitutions in one or two monomers of the zinc finger nuclease. Additionally or alternatively, the mutated zinc finger nuclease may contain Q468E and I499L substitutions in one or two monomers of the zinc finger nuclease. Additionally or alternatively, the mutated zinc finger nuclease may include Q468E and I499L substitutions in the left monomer, and E490K and I538K substitutions in the right monomer of the zinc finger nuclease. Therefore, the mutated zinc finger nuclease may comprise the sequences of SEQ ID NOs. 7 and/or 8.

於進一步的說明性具體實施例中，該突變的鋅指核酸酶可另外或可選地在鋅指核酸酶的右及左單體中包含S418P與K441E取代。因此，突變的鋅指核酸酶可包含SEQ ID NOs. 9、12或13的序列。該鋅指核酸酶已由Guo等人，2010年所描述，並且也稱為「Sharkey變體」。In a further illustrative embodiment, the mutated zinc finger nuclease may additionally or alternatively include S418P and K441E substitutions in the right and left monomers of the zinc finger nuclease. Therefore, the mutated zinc finger nuclease may comprise the sequence of SEQ ID NOs. 9, 12, or 13. This zinc finger nuclease has been described by Guo et al. in 2010, and is also called "Sharkey variant".

於一示例性具體實施例中並且如Guo等人2010年所述，根據Guo等人使用的編號a，Sharkey變體的催化結構域可以在序列位置384至579處具有以下胺基酸序列(SEQ ID NO: 9)，取自Guo等人的補充圖2)。Sharkey切割結構域的相應核酸序列已經保藏於GenBank中，登錄號為HM130522：384 Gln Leu Val Lys Ser Glu 390 Leu Glu Glu Lys Lys Ser Glu Leu Arg His 400 Lys Leu Lys Tyr Val Pro His Glu Tyr Ile 410 Glu Leu Ile Glu IleAla Arg Asn Pro Thr 420 Gln Asp Arg Ile Leu Glu Met Lys Val Met 430 Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg 440 Gly Glu His Leu Gly Gly Ser Arg Lys Pro 450 Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro 460 Ile Asp Tyr Gly Val Ile Val Asp Thr Lys 470 Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile 480 Gly Gln Ala Asp Glu Met Gln Arg Tyr Val 490 Glu Glu Asn Gln Thr Arg Asn Lys His Ile 500 Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro 510 Ser Ser Val Thr Glu Phe Lys Phe Leu Phe 520 Val Ser Gly His Phe Lys Gly Asn Tyr Lys 530 Ala Gln Leu Thr Arg Leu Asn His Ile Thr 540 Asn Cys Asn Gly Ala Val Leu SerVal Glu 550 Glu Leu Leu Ile Gly Gly Glu Met Ile Lys 560 Ala Gly Thr Leu Thr Leu Glu Glu Val Arg 570 Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe 579，其中三字母胺基酸編碼序列對應於下面一個字母的胺基酸編碼序列，QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 9)。In an exemplary embodiment and as described by Guo et al. 2010, according to the number a used by Guo et al., the catalytic domain of the Sharkey variant may have the following amino acid sequence at sequence positions 384 to 579 (SEQ ID NO: 9), taken from Supplementary Figure 2 of Guo et al. The corresponding nucleic acid sequence of the Sharkey cleavage domain has been deposited in GenBank under the accession number HM130522: 384 Gln Leu Val Lys Ser Glu 390 Leu Glu Glu Lys Lys Ser Glu Leu Arg His 400 Lys Leu Lys Tyr Val Pro His Glu Tyr Ile 410 Glu Leu Ile Glu IleAla Arg Asn Pro Thr 420 Gln Asp Arg Ile Leu Glu Met Lys Val Met 430 Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg 440 Gly Glu His Leu Gly Gly Ser Arg Lys Pro 450 Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro 460 Ile Asp Tyr Gly Val Ile Val Asp Thr Lys 470 Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile 480 Gly Gln Ala Asp Glu Met Gln Arg Tyr Val 490 Glu Glu Asn Gln Thr Arg Asn Lys His Ile 500 Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro 510 Ser Ser Val Thr Glu Phe Lys Phe Leu Phe 520 Val Ser Gly His Phe Lys Gly Asn Tyr Lys 530 Ala Gln Leu Thr Arg Leu Asn His Ile Thr 540 Asn Cys Asn Gly Ala Val Leu SerVal Glu 550 Glu Leu Leu Ile Gly Gly Glu Met Ile Lys 560 Ala Gly Thr Leu Thr Leu Glu Glu Val Arg 570 Arg Lys Phe Asn Asn Asn Gly Glu Ile Asn Phe 579, where the three-letter amino acid coding sequence corresponds to the next letter of the amine Base acid coding sequence, QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGE HLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 9).

於以上描述之SEQ ID NO: 9的胺基酸序列中，催化中心的三個胺基酸Asp 450、Asp 467以及Lys 469以粗體、底線以及斜體突顯。同樣地，突變Ser418Pro以及Lys441Glu以粗體、底線以及斜體突顯。In the amino acid sequence of SEQ ID NO: 9 described above, the three amino acids Asp 450, Asp 467, and Lys 469 of the catalytic center are highlighted in bold, underline, and italics. Similarly, the mutations Ser418Pro and Lys441Glu are highlighted in bold, bottom line, and italics.

於本發明方法之其他具體實施例中，該突變的鋅指核酸酶可以另外地或替代地(例如，另外以E490K、I538K、Q468E及/或I499L取代)包含右Fok1 單體中的胺基酸取代S418P、K441E以及H537R，以及左Fok1 單體中的胺基酸取代S418P、K441E以及N496D。因此，突變的鋅指核酸酶可包含SEQ ID NOs. 8、9、10或11中任一個的序列，或可包含與SEQ ID NOs: 10、11、14及/或15的序列具有至少85%，86%，87%，88%，89%，90%，91%，92%，93%，94%，95%，96 %，97%，98%或99%的序列同一性。這些突變/取代在鋅指核酸酶中已由Doyon等人2011描述，並在本文中引用，並且也稱為「增強型Sharkey變體」。於上文描述的SEQ ID NO: 9的胺基酸序列中，位置496 (Asn496)以及537 (His537)的野生型序列以底線以及斜體突顯。因此，突變His537Arg以及Asn496Glu未顯示在SEQ ID NO: 9中。技術人員清楚的是，突變的鋅指核酸酶可以另外地或替代地包含胺基酸取代Q486E、I499L、E490K及/或I538K，亦如Doyon等人描述的，其在本文其他地方引述。In other specific embodiments of the method of the present invention, the mutated zinc finger nuclease may additionally or alternatively (eg, additionally be substituted with E490K, I538K, Q468E, and/or I499L) including the amino acid in the right Fok1 monomer Substitute S418P, K441E and H537R, and amino acids in the left Fok1 monomer replace S418P, K441E and N496D. Therefore, the mutated zinc finger nuclease may comprise the sequence of any one of SEQ ID NOs. 8, 9, 10, or 11, or may comprise at least 85% of the sequence of SEQ ID NOs: 10, 11, 14, and/or 15. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. These mutations/substitutions have been described by Doyon et al. 2011 in zinc finger nucleases and are cited herein, and are also referred to as "enhanced Sharkey variants". In the amino acid sequence of SEQ ID NO: 9 described above, the wild-type sequences at positions 496 (Asn496) and 537 (His537) are underlined and italicized. Therefore, the mutations His537Arg and Asn496Glu are not shown in SEQ ID NO: 9. It is clear to the skilled person that the mutated zinc finger nuclease may additionally or alternatively comprise amino acid substitutions Q486E, I499L, E490K and/or I538K, also as described by Doyon et al., which are cited elsewhere herein.

於進一步的具體實施例中，該突變的鋅指核酸酶(專性異二聚體)可以包含與右側AAVS1同源臂融合的Fok1 單體中的兩個胺基酸取代E490K以及I538K以及與左側同源臂融合的單體中的兩個胺基酸取代Q468E (或T486E)以及I499L。這些取代(與右側AAVS1同源臂融合的Fok1 單體中的E490K、I538K以及兩個胺基酸取代Q468E (或T486E)與I499L)也未在SEQ ID NO: 9中顯示，但是再次以底線及斜體突顯野生型序列在這些序列的位置。應當注意的是，本領域技術人員清楚的是，任何鋅指核酸酶在相應的Fok1 單體中(在同二聚體或異二聚體鋅指核酸酶中)包含上述突變，如只要Fok 單體提供所需的功能以將轉殖基因整合到如本文所述之間質幹細胞群的基因組中，例如，進入AAVS1基因座(存在於人類臍帶襯裡幹細胞的染色體19 q13.3-qter上)。因此，各Fok1 單體的任何功能片段或突變體均可用於本發明之方法中。在該上下文中，技術人員進一步清楚，如本文所述之FokI 中突變的位置均對應於野生型FokI 序列中的各個位置，如SEQ ID NO. 6所示。這表示，例如S418P表示如SEQ ID NO. 6所示的野生型FokI 的418位置的S突變為P。In a further specific embodiment, the mutated zinc finger nuclease (specific heterodimer) may include two amino acid substitutions E490K and I538K in the Fok1 monomer fused to the right AAVS1 homology arm and the left side Two amino acids in the homologous arm-fused monomer replace Q468E (or T486E) and I499L. These substitutions (E490K, I538K in the Fok1 monomer fused to the right AAVS1 homology arm and two amino acid substitutions Q468E (or T486E) and I499L) are also not shown in SEQ ID NO: 9, but again with the bottom line and Italics highlight the position of wild-type sequences in these sequences. It should be noted that it is clear to those skilled in the art that any zinc finger nuclease contains the above mutations in the corresponding Fok1 monomer (in homodimer or heterodimer zinc finger nuclease), as long as the Fok single The body provides the required function to integrate the transferred gene into the genome of the mesenchymal stem cell population as described herein, for example, into the AAVS1 locus (present on chromosome 19 q13.3-qter of human umbilical cord lining stem cells). Therefore, any functional fragment or mutant of each Fok1 monomer can be used in the method of the present invention. In this context, the skilled person is further clear that the positions of mutations in FokI as described herein correspond to the various positions in the wild-type FokI sequence, as shown in SEQ ID NO. 6. This means that, for example, S418P indicates that the S at the 418 position of the wild-type FokI shown in SEQ ID NO. 6 is mutated to P.

根據本發明，任何鋅指蛋白均可存在並用於本文所用之鋅指核酸酶中。例如，該鋅指核酸酶可包含選自具有胺基酸序列基序X2 -Cys-X2,4 -Cys-X12 -His-X3,4,5 -His的類Cys2 His2 鋅指蛋白的一鋅指蛋白，其中X可為任何胺基酸，數字表示殘基數(SEQ ID NO. 16；參閱下表1)，Gag-knuckle鋅指蛋白高音譜號，鋅帶鋅指蛋白或Zn2 /Cys6 鋅指蛋白，僅為舉幾例說明。在本發明方法的進一步說明性具體實施例中，該鋅指蛋白可選自P3鋅指蛋白、E2C (E6)鋅指蛋白、E5鋅指蛋白、E4鋅指蛋白或E3鋅指蛋白(參閱Guo等人，2010年，同上)。According to the present invention, any zinc finger protein can be present and used in the zinc finger nuclease used herein. For example, the zinc finger nuclease may comprise a Cys 2 His 2 zinc selected from amino acid sequence motifs X 2 -Cys-X 2,4 -Cys-X 12 -His-X 3,4,5 -His A zinc finger protein of finger protein, where X can be any amino acid, and the number represents the number of residues (SEQ ID NO. 16; see table 1 below), Gag-knuckle zinc finger protein treble clef, zinc-band zinc finger protein or Zn 2 /Cys 6 zinc finger protein is just a few examples. In a further illustrative embodiment of the method of the present invention, the zinc finger protein may be selected from P3 zinc finger protein, E2C (E6) zinc finger protein, E5 zinc finger protein, E4 zinc finger protein, or E3 zinc finger protein (see Guo Et al., 2010, ibid.).

整合到哺乳動物幹細胞的基因組中可在任何合適的溫度下進行，例如，在37°C，但也可以在更高的溫度(例如，高達42°C)或更低的溫度下進行。於本發明方法的一些具體實施例中，整合反應在約25°C至約32°C的溫度範圍內進行。於示例性具體實施例中，整合反應在約30°C的溫度下進行。Integration into the genome of mammalian stem cells can be performed at any suitable temperature, for example, at 37°C, but can also be performed at a higher temperature (for example, up to 42°C) or lower. In some embodiments of the method of the present invention, the integration reaction is performed at a temperature ranging from about 25°C to about 32°C. In an exemplary embodiment, the integration reaction is performed at a temperature of about 30°C.

在如本文所述之方法中，轉殖基因的整合通常透過轉染進行。在一些具體實施例中，使用單個質體進行轉染，該單個質體遞送例如鋅指核酸酶的兩種單體。在利用單個質體的這些具體實施例中，該單一載體可較佳使用Sharkey或增強型Sharkey AAVS1鋅指核酸酶單體進行轉染。根據上述公開內容，可以使用瞬間低溫進行轉染。In the method as described herein, the integration of the transgene is usually carried out by transfection. In some embodiments, a single plastid is used for transfection, which delivers two monomers such as zinc finger nuclease. In these specific embodiments using a single plastid, the single vector may be preferably transfected using Sharkey or enhanced Sharkey AAVS1 zinc finger nuclease monomers. According to the above disclosure, transient low temperature can be used for transfection.

如上所述，任何所需的轉殖基因可以整合到如本文所述之間質幹細胞群中。通常，轉殖基因可為適合於基因治療的任何核酸，這表示該核酸分子的重組表現可以改變、治療或預防宿主中的疾病，該宿主通常為包括患有(遺傳)功能障礙的包含人類的哺乳動物，該功能障礙導致缺乏產生胜肽，例如激素或任何所需多胜肽。在說明性具體實施例中，該轉殖基因選自編碼凝血因子的基因(核酸分子)以及編碼由內分泌腺分泌的蛋白激素的基因(核酸分子)。該凝血因子可以例如選自包括，但不限於，因子VII、因子VIII以及因子IX的群組。例如，因子VIII可具有SEQ ID NOs. 1、2及/或3中任一個的序列，或可具有與SEQ ID NOs. 1、2及/或3中任一個至少85%，86%，87%，88%，89%，90%，91%，92%，93%，94%，95%，96 %，97%，98%或99%的序列同一性的序列。於其他實例中，該缺陷可能是由內分泌腺分泌的蛋白激素的表現或分泌的缺乏，並且與內分泌缺乏有關。與內分泌缺乏相關的蛋白激素的這種缺乏可以選自胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常。As mentioned above, any desired transgene can be integrated into the mesenchymal stem cell population as described herein. Generally, the transgenic gene can be any nucleic acid suitable for gene therapy, which means that the recombinant performance of the nucleic acid molecule can change, treat or prevent a disease in a host, which usually includes humans including (genetic) dysfunction In mammals, this dysfunction leads to a lack of production of peptides, such as hormones or any desired multiple peptides. In an illustrative embodiment, the transgene is selected from a gene encoding a blood coagulation factor (nucleic acid molecule) and a gene encoding a protein hormone secreted by an endocrine gland (nucleic acid molecule). The coagulation factor may be selected from the group including, but not limited to, factor VII, factor VIII, and factor IX, for example. For example, Factor VIII may have the sequence of any one of SEQ ID NOs. 1, 2 and/or 3, or may have at least 85%, 86%, 87% of any of SEQ ID NOs. 1, 2 and/or 3 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In other examples, the defect may be the lack of expression or secretion of protein hormones secreted by the endocrine glands and is related to endocrine deficiency. This deficiency of protein hormones associated with endocrine deficiency may be selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormalities.

於一具體實施例中，該轉殖基因為編碼嵌合因子VIII多胜肽的基因(核酸分子)。該嵌合因子VIII多胜肽可為一嵌合蛋白，其具有人類區段或結構域以及來自非人類的哺乳動物的區段或結構域。於說明性具體實施例中，該轉殖基因可以編碼包含人類及豬結構域的嵌合因子VIII多胜肽。這種人-豬嵌合蛋白的說明性實例可為編碼嵌合因子VIII多胜肽的轉殖基因，該嵌合因子VIII多胜肽包含或由豬A1與A3結構域、人類訊息胜肽、人類A2結構域，殘留的人類B結構域以及人類C1與C2結構域所組成(參閱Sivalingam等人，2014年)。這種嵌合因子VIII可具有殘留的人類B結構域，其包含B結構域的前266個胺基酸以及8個糖基化位點。作為說明性實施例，該轉殖基因可以編碼具有1709個胺基酸長度的嵌合因子VIII多胜肽「Hybrid FVIII_KON lab」 (SEQ ID NO: 4)。或者，該轉殖基因可以編碼與SEQ ID NO: 4的多胜肽「Hybrid FVIII_KON lab」的序列具有至少85%，86%，87%，88%，89%，90%，91%，92%，93%，94%，95%，96%，97%，98%或99%的序列同一性的多胜肽。於另一具體實施例中，該轉殖基因可編碼稱為「Hybrid FVIII_Doering lab」的分子，其具有1467個胺基酸的長度(SEQ ID NO: 5)，其描述於Doering等人，Molecular Therapy vol. 17 no. 7, 1145–1154，2009年7月。In a specific embodiment, the transferred gene is a gene (nucleic acid molecule) encoding a chimeric factor VIII polypeptide. The chimeric factor VIII polypeptide can be a chimeric protein with a human segment or domain and a segment or domain from a non-human mammal. In an illustrative embodiment, the transgenic gene can encode a chimeric factor VIII polypeptide comprising human and porcine domains. An illustrative example of such a human-porcine chimeric protein may be a transgenic gene encoding a chimeric factor VIII polypeptide, which contains or consists of porcine A1 and A3 domains, human signaling peptides, The human A2 domain, the remaining human B domain, and the human C1 and C2 domains (see Sivalingam et al., 2014). This chimeric factor VIII may have a residual human B domain, which contains the first 266 amino acids of the B domain and 8 glycosylation sites. As an illustrative example, the transgenic gene may encode a chimeric factor VIII polypeptide "Hybrid FVIII_KON lab" (SEQ ID NO: 4) having a length of 1709 amino acids. Alternatively, the transgenic gene may encode a sequence of the multiple peptide "Hybrid FVIII_KON lab" of SEQ ID NO: 4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% of the peptides with sequence identity. In another specific embodiment, the transgene can encode a molecule called "Hybrid FVIII_Doering lab", which has a length of 1467 amino acids (SEQ ID NO: 5), which is described in Doering et al., Molecular Therapy vol. 17 no. 7, 1145–1154, July 2009.

於本文中，應當注意的是，本文所用的「同一性」或「序列同一性」係指測量其相似性或關係的序列的性質。本發明中使用的「序列同一性」或「同一性」等詞係指成對相同殘基的百分比-在本發明多胜肽序列與所述序列之後(同源性)比對-相對於這兩個序列中較長的殘基數。透過將相同殘基的數量除以殘基總數並將產物乘以100來測量同一性。In this article, it should be noted that "identity" or "sequence identity" as used herein refers to the nature of a sequence whose similarity or relationship is measured. The terms "sequence identity" or "identity" used in the present invention refer to the percentage of pairs of identical residues-after the polypeptide sequence of the present invention is aligned with the sequence (homology)-relative to this The number of longer residues in the two sequences. The identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.

根據以上公開內容，本發明提供了攜帶一轉殖基因的間質幹細胞群，其透過本文公開之方法獲得。本文描述的轉殖基因間質幹細胞群可以例如是以冷凍保存的形式存儲。According to the above disclosure, the present invention provides a population of mesenchymal stem cells carrying a transgene, which is obtained by the method disclosed herein. The population of transgenic mesenchymal stem cells described herein can be stored, for example, in the form of cryopreservation.

本發明還提供了透過本發明(第一方面)之方法獲得的轉殖基因間質幹細胞群。The present invention also provides a population of transgenic mesenchymal stem cells obtained by the method of the present invention (first aspect).

例如，如美國專利申請2006/0078993中所述，臍帶羊膜的間質幹細胞具有紡錘形狀並且可以表現以下基因：POU5f1、Bmi-1、白血病抑制因子(leukemia inhibitory factor，LIF)，並分泌活化素A與卵泡抑素。分離的(轉殖基因)間質幹細胞可以例如分化成任何類型的間質細胞，例如，但不限於，脂肪細胞、皮膚纖維母細胞、軟骨細胞、成骨細胞、肌腱細胞、韌帶纖維母細胞、心肌細胞、平滑肌細胞、骨骼肌細胞、產生黏蛋白的細胞、來自內分泌腺的細胞，例如產生胰島素的細胞(例如，β-胰島細胞)或神經外胚層細胞。轉殖基因間質幹細胞群可以在體外分化，以便隨後將分化細胞用於醫學目的。這種方法的一個示例性實例為將攜帶胰島素轉殖基因的轉殖基因間質幹細胞分化為產生胰島素的β-胰島細胞，然後可以例如透過植入將其施用於患有胰島素缺乏症(例如，糖尿病)的患者 (於這方面亦參閱WO2007/046775)。或者，本發明之轉殖基因間質幹細胞可以其未分化狀態用於基於細胞的療法，例如用於傷口癒合目的，例如治療燒傷或慢性糖尿病傷口。在這些治療應用中，本發明之間質幹細胞可以透過與周圍患病組織相互作用促進傷口癒合，或者也可以分化成相應的皮膚細胞(例如，再次參閱WO2007/046775)。For example, as described in US Patent Application 2006/0078993, umbilical cord amniotic mesenchymal stem cells have a spindle shape and can express the following genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF), and secrete activin A With follistatin. The isolated (transgenic gene) mesenchymal stem cells can, for example, differentiate into any type of mesenchymal cells, such as, but not limited to, fat cells, skin fibroblasts, chondrocytes, osteoblasts, tendon cells, ligament fibroblasts, Cardiomyocytes, smooth muscle cells, skeletal muscle cells, mucin-producing cells, cells from endocrine glands, such as insulin-producing cells (eg, β-islet cells), or neuroectodermal cells. The population of transgenic mesenchymal stem cells can be differentiated in vitro so that the differentiated cells can then be used for medical purposes. An illustrative example of such a method is the differentiation of transgenic mesenchymal stem cells carrying an insulin transgene into insulin-producing β-islet cells, which can then be administered to patients with insulin deficiency (eg, by implantation) Patients with diabetes) (see also WO2007/046775 in this regard). Alternatively, the transgenic mesenchymal stem cells of the present invention can be used in cell-based therapies in their undifferentiated state, for example for wound healing purposes, such as treating burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the present invention can promote wound healing by interacting with surrounding diseased tissues, or they can also differentiate into corresponding skin cells (for example, see WO2007/046775 again).

本發明還涉及本發明提供之轉殖基因間質幹細胞群的治療用途。可使用本發明之間質幹細胞群來治療任何疾病，只要透過其重組表現，如上所述，轉殖基因可以在宿主中通常為哺乳動物，包括患有一(遺傳)功能障礙的人類的哺乳動物。The invention also relates to the therapeutic use of the transgenic gene mesenchymal stem cell population provided by the invention. The mesenchymal stem cell population of the present invention can be used to treat any disease as long as through its recombinant expression, as described above, the transgenic gene can be usually a mammal in the host, including a mammal suffering from a (genetic) dysfunction.

該疾病可為與基因缺陷或基因表現缺陷相關的疾病。於這些具體實施例中，所述基因可選自編碼凝血因子的基因以及編碼內分泌腺分泌的蛋白激素的基因。該凝血因子可以，例如，選自因子VII、因子VIII以及因子IX。因此，該疾病可為血友病，例如A型血友病、B型血友病，或C型血友病。可以本發明之轉殖基因幹細胞群治療的另一種示例性疾病為遺傳性血栓性血小板減少性紫癜(Upshaw–Schulman Disease)，其為具有血小板反應蛋白第1型基序的去整合素以及金屬蛋白酶的遺傳形式，成員13 (ADAMTS13)缺乏(參閱本文中的Swystun與Lillicrap Gene Therapy for Coagulation Disorders Circulation Research 2016年；118: 1443-1452)。The disease may be a disease related to a genetic defect or a defective gene expression. In these specific embodiments, the genes may be selected from genes encoding coagulation factors and genes encoding protein hormones secreted by endocrine glands. The coagulation factor may, for example, be selected from factor VII, factor VIII and factor IX. Therefore, the disease may be hemophilia, such as hemophilia A, hemophilia B, or hemophilia C. Another exemplary disease that can be treated by the transgenic gene stem cell population of the present invention is hereditary thrombotic thrombocytopenic purpura (Upshaw-Schulman Disease), which is a deintegrin with a thrombospondin type 1 motif and a metalloprotease The hereditary form of member 13 (ADAMTS13) is lacking (see Swystun and Lillicrap Gene Therapy for Coagulation Disorders Circulation Research 2016; 118: 1443-1452 in this article).

或者，該疾病可能與內分泌缺乏有關。這種疾病可能與內分泌腺分泌的蛋白激素的表現或分泌不足有關。與內分泌缺乏相關的蛋白激素的缺乏可選自胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常。Or, the disease may be related to endocrine deficiencies. This disease may be related to the performance or insufficient secretion of protein hormones secreted by the endocrine glands. The deficiency of protein hormones associated with endocrine deficiency may be selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormalities.

在特定的具體實施例中，將本發明之轉殖基因間質幹細胞應用於傷口，例如，局部應用於傷口。這種傷口可為糖尿病傷口。於此情況下，該轉殖基因間質幹細胞可以，例如，轉殖基因性地分泌胰島素。In a specific embodiment, the transgenic mesenchymal stem cells of the present invention are applied to wounds, for example, applied locally to wounds. Such a wound may be a diabetic wound. In this case, the transgenic mesenchymal stem cells can, for example, transgenicly secrete insulin.

根據以上所述，本發明還提供了治療患有一疾病的患者之方法，該方法包括對該患者施用一轉殖基因間質幹細胞群或含有本文公開的轉殖基因間質幹細胞群之醫藥組合物。According to the above, the present invention also provides a method for treating a patient suffering from a disease, the method comprising administering to the patient a colony of mesenchymal stem cells or a pharmaceutical composition containing the colony of mesenchymal stem cells disclosed herein .

該疾病可為如上所述之任何疾病。為了治療該個體，本發明之轉殖基因間質幹細胞群可以任何合適的方式施用，例如，包括，但不限於，透過植入或注射。轉殖基因間質幹細胞可以例如皮下植入，例如直接植入皮膚下、體內脂肪或腹膜中。為了治療個體，本發明之轉殖基因間質幹細胞群因此可以以任何合適的方式施用，例如，包括，但不限於，局部施用，透過植入或透過注射。例如，該轉殖基因幹細胞群可以直接放置在傷口上，例如，燒傷或糖尿病傷口(參閱國際專利申請WO2007/046775)。或者，該轉殖基因間質幹細胞群也可以皮下植入，例如，直接植入皮膚下、體內脂肪或腹膜中。於一說明性實施例中，可透過腹腔鏡注射到網膜中將轉殖基因間質幹細胞群植入/施用給該患者，如Roth等人，於「Nonviral Transfer of the Gene Encoding Coagulation Factor VIII in Patients with Severe Hemophilia A」， N Engl J Med 2001年; 344:1735-1742中所述。The disease can be any disease as described above. In order to treat the individual, the population of transgenic mesenchymal stem cells of the present invention can be administered in any suitable manner, including, for example, but not limited to, by implantation or injection. Transgenic gene mesenchymal stem cells can be implanted subcutaneously, for example, directly under the skin, in body fat, or in the peritoneum. For the treatment of individuals, the population of transgenic mesenchymal stem cells of the present invention may therefore be administered in any suitable manner, for example, including, but not limited to, local administration, by implantation or by injection. For example, the population of transgenic stem cells can be placed directly on a wound, for example, a burn or a diabetic wound (see international patent application WO2007/046775). Alternatively, the transplanted gene mesenchymal stem cell population can also be implanted subcutaneously, for example, directly under the skin, in body fat, or in the peritoneum. In an illustrative embodiment, a colonic mesenchymal stem cell population can be implanted/administered to the patient through laparoscopic injection into the omentum, such as Roth et al., in "Nonviral Transfer of the Gene Encoding Coagulation Factor VIII in Patients with Severe Hemophilia A", N Engl J Med 2001; 344:1735-1742.

與上述一致，本發明還涉及包含本發明之轉殖基因間質幹細胞群的醫藥組合物。該醫藥組合物可包含任何藥學上可接受的賦形劑，並可配製成任何所需的藥學給藥方式。例如，該醫藥組合物可以適用於全身或局部應用。Consistent with the above, the present invention also relates to a pharmaceutical composition containing the transgenic mesenchymal stem cell population of the present invention. The pharmaceutical composition may contain any pharmaceutically acceptable excipients, and may be formulated into any desired way of pharmaceutical administration. For example, the pharmaceutical composition may be suitable for systemic or local application.

在下表1中顯示已用於本發明之序列： The sequences used in the present invention are shown in Table 1 below:

表1：本申請中使用的序列。Fok1的催化結構域在SEQ ID NO. 6中以粗體字母標示。以粗體、斜體以及底線突顯OH-鋅指核酸酶(ZFN)(E490K以及I538K或Q468E以及I499L)的突變；以紅色突顯的為sharkey突變S418P以及K441E，並以斜體及底線突顯增強型Sharkey突變體N496D與H537R的突變。Table 1: Sequences used in this application. The catalytic domain of Fok1 is indicated in bold letters in SEQ ID NO. The mutation of OH-zinc finger nuclease (ZFN) (E490K and I538K or Q468E and I499L) is highlighted in bold, italics and bottom line; the sharkey mutations S418P and K441E are highlighted in red, and the enhanced type is highlighted in italics and bottom line Sharkey mutants N496D and H537R mutations.

透過以下非限制性實驗實施例還將進一步說明本發明。The invention will be further illustrated by the following non-limiting experimental examples.

實驗的實施例Experimental examples

實施例Examples 11 ：顯示分離本發明之間質幹細胞群之實驗: Shows the experiment of separating the mesenchymal stem cell population of the present invention

1. 製備用於處理來自臍帶組織之 MSCs 的培養基： a. 為了製造500 ml的PTT-6 (培養/生長培養基)，按照以下順序添加以下內容物： i. DMEM，250 ml ii. M171 118 ml iii. DMEM F12 118 ml iv. FBS 12.5 ml (終濃度為2.5%) v. EGF 1ml (終濃度為10 ng/ml) vi. 胰島素0.175 ml (終濃度為5 μg/ml)。 上述含量的組成分i.至vi導致最終體積為499.675 ml的培養基。若不向培養基中加入其他組成分，剩餘的0.325 ml (加至500 ml的體積)可為，例如，組成分i至vi中的任何一種，這表示為DMEM、M171、DMEM/F12或FBS任一種。或者，EGF或胰島素原液的濃度當然可以被調整，使得該培養基的總體積為500 ml。或者，可以加入抗生素如青黴素-鏈黴素-兩性黴素的原液，使終體積為500 ml。也可以向該培養基中加入0.325 ml的一種或多種下列補充物：腺嘌呤、氫皮質酮、3,3’,5-三碘-L-甲狀腺胺酸鈉鹽(T3)，從而達到總體積為500 ml的培養基。 將「PTT6」的瓶子標示培養基的製備日期、起始操作員，以及在「到期日期」的詞彙後寫上到期日期。到期日期為任何組成分中的最早到期日期，或是自製備日起1個月內，以先到者為準。 b. 為了製備沖洗培養基(不含鈣或鎂，含有5% FBS的Hank's緩衝鹽溶液(HBSS))，在一50 ml離心管中加入2.5 ml FBS至47.5 ml的HBSS中。將「沖洗培養基」的管子標示起始操作員與該培養基的製備日期。 c. 使用Bactec Lytic/10-厭氧/F (Becton Dickinson公司)與Bactec Pluc+ 有氧/F (Becton Dickinson公司)對所有培養基進行無菌檢測。將20 ml製備的培養基注入每個瓶子中。 1. Prepare a medium for processing MSCs from umbilical cord tissue : a. In order to manufacture 500 ml of PTT-6 (cultivation/growth medium), add the following contents in the following order: i. DMEM, 250 ml ii. M171 118 ml iii. DMEM F12 118 ml iv. FBS 12.5 ml (final concentration 2.5%) v. EGF 1ml (final concentration 10 ng/ml) vi. Insulin 0.175 ml (final concentration 5 μg/ml). The above-mentioned components i. to vi result in a final volume of 499.675 ml of culture medium. If no other components are added to the medium, the remaining 0.325 ml (to a volume of 500 ml) can be, for example, any one of components i to vi, which is expressed as any of DMEM, M171, DMEM/F12 or FBS. One kind. Alternatively, the concentration of EGF or insulin stock solution can of course be adjusted so that the total volume of the medium is 500 ml. Alternatively, stock solutions of antibiotics such as penicillin-streptomycin-amphotericin can be added to make the final volume 500 ml. It is also possible to add 0.325 ml of one or more of the following supplements to this medium: adenine, hydrocorticosterone, 3,3',5-triiodo-L-thyroxine sodium salt (T3), so that the total volume is 500 ml of medium. Mark the "PTT6" bottle with the date of preparation of the medium, the start operator, and write the expiration date after the word "expiration date". The expiration date is the earliest expiration date in any component, or within 1 month from the preparation date, whichever comes first. b. To prepare a rinse medium (without calcium or magnesium, Hank's buffered salt solution (HBSS) containing 5% FBS), add 2.5 ml FBS to 47.5 ml HBSS in a 50 ml centrifuge tube. Mark the tube of "Flush Medium" with the starting operator and the date of preparation of the medium. c. Use Bactec Lytic/10-Anaerobic/F (Becton Dickinson) and Bactec Pluc+ Aerobic/F (Becton Dickinson) to perform sterility testing on all media. Inject 20 ml of prepared medium into each bottle.

2.分離間質幹細胞 2. Isolation of mesenchymal stem cells

如WO 2006/019357中所述，處理臍帶組織(在母親的知情同意下捐贈臍帶)用於隨後從臍帶的羊膜中分離間質幹細胞。As described in WO 2006/019357, the umbilical cord tissue (donated the umbilical cord with the informed consent of the mother) was processed for subsequent isolation of mesenchymal stem cells from the amniotic membrane of the umbilical cord.

2.1 準備組織處理： a. 臍帶組織處理應在一環境監測(environmentally monitored，EM)的潔淨室中進行。在每次輪班結束時，執行整個房間及通風櫥的清潔。 b. 準備/清潔生物安全櫃。 c. 在該生物安全櫃中工作時執行活體粒子的計數。 d. 在該生物安全櫃內組裝所有必要的用品，檢查每個包裝的損壞與有效日期。當處理注射器、血清移液管、無菌鑷子、解剖刀、組織板，以及針頭時，確保不要接觸到任何會接觸無菌產品的表面。只有注射器針筒、導管、柱塞頭及/或針帽或護套的外部可以安全地處理。若表面已被觸摸或觸及非無菌表面，丟棄該耗材。 e. 記錄要使用的所有試劑及耗材的批號與有效日期(如適用)。 f. 在轉移到生物安全櫃之前，以70%酒精潤濕的無絨抹布清潔小瓶，以接收解凍的小瓶。 g. 使用注射器抽吸針，從小瓶中取出盡可能多的液體。避免抽吸該組織。 h. 使用無菌鑷子，將該組織移轉到一個無菌的100 mm培養皿。 i. 向該組織碎片中加入一5 ml沖洗培養基的等分試樣。 j. 將內容物旋轉15-30秒，然後以移液管或帶有抽吸針的注射器取出沖洗培養基。重複該沖洗過程二次。 k. 添加2 ml沖洗培養基至該組織，以避免該組織乾燥。 2.1 Preparation of tissue treatment: a. Umbilical cord tissue treatment should be carried out in an environmentally monitored (EM) clean room. At the end of each shift, clean the entire room and fume hood. b. Prepare/clean the biological safety cabinet. c. Perform live particle counting while working in the biological safety cabinet. d. Assemble all necessary supplies in the biological safety cabinet and check the damage and expiry date of each package. When handling syringes, serum pipettes, sterile forceps, scalpels, tissue plates, and needles, make sure not to touch any surfaces that will touch sterile products. Only the outside of the syringe barrel, catheter, plunger head, and/or needle cap or sheath can be safely handled. If the surface has been touched or touched a non-sterile surface, discard the consumable. e. Record the batch number and expiration date of all reagents and consumables to be used (if applicable). f. Before transferring to a biosafety cabinet, clean the vial with a lint-free cloth moistened with 70% alcohol to receive the defrosted vial. g. Use a syringe to aspirate the needle and remove as much liquid as possible from the vial. Avoid aspiration of the tissue. h. Using sterile forceps, transfer the tissue to a sterile 100 mm Petri dish. i. Add an aliquot of 5 ml wash medium to the tissue fragments. j. Rotate the contents for 15-30 seconds, then remove the rinse medium with a pipette or syringe with a suction needle. Repeat the rinsing process twice. k. Add 2 ml of rinse medium to the tissue to prevent the tissue from drying out.

2.2. 從組織中啟動 MSC 生長： a. 將一6孔盤的底部標記「生長物1」以及MSC批號或臍帶組織ID，並開始生長日期。如果使用60 mm組織培養皿，則透過在培養皿底部畫一個格柵將培養皿分成4個象限。 b. 使用無菌的一次性鑷子，將一個3×3 mm至5×5 mm的組織放入每個孔中。若使用60 mm組織培養皿，則將組織放置在每個象限的中間以保持組織分離(彼此的距離超過1 cm)。 c. 以3 ml的PTT-6填充每個孔。 d. 使用連接到30 ml注射器的抽吸針，抽出足夠的培養基以幾乎覆蓋該組織。不要傾斜培養盤。不要用抽吸針觸摸孔的底部。 e. 使用倒置光學顯微鏡，每天(24±6小時) 觀察細胞生長。即時細胞培養成像系統可以用來代替光學顯微鏡。 f. 每天更換培養基。使用前務必將培養基平衡至室溫。 i. 吸走該培養基。 ii. 加入3 ml的PTT-6。 iii. 吸出，直到組織幾乎沒有浸在培養基中。 g. 當從組織中觀察到細胞外生時，使用與上述4.a至4.e相同的程序將組織移植至新的6孔盤，除了標記培養盤為「生長物2」之外。維持在「生長物1」盤中的細胞生長，每孔加入2 ml PTT-6。每天觀察匯合。每2-3天更換一次培養基(使用前務必將培養基平衡至室溫)。 h. 當在「生長物2」盤中觀察到細胞生長時，重複步驟4.a至4.e，除了標記板「生長物3」之外。透過向每個孔添加2 ml PTT6維持「生長物2」盤中的細胞生長。每天觀察匯合。每2-3天更換一次培養基(使用前務必將培養基平衡至室溫)。 i. 當在「生長物3」盤中觀察到生長物時，丟棄組織。如果組織非常小，並且似乎不干擾細胞生長，則在繼代時處理該組織。 j. 當細胞達到40-50%匯合時，每天觀察細胞以防止過度擴增。 k. 當細胞達到70-80%匯合時，繼代細胞。不要讓細胞擴張超過80%的匯合。 2.2. Start MSC growth from the tissue : a. Mark the bottom of a 6-well dish with "Growth 1" and the MSC lot number or umbilical cord tissue ID, and start the growth date. If using a 60 mm tissue culture dish, divide the culture dish into 4 quadrants by drawing a grid on the bottom of the culture dish. b. Using sterile disposable forceps, place a 3×3 mm to 5×5 mm tissue into each well. If using a 60 mm tissue culture dish, place the tissue in the middle of each quadrant to keep the tissue separated (the distance between each other exceeds 1 cm). c. Fill each well with 3 ml of PTT-6. d. Using a suction needle connected to a 30 ml syringe, draw enough media to almost cover the tissue. Do not tilt the culture plate. Do not touch the bottom of the hole with the suction needle. e. Use an inverted light microscope to observe cell growth every day (24±6 hours). An instant cell culture imaging system can be used instead of an optical microscope. f. Change the medium every day. Be sure to equilibrate the medium to room temperature before use. i. Aspirate the medium. ii. Add 3 ml of PTT-6. iii. Aspirate until the tissue is barely immersed in the medium. g. When extracellular growth is observed from the tissue, transplant the tissue to a new 6-well plate using the same procedure as in 4.a to 4.e above, except mark the culture plate as "Growth 2". To maintain the growth of cells in the "Growth 1" plate, add 2 ml PTT-6 to each well. Observe confluence every day. Change the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use). h. When cell growth is observed in the "Growth 2" plate, repeat steps 4.a to 4.e except for the marking plate "Growth 3". Maintain cell growth in the "Growth 2" plate by adding 2 ml PTT6 to each well. Observe confluence every day. Change the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use). i. When growth is observed in the "Growth 3" plate, discard the tissue. If the tissue is very small and does not seem to interfere with cell growth, then treat the tissue at passage. j. When the cells reach 40-50% confluence, observe the cells daily to prevent excessive expansion. k. When the cells reach 70-80% confluence, subculture the cells. Don't let cells expand more than 80% of confluence.

由於組織外植體的大小約為1-3 mm，並且組織外植體/細胞培養在175 mm平方的培養皿中進行，從外植體收穫的間質幹細胞的平均數通常為約4,000-6,000個細胞/外植體。因此，當從48個外植體中同時生長間質幹細胞時，可以在收穫時獲得約300,000個細胞。然後將這些從外植體收集的300,000個間質幹細胞透過接種如下面的實施例2.3中所述之這種300,000個細胞的175 cm2 細胞培養瓶進行繼代培養(這可稱為第一代)。然後可使用從第1代獲得的間質幹細胞再次種於175 cm2 培養瓶(第2代)，並如下面實施例2.5中所述使細胞擴張。從第一代與第二代獲得的細胞可以透過冷凍保存「儲存」，第二代之後獲得的間質幹細胞被認為代表用於進一步擴增間質幹細胞的主要細胞庫，例如，在下面的實施例2.5中所述之生物反應器中。Since the size of tissue explants is about 1-3 mm, and tissue explants/cell cultures are carried out in 175 mm square petri dishes, the average number of mesenchymal stem cells harvested from explants is usually about 4,000-6,000 Cells/explants. Therefore, when mesenchymal stem cells are simultaneously grown from 48 explants, approximately 300,000 cells can be obtained at harvest. These 300,000 mesenchymal stem cells collected from the explants were then subcultured through a 175 cm 2 cell culture flask seeded with 300,000 cells as described in Example 2.3 below (this can be called the first generation ). The mesenchymal stem cells obtained from the first generation can then be used to replant in 175 cm 2 culture flasks (second generation) and the cells expanded as described in Example 2.5 below. Cells obtained from the first and second generations can be "stored" by cryopreservation. Mesenchymal stem cells obtained after the second generation are considered to represent the main cell bank for further expansion of mesenchymal stem cells. For example, in the following implementation In the bioreactor described in Example 2.5.

2.3. MSC 在細胞培養皿中繼代培養 a. 在生物安全櫃中工作時執行監視活性粒子。使用前將所有培養基平衡至室溫。 b. 當細胞生長達到約70-80%匯合時，繼代培養細胞。 i. 從培養皿中取出PTT-6。 ii. 用不含鈣或鎂的HBSS沖洗。 iii. 加入0.2 ml 1X TrypLE-EDTA並以渦旋混合1-2分鐘。 iv. 傾斜該培養皿30-45°，讓細胞透過引力流向下移動。輕輕敲擊培養盤的一側，使其加速脫離。 v. 加入1 ml的PTT-6。輕輕地上下移液，然後將細胞轉移到15 ml離心管中。每個孔井使用乾淨的吸頭。來自所有6個孔的細胞可以合併到單個15 ml管中。 vi. 以1,200 rpm離心10分鐘。 vii. 去除上清液並用5 ml PTT6重新懸浮細胞。 c. 間質幹細胞(MSC)的繼代培養 i. 分裝50 μl的細胞懸浮液並透過台盼藍排除分析測定TNC與細胞存活率。 ii. 以血球細胞計數器計數細胞。預計數20-100個細胞/平方。如果計數高於100，則將原始樣品稀釋1：5，並以血球細胞計數器重複台盼藍法 iii. 計算活細胞/ml與總活細胞數： 1. 活細胞數/ml = 活細胞數×稀釋倍數×104 2. 總活細胞 = 活細胞數×稀釋因子×總體積×104 iv. 計算%存活率： 1. 活力% = 活細胞數×100 /(活細胞數+死細胞數) v. 將細胞懸浮液稀釋至1.0×106 個細胞/ml： 1. 「X」體積 = 總活細胞/106 個細胞/ml 2. 例如，如果總活細胞數為1.0×107 ； 3. 「X」= 107 /106 個細胞/ml或10 ml，因此，透過加入5 ml到該細胞懸浮液(即在5 ml)，總細胞體積將達到10 ml。 vi. 如果細胞懸浮液小於106 /ml，則確定每個150 mm培養皿或175 cm2 培養瓶接種2x106 個細胞所需的體積。 1. 2×106 個細胞的體積 = 2×106 個細胞 ÷ 活細胞/ml 2. 例如，如果活細胞/ml為8×105 細胞/ml，則需要2×106 個細胞÷8×105 個細胞/ml或2.5ml。 vii. 留出0.5 ml進行MSC標記分析。 viii. 接種2×106 細胞到每個具有30 ml PTT-6的150 mm培養皿或175 ml培養瓶。 ix. 每三天觀察細胞的附著、集落形成，以及匯合。當細胞達到40-50%匯合時，每隔一天觀察一次細胞以防止過度擴增。不要讓細胞擴張超過80%的匯合。可以使用即時細胞培養監測系統代替光學顯微鏡。 x. 每2-3天更換一次培養基。 2.3. Subculture of MSC in cell culture dishes a. Perform monitoring of active particles while working in a biological safety cabinet. Equilibrate all media to room temperature before use. b. When the cell growth reaches about 70-80% confluence, subculture the cells. i. Remove PTT-6 from the Petri dish. ii. Flush with HBSS without calcium or magnesium. iii. Add 0.2 ml 1X TrypLE-EDTA and mix by vortexing for 1-2 minutes. iv. Tilt the Petri dish 30-45° to allow the cells to move downward through gravity flow. Gently tap on the side of the culture plate to accelerate it off. v. Add 1 ml of PTT-6. Pipette up and down gently, then transfer the cells to a 15 ml centrifuge tube. Use clean tips for each well. Cells from all 6 wells can be combined into a single 15 ml tube. vi. Centrifuge at 1,200 rpm for 10 minutes. vii. Remove the supernatant and resuspend the cells with 5 ml PTT6. c. Subculture of mesenchymal stem cells (MSC) i. Aliquot 50 μl of cell suspension and determine TNC and cell survival rate by trypan blue exclusion analysis. ii. Count the cells with a hemocytometer. The estimated number is 20-100 cells/square. If the count is higher than 100, dilute the original sample 1:5 and repeat the trypan blue method with a hemacytometer iii. Calculate the number of viable cells/ml and total viable cells: 1. Number of viable cells/ml = number of viable cells × Dilution factor × 10 4 2. Total viable cells = viable cells × dilution factor × total volume × 10 4 iv. Calculate% survival rate: 1. Viability% = viable cells × 100 / (viable cells + dead cells) v. Dilute the cell suspension to 1.0×10 6 cells/ml: 1. “X” volume = total living cells/10 6 cells/ml 2. For example, if the total number of living cells is 1.0×10 7 ; 3 . "X" = 10 7 /10 6 cells/ml or 10 ml, therefore, by adding 5 ml to the cell suspension (ie at 5 ml), the total cell volume will reach 10 ml. vi. If the cell suspension is less than 10 6 /ml, determine the volume required to inoculate 2x10 6 cells per 150 mm Petri dish or 175 cm 2 flask. Volume 1. 2 × 10 6 cells was = 2 × 10 6 cells ÷ viable cells / ml 2. For example, if viable cells / ml of 8 × 10 5 cells / ml, you need to 2 × 10 6 cells ÷ 8 ×10 5 cells/ml or 2.5ml. vii. Set aside 0.5 ml for MSC label analysis. viii. Inoculate 2×10 6 cells into each 150 mm Petri dish or 175 ml culture flask with 30 ml PTT-6. ix. Observe cell attachment, colony formation, and confluence every three days. When the cells reach 40-50% confluence, observe the cells every other day to prevent excessive expansion. Don't let cells expand more than 80% of confluence. Instead of an optical microscope, an instant cell culture monitoring system can be used. x. Change the medium every 2-3 days.

2.4 冷凍保存 MSC 細胞 a. 在生物安全櫃中工作時執行監測活性粒子。 b. 當細胞達到70-80%匯合時，針對每個150 mm培養皿或175 cm2 培養瓶，使用2 ml 1X TrypLE-EDTA分離細胞。 i. 從培養皿中取出PTT6。 ii. 以5 ml HBSS或不含鈣或鎂的PBS洗滌。 iii. 加入2 ml 1X TrypLE-EDTA並渦旋混合1-2分鐘。 iv. 傾斜培養皿30-45°，讓細胞透過引力流向下移動。輕輕拍打培養皿一側有助於加速分離。 v. 加入10 ml PTT6以不活化TrypLE。充分混合以解離細胞團塊。 vi. 使用巴斯德移液管將細胞轉移到15 ml離心管中。 vii. 以1,200 rpm離心10分鐘。 viii. 以10 ml PTT6吸取培養基並重新懸浮。 ix. 分裝50 μl，並如上所述測定總活細胞數量以及 %存活率。細胞計數將需要在15分鐘內進行，因為細胞可能開始結塊。 c. 準備細胞以進行冷凍保存 i. 準備細胞懸浮液培養基以及低溫保存培養基並冷凍細胞。 2.4 Cryopreservation of MSC cells a. Perform active particle monitoring while working in a biological safety cabinet. b. When the cells reach 70-80% confluence, use 2 ml 1X TrypLE-EDTA to separate the cells for each 150 mm Petri dish or 175 cm 2 flask. i. Remove PTT6 from the Petri dish. ii. Wash with 5 ml HBSS or PBS without calcium or magnesium. iii. Add 2 ml 1X TrypLE-EDTA and vortex to mix for 1-2 minutes. iv. Tilt the Petri dish 30-45° to allow the cells to move downward through the gravitational flow. Gently tapping the side of the Petri dish helps to speed up the separation. v. Add 10 ml PTT6 to not activate TrypLE. Mix thoroughly to dissociate cell clumps. vi. Use a Pasteur pipette to transfer the cells to a 15 ml centrifuge tube. vii. Centrifuge at 1,200 rpm for 10 minutes. viii. Aspirate the medium with 10 ml PTT6 and resuspend. ix. Aliquot 50 μl and determine the total viable cell number and% survival rate as described above. The cell count will need to be performed within 15 minutes because the cells may start to clump. c. Prepare the cells for cryopreservation i. Prepare the cell suspension medium and cryopreservation medium and freeze the cells.

2.5. 在 Quantum 生物反應器 (Terumo BTC 公司 ) 中進行間質幹細胞 (MSC) 的培養 ( 擴增 ) 也能使用Quantum生物反應器來擴大間質幹細胞(MSC)。Quantum生物反應器中擴增的起始細胞數量應為每次運行20至30百萬個細胞。每輪收穫的典型產量為300至700百萬間質幹細胞(MSC)。生物反應器按照製造商之方法運行。如此獲得的間質幹細胞通常是低溫保存的(參閱下文)並可作為一用於產生本發明之轉殖基因間質幹細胞群的工作細胞庫。材料 / 試劑： 1. Quantum擴展套組 2. Quantum垃圾袋 3. Quantum培養基袋 4. Quantum入口袋 5. PTT6 6. PBS 7. 纖維連接蛋白 8. TrypLE 9. 3 ml注射器 10. 葡萄糖試紙 11. 乳酸鹽試紙 12. 60 ml細胞培養盤或等同物 13. 醫用級5%CO2 氣體混合物 14. 50 ml Combi-tip分注吸管。設備： 1. 生物安全櫃 2. 血糖儀(Bayer Healthcare公司/Ascensia Contour血糖儀) 3. Lactate Plus血乳酸分析儀(Nova Biomedical公司) 4. 蠕動幫浦 5. 離心機，Eppendorf 5810 6. 無菌管連接器 7. M4重複移液器 8. 射頻封口機程序： 1. 準備 Quantum生物反應器 a) 啟動Quantum生物反應器 b) 塗覆該生物反應器： 1) 在生物安全櫃中準備纖維連接蛋白溶液。 1) 使凍乾的纖維連接蛋白回溫至室溫(室溫下≥15分鐘) 2) 加5 ml無菌蒸餾水；不要以漩渦混合或攪動 3) 使纖維連接蛋白進入溶液30分鐘。 4) 使用裝有18g針頭的10 ml注射器，將纖維連接蛋白溶液轉移到含有95 ml PBS的細胞入口袋中。 2) 將袋子連接到「試劑」線上 3) 檢查氣泡(使用「移除IC空氣」或「移除EC空氣」並使用「清洗」作為入口源可能會除去氣泡)。 4) 打開或設置生物反應器塗層程序(圖1. 步驟3-5)。 5) 運行該程序 6) 當程序運行以塗覆生物反應器時，準備一個培養基袋與4 L的PTT6培養基。 7) 使用一無菌管連接器將培養基袋連接到IC培養基線。 8) 當生物反應器塗覆步驟完成時，使用RF封口機拆下用於纖維連接蛋白溶液的細胞入口袋。 c) 洗掉多餘的纖維連接蛋白 d) 以培養基調節該生物反應器 2.5. Culture ( expansion ) of mesenchymal stem cells (MSC) in Quantum bioreactor (Terumo BTC company ) The Quantum bioreactor can also be used to expand mesenchymal stem cells (MSC). The initial number of cells expanded in the Quantum bioreactor should be 20 to 30 million cells per run. The typical yield for each round of harvest is 300 to 700 million mesenchymal stem cells (MSC). The bioreactor is operated according to the manufacturer's method. The mesenchymal stem cells thus obtained are usually cryopreserved (see below) and can be used as a working cell bank for generating the population of transgenic mesenchymal stem cells of the present invention. Materials / Reagents: 1. Quantum expansion kit 2. Quantum garbage bag 3. Quantum culture bag 4. Quantum inlet bag 5. PTT6 6. PBS 7. Fibronectin 8. TrypLE 9. 3 ml syringe 10. Glucose test paper 11. Lactate test paper 12. 60 ml cell culture dish or equivalent 13. Medical grade 5% CO 2 gas mixture 14. 50 ml Combi-tip dispensing pipette. Equipment: 1. Biosafety cabinet 2. Blood glucose meter (Bayer Healthcare/Ascensia Contour blood glucose meter) 3. Lactate Plus blood lactate analyzer (Nova Biomedical) 4. Peristaltic pump 5. Centrifuge, Eppendorf 5810 6. Sterile tube Connector 7. M4 repeat pipette 8. RF sealer procedure: 1. Prepare Quantum bioreactor a) Start Quantum bioreactor b) Coat the bioreactor: 1) Prepare fibronectin in the biosafety cabinet Solution. 1) Warm the lyophilized fibronectin to room temperature (≥15 minutes at room temperature) 2) Add 5 ml of sterile distilled water; do not mix or agitate in a vortex 3) Put fibronectin into the solution for 30 minutes. 4) Using a 10 ml syringe equipped with an 18g needle, transfer the fibronectin solution to the cell inlet bag containing 95 ml PBS. 2) Connect the bag to the "Reagent" line 3) Check for air bubbles (using "Remove IC Air" or "Remove EC Air" and using "Clean" as the inlet source may remove air bubbles). 4) Open or set the bioreactor coating program (Figure 1. Steps 3-5). 5) Run the program 6) When the program is run to coat the bioreactor, prepare a culture bag and 4 L of PTT6 medium. 7) Use a sterile tube connector to connect the culture bag to the IC culture line. 8) When the bioreactor coating step is completed, use an RF sealer to remove the cell inlet bag for the fibronectin solution. c) Wash off excess fibronectin d) Adjust the bioreactor with medium

2.5.1 在Quantum生物反應器中培養細胞 e) 以均勻懸浮液加載並附著該細胞： f) 餵養並培養該細胞 1) 選擇培養基流速以餵養該細胞。 2) 每天取乳酸即葡萄糖樣品。 3) 隨著乳酸鹽含量的增加，調整培養基的流速。實際最大可耐受的乳酸鹽濃度將由細胞起源的培養瓶培養物來定義。確定培養基袋中是否有足夠的PTT-6培養基。如有必要，請以新的PTT-6培養基袋來更換PTT6培養基袋。 4) 當流量達到所需值時，每8-12小時測量一次乳酸含量。若乳酸含量不降低或乳酸含量繼續增加，則收穫細胞。2.5.1 Cultivating cells in Quantum bioreactor e) Load and attach the cells in a uniform suspension: f) Feed and cultivate the cell 1) Select the medium flow rate to feed the cells. 2) Take lactic acid or glucose samples every day. 3) As the content of lactate increases, adjust the flow rate of the medium. The actual maximum tolerable lactate concentration will be defined by the culture of the flask of cell origin. Determine whether there is enough PTT-6 medium in the medium bag. If necessary, replace the PTT6 medium bag with a new PTT-6 medium bag. 4) When the flow rate reaches the desired value, measure the lactic acid content every 8-12 hours. If the lactic acid content does not decrease or the lactic acid content continues to increase, the cells are harvested.

2.5.2 自Quantum生物反應器收穫細胞 g) 當乳酸濃度不降低時，最後一次採樣乳酸與葡萄糖後收穫細胞。 h) 收穫細胞： 1) 使用無菌管連接器將裝有100 ml TrypLE的細胞入口袋連接到「試劑」生產線。 2) 確認PBS袋中有足夠的PBS。如果沒有，使用無菌管連接器將一個至少有1.7 L PBS的新袋子連接到「清洗」線上。 3) 運行收穫程序。2.5.2 Harvesting cells from Quantum bioreactor g) When the concentration of lactic acid does not decrease, the cells are harvested after the last sampling of lactic acid and glucose. h) Harvest cells: 1) Use a sterile tube connector to connect the cell inlet bag containing 100 ml TrypLE to the "Reagent" production line. 2) Make sure there is enough PBS in the PBS bag. If not, use a sterile tube connector to connect a new bag with at least 1.7 L of PBS to the "cleaning" line. 3) Run the harvesting program.

使用不同培養基從臍帶組織分離的間質臍帶襯裡幹細胞群中幹細胞標記表現的分析Analysis of the expression of stem cell markers in interstitial umbilical cord lining stem cell populations isolated from umbilical cord tissue using different media

進行流式細胞儀實驗以分析從臍帶分離的間質幹細胞用於表現間質幹細胞標記CD73、CD90以及CD105。Flow cytometry experiments were performed to analyze mesenchymal stem cells isolated from the umbilical cord for expression of mesenchymal stem cell markers CD73, CD90, and CD105.

對於這些實驗，透過在三種不同培養基中培養臍帶組織，以從臍帶組織中分離間質幹細胞，然後如實施例2中所述在各培養基中繼代培養間質幹細胞。For these experiments, the mesenchymal stem cells were separated from the umbilical cord tissue by culturing the umbilical cord tissue in three different culture media, and then the mesenchymal stem cells were subcultured in each medium as described in Example 2.

在這些實驗中使用以下三種培養基：a) 90% (v/v/補充有10% (v/v) FBS的DMEM)，b) 美國專利申請案2006/0078993以及相應的國際專利申請案WO2006/019357中描述的培養基PTT-4，其係由90% (v/v) CMRL1066以及10% (v/v) FBS組成(參閱WO2006/019357的段落[0183])，以及c) 本發明之培養基PPT-6，在此描述了其組成分。在該流式細胞儀分析中，對三種所用培養基中的每一種分析了兩種不同的臍帶間質幹細胞群(cord lining mesenchymal stem cell ，CLMC)樣品。The following three media were used in these experiments: a) 90% (v/v/DMEM supplemented with 10% (v/v) FBS), b) US Patent Application 2006/0078993 and the corresponding international patent application WO2006/ The medium PTT-4 described in 019357 is composed of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO2006/019357), and c) the medium PPT of the present invention -6, the composition of which is described here. In this flow cytometry analysis, two different cord lining mesenchymal stem cell (CLMC) samples were analyzed for each of the three media used.

以下方案係用於流式細胞儀分析。材料與方法

Figure 108112053-A0304-0001
Figure 108112053-A0304-0002
 The following protocol is used for flow cytometry analysis. Materials and Methods
Figure 108112053-A0304-0001
Figure 108112053-A0304-0002

程序 a) 從臍帶襯膜分離與培養細胞 1. 將外植體組織樣品在細胞培養盤中培養並浸沒在各自的培養基中，然後如實施例2所述將其保持在37°C的CO2 培養箱中。 2. 培養基每3天更換一次。 3. 在光學顯微鏡下監測來自組織培養外植體的細胞生長。 4. 在約70%的匯合時，透過胰蛋白酶消化(0.0125%胰蛋白酶/0.05% EDTA)從培養皿中分離細胞並用於流式細胞儀實驗。 b) 用於實驗的細胞胰蛋白酶消化作用 1. 自細胞培養盤中移除培養基 2. 以無菌1X PBS輕輕沖洗以除去FBS的殘跡，因為FBS會干擾胰蛋白酶的酵素作用。 3. 將1X胰蛋白酶加入細胞培養盤中，並於37°C下培養3-5分鐘。 4. 在顯微鏡下觀察細胞，確認其脫落。透過加入含有FBS (含10% FBS的DMEM)的完全培養基以中和胰蛋白酶。 5. 使用移液器透過將培養基中的細胞吸移到培養皿的壁上來分解細胞團塊。收集細胞懸浮液並將其轉移到50 ml離心管中 6. 加入無菌1X PBS至細胞盤中並沖洗，收集細胞懸浮液至同一個離心管。 7. 以1800 rpm離心10分鐘。 8. 去除上清液，以PBA培養基重新懸浮細胞沉澱物。 c) 計數細胞 1. 確認血球細胞計數器與蓋玻片清潔乾燥，最好以70%乙醇清洗，並擦乾，然後以Kim wipes(無絨紙)擦拭。 2. 將懸浮的少量細胞等分於微量離心管中，並從BSC抽風櫥中取出。 3. 用等體積的台盼藍將懸浮細胞染色，向500 μl懸浮液中加入500 μl台盼藍(稀釋因子 = 2X，產生0.2%台盼藍溶液)。 4. 避免將細胞暴露於台盼藍超過30分鐘，因為台盼藍是有毒的，會導致死細胞的增加，進而導致錯誤的細胞計數結果。 5. 將20 μl細胞懸浮液混合物加入血球細胞計數器的每個腔室內，並在光學顯微鏡下觀察。 a. 在血球細胞計數器的每個象限中計數活細胞的數量(明亮的細胞；死細胞很容易吸收台盼藍，因此呈現暗色的)，在上下腔室中共有8個象限。 總細胞計數為(細胞/象限的平均數)×104 個細胞/ml。 d) 染色細胞 i. 細胞染色前的準備 • 將細胞懸浮液分裝至3個試管(CD73、CD90以及CD105)中，二重複，且二管陰性對照，每根試管中含有50,000個細胞。 ii. 以一級抗體(Ab)染色 • 向100 ul細胞懸浮液中加入1 μl [0.5 mg/ml Ab]一級抗體。在4°C下培養45分鐘。 • 以PBA補足1 ml體積。 • 在4°C下以8000 rpm離心5分鐘。 • 移除上清液。 • 加入1 ml PBA，重新懸浮沉澱 • 在4°C下以8000 rpm離心5分鐘。 • 移除上清液。 • 重新懸浮於100 ul PBA。 iii. 以二級抗體染色-在黑暗中 • 向100 ul細胞懸浮液中加入1ul [0.5 mg/ml ab]二級抗體。在4°C下培養30分鐘。 • 以PBA補足1ml體積。 • 在4°C下以8000 rpm離心5分鐘。 • 移除上清液。 • 加入1 ml PBA，重新懸浮沉澱 • 在4°C下以8000 rpm離心5分鐘。 • 移除上清液 • 重新懸浮於200-300 ul PBA以進行流式細胞儀分析 • 將細胞轉移到FACS管中以在BD FACS CANDO流式細胞儀中讀取數值。 Procedure a) Isolation and culture of cells from the umbilical cord lining membrane 1. The explant tissue samples were cultured in cell culture dishes and immersed in the respective culture medium, and then maintained at 37°C CO 2 as described in Example 2. In the incubator. 2. The medium is changed every 3 days. 3. Monitor cell growth from tissue culture explants under an optical microscope. 4. At about 70% confluence, separate the cells from the culture dish by trypsin digestion (0.0125% trypsin/0.05% EDTA) and use them for flow cytometry experiments. b) Trypsin digestion of cells used in the experiment 1. Remove the culture medium from the cell culture plate 2. Rinse gently with sterile 1X PBS to remove the residue of FBS, because FBS will interfere with the enzyme effect of trypsin. 3. Add 1X trypsin to the cell culture dish and incubate at 37°C for 3-5 minutes. 4. Observe the cells under a microscope to confirm their shedding. Trypsin was neutralized by adding complete medium containing FBS (DMEM with 10% FBS). 5. Use a pipette to break up cell clumps by pipetting the cells in the culture medium onto the walls of the Petri dish. Collect the cell suspension and transfer it to a 50 ml centrifuge tube. 6. Add sterile 1X PBS to the cell dish and rinse, collect the cell suspension into the same centrifuge tube. 7. Centrifuge at 1800 rpm for 10 minutes. 8. Remove the supernatant and resuspend the cell pellet in PBA medium. c) Counting cells 1. Make sure that the hemacytometer and coverslip are clean and dry, preferably washed with 70% ethanol and wiped dry, and then wiped with Kim wipes. 2. Aliquot a small amount of suspended cells into microcentrifuge tubes and remove them from the BSC fume hood. 3. Stain the suspension cells with an equal volume of trypan blue, add 500 μl of trypan blue to 500 μl of suspension (dilution factor = 2X, resulting in 0.2% trypan blue solution). 4. Avoid exposing the cells to trypan blue for more than 30 minutes, because trypan blue is toxic and can lead to an increase in dead cells, which can lead to erroneous cell count results. 5. Add 20 μl of the cell suspension mixture to each chamber of the hemacytometer and observe under an optical microscope. a. Count the number of live cells in each quadrant of the hemocytometer (bright cells; dead cells easily absorb trypan blue, so it appears dark), there are 8 quadrants in the upper and lower chambers. The total cell count is (average number of cells/quadrant)×10 4 cells/ml. d) Stained cells i. Preparation before cell staining • Dispense the cell suspension into 3 test tubes (CD73, CD90, and CD105), repeat in duplicate, and two tubes of negative control, each test tube contains 50,000 cells. ii. Stain with primary antibody (Ab) • Add 1 μl [0.5 mg/ml Ab] primary antibody to 100 ul of cell suspension. Incubate at 4°C for 45 minutes. • Make up to 1 ml volume with PBA. • Centrifuge at 8000 rpm for 5 minutes at 4°C. • Remove the supernatant. • Add 1 ml PBA and resuspend the pellet • Centrifuge at 8000 rpm for 5 minutes at 4°C. • Remove the supernatant. • Resuspend in 100 ul PBA. iii. Stain with secondary antibody-in the dark • Add 1 ul [0.5 mg/ml ab] secondary antibody to 100 ul of cell suspension. Incubate at 4°C for 30 minutes. • Make up 1ml volume with PBA. • Centrifuge at 8000 rpm for 5 minutes at 4°C. • Remove the supernatant. • Add 1 ml PBA and resuspend the pellet • Centrifuge at 8000 rpm for 5 minutes at 4°C. • Remove supernatant • Resuspend in 200-300 ul PBA for flow cytometry analysis • Transfer cells to FACS tubes to read values in BD FACS CANDO flow cytometer.

流式細胞儀分析的結果如圖 6a 至圖 6c 所示。圖 6a 所示為從臍帶組織中分離並在DMEM/10%FBS中培養後表現幹細胞標記CD73、CD90以及CD105的分離的間質襯裡幹細胞的百分比，圖 6b 所示為分離的間質襯裡幹細胞在從臍帶組織中分離並在PTT-4中培養後表現幹細胞標記CD73、CD90以及CD105的百分比，圖 6c 所示為從臍帶組織中分離並在PTT-6中培養後表現幹細胞標記CD73、CD90以及CD105的分離的間質襯裡幹細胞的百分比。從圖 6a 可以看出，使用DMEM/10% FBS作為培養基的細胞群具有約75%的CD73+細胞、78%的CD90+細胞，以及80%的CD105+細胞(兩次實驗的平均值)，而在分離/使用PPT-4培養基培養的臍帶組織(參閱圖 6b )，CD73-陽性，CD90-陽性，以及CD105-陽性的間質幹細胞的數量為約87% (CD73+細胞)，93% (CD90+細胞)以及86% (CD105+細胞)，兩次實驗的平均值。透過在本發明之PTT-6培養基中培養獲得的間質幹細胞群的純度相對於全部三種標記(CD73、CD90以及CD105)為至少99.0%，這表示該細胞群的純度顯著高於以PPT-4培養基或DMEM/10%FBS培養者。此外，甚至更重要的是，透過在PTT-6中培養獲得的間質幹細胞群基本上是100%純度且確定的幹細胞群。這使得本發明之幹細胞群成為幹細胞療法的理想候選者。因此，這種間質臍帶襯裡幹細胞群可能成為這種基於幹細胞的治療方法的黃金標準，包括作為產生如本文所述之轉殖基因間質幹細胞的起始材料。The results of flow cytometry analysis as shown in FIGS. 6a to 6c. Fig. 6a shows the percentage of isolated mesenchymal lining stem cells isolated from umbilical cord tissue and cultured in DMEM/10% FBS, showing stem cell markers CD73, CD90 and CD105 . Fig. 6b shows the isolated mesenchymal lining stem cells in The percentage of stem cell markers CD73, CD90 and CD105 after being isolated from umbilical cord tissue and cultured in PTT-4 . Figure 6c shows the stem cell markers CD73, CD90 and CD105 after being isolated from umbilical cord tissue and cultured in PTT-6 The percentage of isolated mesenchymal lining stem cells. As can be seen from Fig. 6a , the cell population using DMEM/10% FBS as the culture medium has about 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (the average of the two experiments), while the separation / The number of umbilical cord tissue cultured with PPT-4 medium (see Figure 6b ), CD73-positive, CD90-positive, and CD105-positive mesenchymal stem cells is about 87% (CD73+ cells), 93% (CD90+ cells) and 86% (CD105+ cells), the average of two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0% relative to all three markers (CD73, CD90, and CD105), which means that the purity of the cell population is significantly higher than that of the PPT-4 Medium or DMEM/10% FBS culture. Furthermore, even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is basically a 100% pure and definite stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell therapy. Therefore, this mesenchymal umbilical cord-lined stem cell population may become the gold standard for this stem cell-based treatment, including as a starting material for the generation of transgenic mesenchymal stem cells as described herein.

圖6所示的發現進一步透過圖 7a 與圖 7b 所示的流式細胞儀分析的結果得到證實。圖 7a 所示為從臍帶組織分離後並且在PTT-6培養基中培養，表現幹細胞標記CD73、CD90以及CD105並且缺乏CD34，CD45與HLA-DR的分離的間質臍帶襯裡幹細胞(臍帶羊膜的間質幹細胞)的百分比。如圖 7a 所示，間質幹細胞群含有97.5%的活細胞，其中100%分別表現CD73、CD90以及CD105 (參閱「CD73+CD90+」與「CD73+CD105+」的行列)，而99.2%幹細胞群不表現CD45，且100%的幹細胞群不表現CD34與HLA-DR (參閱「CD34-CD45-」與「CD34-HLA-DR-」的行列)。因此，透過在PTT-6培養基中培養而獲得的間質幹細胞群基本上為100%純度且確定的幹細胞群，其符合將間質幹細胞用於細胞療法的標準(95%或更多幹細胞群表現CD73、CD90以及CD105，且98%或更多的幹細胞群缺乏CD34、CD45以及HLA-DR的表現，參閱Sensebe等人「Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review」，同上)。這裡注意到，本發明之羊膜的間質幹細胞在標準培養條件下黏附於塑料，並在體外分化為成骨細胞、脂肪細胞與成軟骨細胞，參閱美國專利9,085,755、美國專利8,287,854或WO2007/046775，因此符合通常被接受在細胞療法中使用間質幹細胞的標準。It found that the results shown in Figure 6 the analysis of the flow cytometer shown in FIG. 7b further confirmed through FIG. 7a. Figure 7a shows the isolated interstitial umbilical cord lining stem cells (umbilical cord amniotic interstitium) isolated from umbilical cord tissue and cultured in PTT-6 medium, showing stem cell markers CD73, CD90 and CD105 and lacking CD34, CD45 and HLA-DR Stem cells). As shown in Figure 7a, mesenchymal stem cells contained 97.5% viable cells, wherein 100% respectively showed CD73, CD90 and of CD105 (see "CD73 + CD90 +" and "CD73 + CD105 +" ranks), while 99.2% of stem cells is not Express CD45, and 100% of the stem cell population does not express CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-"). Therefore, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is basically a 100% pure and defined stem cell population, which meets the criteria for using mesenchymal stem cells for cell therapy (95% or more stem cell population performance CD73, CD90 and CD105, and 98% or more of the stem cell population lacks the performance of CD34, CD45 and HLA-DR, see Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", ibid.) . It is noted here that the mesenchymal stem cells of the amniotic membrane of the present invention adhere to plastics under standard culture conditions and differentiate into osteoblasts, adipocytes and chondrocytes in vitro, see US Patent 9,085,755, US Patent 8,287,854 or WO2007/046775, It therefore meets the standards generally accepted for the use of mesenchymal stem cells in cell therapy.

圖 7b 所示為表現CD73、CD90以及CD105並缺乏CD34、CD45以及HLA-DR表現的分離的骨髓間質幹細胞的百分比。如圖 7b 所示，骨髓間質幹細胞群含有94.3%活細胞，其中100%分別表現CD73、CD90以及CD105(見「CD73+CD90+」與「CD73+CD105+」的行列)，而僅有62.8%的骨髓幹細胞群缺乏CD45的表現，並且99.9%的幹細胞群缺乏CD34與HLA-DR的表現(參閱「CD34-CD45-」與「CD34-HLA-DR-」的行列)。因此，現被認為是間質幹細胞的黃金標準之骨髓間質幹細胞在幹細胞標記方面，遠低於本發明之(臍帶的羊膜的)間質幹細胞群。這一發現還顯示，本發明之幹細胞群可能是基於幹細胞療法的理想候選者，包括基於轉殖基因幹細胞療法的起始材料，並且可能成為基於幹細胞的治療方法的黃金標準，包括基因治療方法，其中將本發明之轉殖基因幹細胞群施用於個體，以例如產生個體缺乏的蛋白質。 Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking CD34, CD45 and HLA-DR. As shown in Figure 7b, the mesenchymal stem cell population contained 94.3% viable cells, wherein 100% respectively showed CD73, CD90 and of CD105 (see "CD73 + CD90 +" and "CD73 + CD105 +" ranks), while only 62.8% of The bone marrow stem cell population lacks the performance of CD45, and 99.9% of the stem cell population lacks the performance of CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-"). Therefore, bone marrow mesenchymal stem cells, which are now considered to be the gold standard for mesenchymal stem cells, are far lower than the mesenchymal stem cell population (of the amniotic membrane of the umbilical cord) of the present invention in terms of stem cell labeling. This finding also shows that the stem cell population of the present invention may be an ideal candidate for stem cell-based therapy, including starting materials for stem cell-based therapy based on transgenic genes, and may become the gold standard for stem cell-based treatment methods, including gene therapy methods, Among them, the population of transgenic stem cells of the present invention is applied to an individual, for example, to produce a protein that the individual lacks.

實施例Examples 22 ：關於:on AAVS1AAVS1 鋅指核酸酶Zinc finger nuclease (ZFN)(ZFN) 構築之實驗Construction Experiment

實施例Examples 2.12.1

在這組實驗中，對於負擔得起的以及可接近的FVIII替換的未滿足的需求促使本案發明人使用鋅指核酸酶(ZFN)調節的FVIII轉殖基因整合在原代人類細胞的AAVS1基因座中開發自體細胞療法。該基因座被認為是一個基因組安全港(Smith等人，2008年；DeKelver等人，2010年；Rio等人，2014年)，但尚未得到嚴格證明，主要是因為內源性絕緣子元素(Ogata等人，2003年)有利於在不同細胞類型中持久轉殖基因表現而不反轉活化鄰近基因(Smith等人，2008年；Hockemeyer等人，2009年；DeKelver等人，2010年；Zou等人，2011年；Coluccio等人，2013年)。透過使用一套不同的技術結合基因組以及轉錄組的生物資訊指導及無偏見的詢問，本文顯示具有分子特徵的AAVS1 鋅指核酸酶(ZFN)有利於精確整合並增強的核酸酶活性、整合FVIII轉殖基因，並透過誘導原代人類臍帶的上皮幹細胞，其具有可忽略的偏離目標效應，而持久分泌FVIII。這些細胞可以如上所述以及如Sivalingam, J.等人，2016年，同上，所述製備。雖然為了說明之目的，實施例2.1中的這些實驗已經用從臍帶的羊膜中分離的上皮幹細胞進行，該臍帶為本發明之轉殖基因間質幹細胞群(例如，幹細胞群，其至少約97%或更多細胞表現以下標記中的每一種：CD73、CD90以及CD105以及缺乏CD34、CD45以及HLA-DR的表現也可以相同的方式產生。In this set of experiments, the unmet need for affordable and accessible FVIII replacement prompted the inventors of this case to use zinc finger nuclease (ZFN) regulated FVIII transgenes to integrate in the AAVS1 locus of primary human cells Development of autologous cell therapy. The locus is considered to be a genomic safe haven (Smith et al. 2008; DeKelver et al. 2010; Rio et al. 2014) but has not been rigorously proven, mainly because of endogenous insulator elements (Ogata et al. Human, 2003) Facilitates long-term transgene expression in different cell types without reversing the activation of neighboring genes (Smith et al., 2008; Hockemeyer et al., 2009; DeKelver et al., 2010; Zou et al., 2011; Coluccio et al., 2013). Through the use of a different set of techniques combined with bioinformatics guidance and unbiased interrogation of the genome and transcriptome, this article shows that the AAVS1 zinc finger nuclease (ZFN) with molecular characteristics is conducive to precise integration and enhanced nuclease activity Reproduction of genes, and by inducing primary human umbilical cord epithelial stem cells, it has a negligible off-target effect, while persistently secreting FVIII. These cells can be prepared as described above and as described in Sivalingam, J. et al., 2016, supra. Although for illustrative purposes, the experiments in Example 2.1 have been performed with epithelial stem cells isolated from amniotic membrane of the umbilical cord, which is the population of transgenic mesenchymal stem cells of the invention (eg, stem cell population, which is at least about 97% Or more cells expressing each of the following markers: CD73, CD90 and CD105 and the lack of CD34, CD45 and HLA-DR can also be produced in the same way.

結果result

圖14所示為本發明中使用的所有質體構築。Fig. 14 shows all plastid structures used in the present invention.

於in K562K562 細胞中評估Intracellular evaluation AAVS1AAVS1 鋅指核酸酶Zinc finger nuclease (ZFN)(ZFN) 構築Build

測試了三種AAVS1 鋅指核酸酶(ZFN)構築：專性異二聚體(OH)；Sharkey以及增強型Sharkey (於材料及方法中描述)量化pZDonor (50-bp)在K562細胞中的整合。本案發明人在次低溫(30°C)(Doyon等人，2010年)以及37°C的條件下進一步比較了每種構築。Sharkey與增強型Sharkey 鋅指核酸酶(ZFNs)在30°C誘導供體DNA整合的效率最高(分別為44.5%以及47.9%)，透過限制性片段長度多態性(RFLP)評估(圖8)。在所有後續實驗中，增強型Sharkey鋅指核酸酶(ZFN)在30°C下使用。在這些條件下，透過RFLP和整合連接PCR證實了50-bp、3.75-kb，以及9.1-kb供體DNA整合到K562細胞的AAVS1基因座中。整合連接PCR擴增子的定序證實供體DNA精確整合到PPP1R12C 的內含子1中(圖9B、C)。在不存在鋅指核酸酶(ZFN)的情況下單獨以供體DNA電穿孔的細胞未顯示透過整合連接PCR與RFLP的轉殖基因整合的證據(圖9A、C)。該9.1-kb供體DNA遞送雜合的人-豬B結構域缺失的FVIII cDNA (Sivalingam等人，2014年)(圖25；參閱材料及方法)。Three AAVS1 zinc finger nuclease (ZFN) constructs were tested: obligate heterodimer (OH); Sharkey and enhanced Sharkey (described in Materials and Methods) to quantify pZDonor (50-bp) integration in K562 cells. The inventor of the present case further compared each structure under the conditions of sub-low temperature (30°C) (Doyon et al., 2010) and 37°C. Sharkey and enhanced Sharkey zinc finger nucleases (ZFNs) were the most efficient at inducing donor DNA integration at 30°C (44.5% and 47.9%, respectively), and were evaluated by restriction fragment length polymorphism (RFLP) (Figure 8) . In all subsequent experiments, enhanced Sharkey zinc finger nuclease (ZFN) was used at 30°C. Under these conditions, integration of 50-bp, 3.75-kb, and 9.1-kb donor DNA into the AAVS1 locus of K562 cells was confirmed by RFLP and integration ligation PCR. The sequencing of the integrated ligation PCR amplicon confirmed that the donor DNA was accurately integrated into intron 1 of PPP1R12C (Figure 9B, C). Cells electroporated with donor DNA alone in the absence of zinc finger nuclease (ZFN) showed no evidence of integration of the transfer gene with RFLP through integration link PCR (Figure 9A, C). This 9.1-kb donor DNA delivers a hybrid human-porcine B domain-deleted FVIII cDNA (Sivalingam et al., 2014) (Figure 25; see materials and methods).

CLECsCLECs 中的middle AAVS1AAVS1 鋅指核酸酶Zinc finger nuclease (ZFN)(ZFN) 活性active (( 上皮臍帶襯裡幹細胞Epithelial umbilical cord lining stem cells ))

如WO 2006/019357中所述分離上皮臍帶襯裡幹細胞。在以AAVS1鋅指核酸酶(ZFN)質體電穿孔臍帶襯裡上皮細胞(CLECs)後8-48小時，RT-PCR顯示最高含量的鋅指核酸酶(ZFN)表現(圖15A)。研究了兩個因素對透過CEL-1測定評估的鋅指核酸酶(ZFN)活性的影響(報告透過非同源末端連接修復DNA斷裂)，整合連接PCR與RFLP (位點特異性供體整合的證據)。CEL-1測定結果顯示，與作為兩種構築遞送的鋅指核酸酶(ZFN)單體(35%±1.8%)相比，右側及左側AAVS1 鋅指核酸酶(ZFN)單體作為單一構築遞送時(43%±1.9%)具有顯著更高的鋅指核酸酶(ZFN)活性(P = 0.045) (圖16A)。在轉染後經歷瞬間次低溫的臍帶襯裡上皮細胞(CLECs)中鋅指核酸酶(ZFN)蛋白表現也更高(37°C持續1天，然後30°C持續2天)(圖15B)。因此，使用遞送增強型Sharkey AAVS1 鋅指核酸酶(ZFN)單體以及瞬間低溫的單一質體進行後續實驗。在這些條件下，整合連接PCR與RFLP分析顯示在僅以pZDonor電穿孔的臍帶襯裡上皮細胞(CLECs)中沒有供體DNA整合(圖16B)。The epithelial umbilical cord lining stem cells were isolated as described in WO 2006/019357. 8-48 hours after electroporation of umbilical cord lined epithelial cells (CLECs) with AAVS1 zinc finger nuclease (ZFN) plastids, RT-PCR showed the highest content of zinc finger nuclease (ZFN) performance (Figure 15A). The effect of two factors on zinc finger nuclease (ZFN) activity assessed by the CEL-1 assay (reporting repair of DNA breaks through non-homologous end ligation), integration of ligation PCR and RFLP (site-specific donor integration) evidence). CEL-1 measurement results show that the right and left AAVS1 zinc finger nuclease (ZFN) monomers are delivered as a single construct compared to the zinc finger nuclease (ZFN) monomers (35% ± 1.8%) delivered as two constructs When (43%±1.9%) had significantly higher zinc finger nuclease (ZFN) activity (P = 0.045) (Figure 16A). Zinc finger nuclease (ZFN) protein in umbilical cord lining epithelial cells (CLECs) that experienced transient hypothermia after transfection also performed higher (37°C for 1 day, then 30°C for 2 days) (Figure 15B). Therefore, the follow-up experiments were performed using the delivery-enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) monomer and a single plastid with an instant low temperature. Under these conditions, integrated ligation PCR and RFLP analysis revealed no donor DNA integration in umbilical cord lined epithelial cells (CLECs) electroporated with pZDonor only (Figure 16B).

測試不同劑量的AAVS1鋅指核酸酶(ZFN)以及供體DNA以確定誘導由細胞活力及磷酸化組蛋白H2AX定量的最小細胞毒性之條件。總體結果顯示，5-10 μgAAVS1鋅指核酸酶(ZFN)與10 μg供體DNA的共電穿孔誘導最小的細胞毒性(圖17)。Different doses of AAVS1 zinc finger nuclease (ZFN) and donor DNA were tested to determine the conditions that induce minimal cytotoxicity quantified by cell viability and phosphorylated histone H2AX. Overall results showed that co-electroporation of 5-10 μg AAVS1 zinc finger nuclease (ZFN) with 10 μg donor DNA induced minimal cytotoxicity (Figure 17).

FVIIIFVIII 轉殖基因的基因陷阱整合Transgenic gene trap integration

由於FVIII轉殖基因供體DNA的大小相對較大(9 kb)，且因為準確整合是最重要的目標，本案發明人首先使用基因捕獲供體載體AAVS1 SA-2A-puromycin-pA以及AAV-CAGGS-EGFP (Hockemeyer等人，2009年)，其提供較小尺寸的供體DNA，以評估臍帶襯裡上皮細胞(CLECs)中基因組目標的準確性。嘌呤黴素抗性臍帶襯裡上皮細胞(CLECs)的PCR分別使用與預期整合位點重疊的基因組特異性引子證明了1-kb與4.2-kb供體的整合(圖18與19)。PCR擴增子的定序證實1-kb供體完全整合。大多數整合的4.2-kb供體被擴增和定序(除了CAGGS啟動子內1-kb富含GC的區域)，且無顯示***、缺失或重排。 整合連接與供體DNA的PCR在未處理的臍帶襯裡上皮細胞(CLECs)中呈現陰性。Because the size of the FVIII transgenic gene donor DNA is relatively large (9 kb), and because accurate integration is the most important goal, the inventors of this case first used the gene capture donor vectors AAVS1 SA-2A-puromycin-pA and AAV-CAGGS -EGFP (Hockemeyer et al., 2009), which provides donor DNA of smaller size to assess the accuracy of genomic targets in umbilical cord lined epithelial cells (CLECs). PCR of puromycin-resistant umbilical cord liner epithelial cells (CLECs) demonstrated the integration of 1-kb and 4.2-kb donors using genome-specific primers that overlapped the expected integration sites (Figures 18 and 19). The sequencing of the PCR amplicon confirmed the complete integration of the 1-kb donor. Most integrated 4.2-kb donors were amplified and sequenced (except for the 1-kb GC-rich region within the CAGGS promoter), and showed no insertions, deletions, or rearrangements. PCR that integrates ligation with donor DNA is negative in untreated umbilical cord lining epithelial cells (CLECs).

使用相同的基因捕獲策略，與FVIII轉殖基因供體DNA以及增強型Sharkey AAVS1 鋅指核酸酶(ZFN)共電穿孔的臍帶襯裡上皮細胞(CLECs)的整合連接與長PCR顯示完整供體在PPP1R12C 的內含子1中的整合(圖10A、B)。AAVS1位點特異性PCR與定序證實了完整FVIII轉殖基因的***。對嘌呤黴素篩選的臍帶襯裡上皮細胞(CLECs)具有抗性，該臍帶襯裡上皮細胞(CLECs)已穩定整合FVIII轉殖基因(puro-CLEC)。電穿孔後37天這些細胞的FVIII分泌為2,131±17 mU/106 細胞/24小時，而電穿孔後1天為952±8 mU/106 細胞/24小時(圖10D)。Using the same gene capture strategy, integration of umbilical cord lining epithelial cells (CLECs) co-electroporated with FVIII transgenic gene donor DNA and enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) and long PCR showed the complete donor at PPP1R12C Integration in intron 1 (Figure 10A, B). AAVS1 site-specific PCR and sequencing confirmed the insertion of the complete FVIII transgene. It is resistant to puromycin-selected umbilical cord lining epithelial cells (CLECs) that have stably integrated the FVIII transgene (puro-CLEC). FVIII secretion of these cells 37 days after electroporation was 2,131±17 mU/10 6 cells/24 hours, and 1 day after electroporation was 952±8 mU/10 6 cells/24 hours (FIG. 10D ).

量化目標與非目標Quantifying goals and non-goals FVIIIFVIII 轉殖基因整合Transgene integration

透過整合連接擴增子(正確目標)與載體特異性擴增子(正確目標與偏離目標整合)的數位微滴式PCR定量正確目標與偏離目標的轉殖基因整合，顯示總載體的複製數無顯著差異(300±61複製數/μl)與左連結點(312±38 複製數/μl；P = 0.835)以及右連結點(360±30 複製數/μl；P = 0.337)擴增子相比，顯示最小的偏離目標整合(圖10E)。與未處理的臍帶襯裡上皮細胞(CLECs)相比，RT-PCR顯示puro-CLEC中PPP1R12C 表現低2倍，與單等位基因轉殖基因整合一致(圖10F)。Quantitative integration of transfected genes with correct target and off-target is quantified by the integration of digital droplet PCR connecting the amplicon (correct target) and vector-specific amplicon (correct target and off-target integration). Significant difference (300±61 copies/μl) compared to the left junction (312±38 copies/μl; P = 0.835) and right junction (360±30 copies/μl; P=0.337) amplicons , Showing the smallest off-target integration (Figure 10E). Compared with untreated umbilical cord lining epithelial cells (CLECs), RT-PCR showed that PPP1R12C in puro-CLEC was 2 times lower, consistent with the integration of single allele transfer genes (Figure 10F).

分析電腦預測的偏離目標位點Analyze computer predictions of deviations from the target site

針對預測的10個最可能的偏離目標位點(OT1-OT10)的目標深度定序(Hockemeyer等人，2009年)顯示僅在OT1中低頻率(分別為1.36%以及1.37%)的4-bp以及1-bp缺失，OT1為在8q24.3的基因間位點。 OT2至OT10中沒有其他特異於puro-CLEC的***或缺失(indel)被檢測到(圖26)。***野生型AAVS1擴增子與AAVS1靶位點處具有5-bp缺失的合成擴增子，莫爾比為1:10至1：1000 (突變體：野生型)，確定***缺失檢測的靈敏度。深度定序檢測到所有比例的加標缺失，加標與觀察含量之間存在高度線性關係(R2 = 0.999)，檢測靈敏度為0.1% (圖20)。Target depth sequencing (Hockemeyer et al., 2009) for the predicted 10 most likely off-target sites (OT1-OT10) shows only 4-bp at low and medium frequencies (1.36% and 1.37%, respectively) in OT1 As well as the 1-bp deletion, OT1 is an intergenic locus at 8q24.3. No other insertions or deletions (indel) specific to puro-CLEC were detected in OT2 to OT10 (Figure 26). Insert a wild-type AAVS1 amplicon and a synthetic amplicon with a 5-bp deletion at the AAVS1 target site, with a molar ratio of 1:10 to 1:1000 (mutant: wild-type), to determine the sensitivity of insertion deletion detection. Deep sequencing detected all proportions of missing spikes, and there was a highly linear relationship between spiked and observed content (R2 = 0.999), with a detection sensitivity of 0.1% (Figure 20).

全基因組定序Genome-wide sequencing

VarScan分析(Koboldt等人，2012年)的全基因組定序(WGS)數據產生2736個特異於puro-CLEC的高可信度***缺失，但沒有一個是電腦預測的偏離目標位點(Cradick等人，2011年；Reyon等人，2011年；Fine等人，2014年)。本案發明人只考慮含有多個***或缺失(indel)的基因座為真正的偏離目標事件(Gupta等人，2011年)。高信度***缺失的基因組坐標顯示196個基因座，每個基因座有2-5個***缺失。所有都在重複的DNA區域。3個偏離目標基因座具有最高的***或缺失(indel)以及SNP組合數(4-6)，且映射讀數的比例(33-50%)高於正確目標***(19-26%)。透過將所有讀數與參考AAVS1基因座的980鹼基序列(hg19)比對以鑑定正確目標的***缺失。鋅指核酸酶(ZFN)切割位點位於該序列的位置401-430之間。21個映射讀數中的4個(19%)在該序列的400位顯示單個A缺失。50個映射讀數中的23個(46%)顯示位置407處的單個T缺失或單個C***。推定的偏離目標事件映射到chr2_237939600 (indel 1)中的基因間區域；chr21_15213700 (indel 2)；以及chr8_1365500 (indel 3)。(基因組坐標參閱hg19。)所有皆缺乏潛在的AAVS1 鋅指核酸酶(ZFN)結合位點。最接近的蛋白質編碼基因為COPS8 (來自indel 1的54 kb)、C21orf15 (來自indel 2的1.7 kb)，以及DLGAP2 (來自indel 3的84 kb)。indel 1的序列數據顯示其為偽陽性的***或缺失(indel)，因為它僅由兩個單鹼基取代組成而沒有***或缺失(圖11A)。indel 3包含兩個不同衛星DNA的***。較大的***(1489 bp)為衛星DNA的15倍擴增，而較小的***(186 bp)為不同衛星的2倍擴增(圖11B)。(由於高度重複的序列基序，indel 2無法連續定序。)串聯重複本質上是不穩定的，因為在有絲***期間複製滑動以及不均等的姐妹染色單體交換(George與Alani，2012年)。Fok1 的偏離目標切割可能透過股(strand)入侵促進重複擴增(Buard與Jeffreys，1997年)。本案發明人的數據顯示，由Fok1 二聚化引起的偏離目標事件不依賴於鋅指核酸酶(ZFN)結合是很罕見的。VarScan analysis (Koboldt et al., 2012) of genome-wide sequencing (WGS) data produced 2,736 high-confidence insertion deletions specific to puro-CLEC, but none were computer-predicted off-target sites (Cradick et al. , 2011; Reyon et al., 2011; Fine et al., 2014). The inventors in this case only considered loci containing multiple insertions or deletions (indels) as true off-target events (Gupta et al., 2011). The genomic coordinates of high-confidence indels show 196 loci, with 2-5 indels per locus. All are in the repeated DNA region. The three off-target loci have the highest insertion or deletion (indel) and the number of SNP combinations (4-6), and the ratio of mapped reads (33-50%) is higher than the correct target insertions (19-26%). Identify the correct target insertion deletion by aligning all reads with the 980 base sequence (hg19) of the reference AAVS1 locus. The zinc finger nuclease (ZFN) cleavage site is located between positions 401-430 of the sequence. Four of the 21 mapping reads (19%) showed a single A deletion at 400 in this sequence. Twenty-three of the 50 mapped readings (46%) showed a single T deletion or a single C insertion at position 407. The putative off-target events are mapped to intergenic regions in chr2_237939600 (indel 1); chr21_15213700 (indel 2); and chr8_1365500 (indel 3). (Refer to hg19 for genome coordinates.) All lack the potential AAVS1 zinc finger nuclease (ZFN) binding site. The closest protein coding genes are COPS8 (54 kb from indel 1), C21orf15 (1.7 kb from indel 2), and DLGAP2 (84 kb from indel 3). The sequence data of indel 1 shows that it is a false positive insertion or deletion (indel) because it consists of only two single-base substitutions without insertion or deletion (Figure 11A). indel 3 contains the insertion of two different satellite DNAs. The larger insertion (1489 bp) is a 15-fold amplification of satellite DNA, while the smaller insertion (186 bp) is a 2-fold amplification of different satellites (Figure 11B). (Indel 2 cannot be sequenced continuously due to highly repetitive sequence motifs.) Tandem repeats are inherently unstable because of replication slips and unequal sister chromatid exchanges during mitosis (George and Alani, 2012). Fok1 's off-target cutting may promote repeated amplification through strand invasion (Buard and Jeffreys, 1997). The data of the inventors of this case show that off-target events caused by Fok1 dimerization do not depend on zinc finger nuclease (ZFN) binding is very rare.

全基因組定序(WGS)分析顯示存在四種染色體重排，均具有不平衡的基因組複製數。三個為染色體間的，一個為染色體內結構變體(圖11C)。基因組PCR反複檢測不到所有推定的異常染色體連接，顯示重排為另一種偽陽性結果。然而，由於確定鋅指核酸酶(ZFN)處理是否已誘導染色體結構變化很重要，本案發明人採用了基於相對基因組複製數分析的不同驗證方法。由於預測四種結構變體中的所有斷裂位點具有不平衡的基因組複製數，本案發明人使用定量PCR來確定puro-CLEC的基因組DNA中的每個斷點基因座的複製數相對於野生型臍帶襯裡上皮細胞(CLECs) (wt-CLEC)中的相同斷裂點基因座。每個斷裂點基因座處的相對複製數表示為puro-以及野生型-CLEC的標準化C T 值的比率。標準化是必要的，因為SV1-SV4斷點基因座在不同的黏合溫度下被擴增以實現擴增的特異性。在相同實驗中測定的肌動蛋白基因座擴增的C T 值用於標準化每個斷裂點基因座的C T 值。在每個斷裂點處puro-CLEC與野生型-CLECC T 值的比率計算為[puro-CLEC斷點基因座C T /肌動蛋白C T 除以野生型-CLEC斷點C T /肌動蛋白C T ](圖11D)。SV1的斷點2基因座無法擴增。SV4的候選斷裂點位點間隔8 bp並作為單個基因座擴增。在所分析的所有候選斷裂點處puro-CLEC與野生型-CLEC基因組複製數的比率為0.97-1.07與具有不平衡複製數的結構改變的實質頻率不一致(圖11D)。基於透過PCR擴增不存在異常染色體連接並且透過實驗驗證在斷裂點基因座處沒有異常基因組複製數，鋅指核酸酶(ZFN)處理不太可能誘導具有生物學意義的染色體重排。Whole genome sequencing (WGS) analysis revealed that there are four chromosomal rearrangements, all with unbalanced genome replication numbers. Three are inter-chromosomal, and one is an intra-chromosomal structural variant (Figure 11C). Genomic PCR repeatedly failed to detect all the putative abnormal chromosomal connections, indicating that the rearrangement was another false positive result. However, since it is important to determine whether zinc finger nuclease (ZFN) treatment has induced changes in chromosome structure, the inventors of the present case adopted different verification methods based on relative genome copy number analysis. Since it is predicted that all break sites in the four structural variants have an unbalanced genomic copy number, the inventors of the present case used quantitative PCR to determine the copy number of each break point locus in the genomic DNA of puro-CLEC relative to the wild type The same breakpoint locus in umbilical cord lining epithelial cells (CLECs) (wt-CLEC). Relative copy number at each locus breaking point and expressed as the ratio puro- normalized C T value of the wild-type -CLEC. Standardization is necessary because the SV1-SV4 breakpoint loci are amplified at different bonding temperatures to achieve specificity of amplification. Actin locus amplification C T value measured in the same experiment for normalization C T value of each breakpoint locus. The ratio of puro-CLEC to wild-type-CLEC C T value at each breakpoint is calculated as [puro-CLEC breakpoint locus C T /actin C T divided by wild-type-CLEC break point C T /actin Protein C T ] (Figure 11D). The breakpoint 2 locus of SV1 cannot be amplified. The candidate breakpoints of SV4 are separated by 8 bp and amplified as a single locus. The ratio of puro-CLEC to wild-type-CLEC genome copy numbers at all candidate breakpoints analyzed was 0.97-1.07, which was inconsistent with the substantial frequency of structural changes with unbalanced copy numbers (Figure 11D). Based on the absence of abnormal chromosomal junctions through PCR amplification and the experimental verification that there is no abnormal genomic copy number at the breakpoint locus, zinc finger nuclease (ZFN) treatment is unlikely to induce biologically significant chromosomal rearrangements.

RNARNA 定序Sequencing

野生型-CLEC與puro-CLEC的RNA定序總共鑑定了17,751個轉錄物，其中只有57個(0.3%)過度表現，33個(0.2%)在puro-CLEC中表現不足為至少2倍(圖12)。FVIII為過度表現的轉錄物之一。途徑分析顯示10個失調的轉錄物透過DAVID分析(Huang等人，2009年)(Benjamini校正的P = 0.011)(圖21)與PPP1R12C 在發炎反應中的作用(Bannert等人，2003年)一致，映射到細胞因子-細胞因子受體相互作用。雖然7個失調的基因為超過1,600個致癌基因的綜合目錄中潛在的原癌基因(http://www.bushmanlab.org/links/genelists)，但沒有一個基因定位於KEGG的任何經典癌症途徑(http：//www.kegg.jp/kegg-bin/show_pathway?map=hsa05200&show_description=show)。A total of 17,751 transcripts were identified by RNA sequencing of wild-type CLEC and puro-CLEC, of which only 57 (0.3%) were over-expressed and 33 (0.2%) under-purified at least 2-fold in puro-CLEC (Figure 12). FVIII is one of the overexpressed transcripts. Pathway analysis showed that the 10 dysregulated transcripts were consistent with the role of PPP1R12C in the inflammatory response (Bannert et al., 2003) by DAVID analysis (Huang et al., 2009) (Benjamini corrected P = 0.011) (Figure 21). Mapping to cytokine-cytokine receptor interaction. Although the 7 dysregulated genes are potential proto-oncogenes in the comprehensive catalog of more than 1,600 oncogenic genes (http://www.bushmanlab.org/links/genelists), none of the genes is located in any of the classic cancer pathways of KEGG ( http: //www.kegg.jp/kegg-bin/show_pathway?map=hsa05200&show_description=show).

鑑於AAVS1 鋅指核酸酶(ZFN)在轉錄組上的最小足跡，本案發明人擴展了其分析，以研究單倍體不足的PPP1R12C 表現對其相互作用蛋白質配偶體以及下游效應物的可能間接影響。其他74種蛋白磷酸酶，50種肌球蛋白相關以及下游基因的RNA定序數據，29種已知的蛋白質相互作用配偶體以及以AAVS1整合位點為中心的1 Mb內的PPP1R12C 的43個相鄰基因僅顯示出DUSP6的失調，DUSP6為PPP1R12C相互作用的配偶體，其puro-CLEC中的表現高5.5倍。定量RT-PCR證實puro-CLEC中DUSP6表現增加4.2倍(圖5)。DUSP6負調節ERK1/2 (Zhang等人，2010年)，高表現損害上皮-間質轉化及致瘤性(Wong等人，2012年)。puro-CLEC的增殖略有但沒有顯著降低(圖23)，可能反映了高DUSP6表現與PPP1R12C 單倍不足的綜合影響，後者為完成有絲***及胞質***所必需的(Banko等人，2011年)。In view of the smallest footprint of AAVS1 zinc finger nuclease (ZFN) on the transcriptome, the inventors of the present case expanded their analysis to investigate the possible indirect effect of haploid deficient PPP1R12C performance on its interacting protein partners and downstream effectors. 74 protein phosphatases, 50 myosin-related and downstream gene RNA sequencing data, 29 known protein interaction partners, and 43 phases of PPP1R12C within 1 Mb centered on the AAVS1 integration site The neighbor genes only showed dysregulation of DUSP6, which is a partner of PPP1R12C interaction, and its performance in puro-CLEC is 5.5 times higher. Quantitative RT-PCR confirmed that the performance of DUSP6 in puro-CLEC increased 4.2-fold (Figure 5). DUSP6 negatively regulates ERK1/2 (Zhang et al., 2010), high expression impairs epithelial-mesenchymal transition and tumorigenicity (Wong et al., 2012). The proliferation of puro-CLEC is slightly but not significantly reduced (Figure 23), which may reflect the combined effect of high DUSP6 performance and PPP1R12C haploinsufficiency , which is necessary to complete mitosis and cytokinesis (Banko et al., 2011) .

在AAVS1基因座處的鋅指核酸酶(ZFN)調節的切割也容易在原代人類真皮纖維母細胞，人類骨髓及脂肪組織衍生的基質細胞中誘導，總效率為20%或更高(圖24)。使用在臍帶襯裡上皮細胞(CLECs)中整合FVIII轉殖基因的相同條件，還可以誘導原代人類真皮纖維母細胞、骨髓及脂肪組織衍生的基質細胞整合併分泌轉殖基因FVIII (圖24)。Zinc finger nuclease (ZFN)-mediated cleavage at the AAVS1 locus is also easily induced in primary human dermal fibroblasts, human bone marrow and adipose tissue-derived stromal cells, with a total efficiency of 20% or higher (Figure 24) . Using the same conditions for integration of FVIII transgenes in umbilical cord lining epithelial cells (CLECs), primary human dermal fibroblasts, bone marrow, and adipose tissue-derived stromal cells can also be induced to integrate and secrete the transgene FVIII (Figure 24).

討論discuss

最近家族性脂蛋白脂肪酶缺乏症以及B型血友病基因治療的成功(Buning，2013年；Nathwani等人，2014年)已經做了很多工作來推進一個大約在五十年前首次被提出的想法(Tatum，1966年)，該想法為基因替代對於某些疾病可能既有幫助又對臨床可行。然而，回顧臨床採用時間表的初步預測是樂觀的(Friedman，1992年)。在過去十年中，基因治療臨床試驗的致癌併發症使得安全性成為最關鍵的臨床標準，特別是對於已經存在非遺傳治療且非緊急生命限制的疾病，例如癌症。長期以來，γ反轉錄病毒載體一直是整合治療性轉殖基因的有利工具。最初認為***性腫瘤發生在很大程度上是理論風險，因為早期研究顯示非人類靈長類動物以及少數人類個體沒有不良併發症(Anderson，1992年)。X連結的嚴重聯合免疫缺陷(X-linked severe combined immunodeficiency, SCID-X1)(Hacein-Bey-Abina等人，2003年；Hacein-Bey-Abina等人，2008年；Howe等人， 2008年)、慢性肉芽腫病(Stein等人，2010年)，以及Wiskott-Aldrich症候群(Avedillo Diez等人，2011年) 的γ反轉錄病毒基因治療試驗中白血病以及前白血病的發生消除了這種看法，並重點強調生物安全性。對白血病細胞的分析揭示了在幾種致癌基因(LMO2 、BMI1 、CCND2 、PRDM16 、SETBP1 以及MDS-EVI1 )附近的γ反轉錄病毒載體整合，導致癌基因過度表現。然而，由於SCID-X1患者使用的相同載體在腺苷脫氨酶缺乏症的臨床試驗中沒有誘導白血病(Cassani等人，2009年)，因此這些試驗中的精確白血病生成機制仍然不明確，這顯示疾病背景本身設定了致癌轉化的閾值，其需要超過單個基因的失調(Modlich等人，2005年；Dave等人，2009年)。正在探索非病毒遞送的可編程核酸酶作為實現安全有效的治療性轉殖基因整合的平行策略。理論上，基因組編輯技術非常適合於此目的，因為設計避免在潛在危險區域(即調節元件、轉錄起始位點以及轉錄單元內)中整合的構築應該是可行的。目前，鋅指核酸酶(ZFN)技術的臨床前研究經驗多於類轉錄活化劑效應子核酸酶(TALENs)以及RNA引導聚類的規則間隔短回文重複CRISPR/Cas9系統。事實上，鋅指核酸酶(ZFN)針對HIV感染的五項臨床試驗已經完成或正在進行中(www.clinicaltrials.gov；於2015年6月8日訪問)。一項針對12名HIV感染患者的完整試驗未報告在單次輸注自體T細胞後36週內由鋅指核酸酶(ZFN)引起的不良併發症，該自體T細胞已經以CCR5特異性鋅指核酸酶(ZFN)離體轉導。鑑於致癌作用可能僅在更長的時期內出現，該試驗的個體將被觀察10年(Tebas等人，2014年)。Recent familial lipoprotein lipase deficiency and the success of gene therapy for hemophilia B (Buning, 2013; Nathwani et al., 2014) have done a lot of work to advance a proposal that was first proposed about fifty years ago The idea (Tatum, 1966), the idea is that gene replacement may be both helpful and clinically feasible for certain diseases. However, reviewing the preliminary predictions of the clinical adoption schedule is optimistic (Friedman, 1992). In the past decade, the carcinogenic complications of gene therapy clinical trials have made safety the most critical clinical standard, especially for diseases that already have non-genetic therapy and non-emergency life limitations, such as cancer. For a long time, γ-retroviral vectors have been an advantageous tool for integrating therapeutic transgenes. It was originally believed that the occurrence of insertional tumors was largely a theoretical risk, because early studies showed that non-human primates and a few human individuals have no adverse complications (Anderson, 1992). X-linked severe combined immunodeficiency (SCID-X1) (Hacein-Bey-Abina et al., 2003; Hacein-Bey-Abina et al., 2008; Howe et al., 2008), Chronic granulomatosis (Stein et al., 2010), and Wiskott-Aldrich syndrome (Avedillo Diez et al., 2011) have eliminated this perception and focused on the occurrence of leukemia and pre-leukemia in the γ-retroviral gene therapy trial of Wiskott-Aldrich syndrome (Avedillo Diez et al., 2011). Emphasize biosecurity. Analysis of leukemia cells revealed that the integration of γ-retroviral vectors near several oncogenes ( LMO2 , BMI1 , CCND2 , PRDM16 , SETBP1, and MDS-EVI1 ) resulted in overexpression of oncogenes. However, since the same carrier used in SCID-X1 patients did not induce leukemia in clinical trials of adenosine deaminase deficiency (Cassani et al., 2009), the precise mechanism of leukemia generation in these trials is still unclear, which shows The disease background itself sets a threshold for carcinogenic transformation, which needs to exceed the dysregulation of a single gene (Modlich et al., 2005; Dave et al., 2009). Programmable nucleases for non-viral delivery are being explored as a parallel strategy to achieve safe and effective integration of therapeutic transfer genes. In theory, genome editing techniques are very suitable for this purpose, because it should be feasible to design constructs that avoid integration in potentially dangerous regions (ie, regulatory elements, transcription initiation sites, and transcription units). Currently, zinc finger nuclease (ZFN) technology has more experience in preclinical research than transcriptional activator effector nucleases (TALENs) and RNA-guided clustering with regular interval short palindromic repeat CRISPR/Cas9 systems. In fact, five clinical trials of zinc finger nuclease (ZFN) against HIV infection have been completed or are in progress (www.clinicaltrials.gov; accessed on June 8, 2015). A complete trial of 12 HIV-infected patients did not report adverse complications caused by zinc finger nuclease (ZFN) within 36 weeks after a single infusion of autologous T cells, which had been treated with CCR5-specific zinc Refers to nuclease (ZFN) transduction in vitro. Given that the carcinogenic effect may only occur for a longer period of time, individuals in this trial will be observed for 10 years (Tebas et al., 2014).

被認為足以做出臨床決策的對鋅指核酸酶(ZFN)以及其他基因組修飾方法的遺傳毒性風險所進行的可靠性評估是一個令人煩惱的挑戰。目前尚未就如何檢查及分析修飾的基因組以進行非預期的潛在遺傳毒性偏離目標改變達成共識。此外，基因組編輯的每個應用必須獨立評估，因為許多技術及生物因素影響基因組編輯的準確性(Porteus與Baltimore，2003年；Miller等人，2007年；Beumer等人，2008年；Pruett-Miller等人，2008年；Doyon等人，2010年；Guo等人，2010年；Jantz與Berg，2010年；Doyon等人，2011年；Ramirez等人，2012年；Wang等人，2012年)。此外，表觀遺傳狀態可以解釋切割效率的細胞株依賴性變異(Handel等人，2009年；Lombardo等人，2011年)。Reliability assessment of the risk of genotoxicity of zinc finger nucleases (ZFN) and other genome modification methods, considered to be sufficient for clinical decision-making, is an annoying challenge. There is currently no consensus on how to examine and analyze modified genomes for unintended potential genotoxicity deviations from the target. In addition, each application of genome editing must be independently evaluated because many technical and biological factors affect the accuracy of genome editing (Porteus and Baltimore, 2003; Miller et al., 2007; Beumer et al., 2008; Pruett-Miller et al. People, 2008; Doyon et al., 2010; Guo et al., 2010; Jantz and Berg, 2010; Doyon et al., 2011; Ramirez et al., 2012; Wang et al., 2012). In addition, the epigenetic state can explain cell line-dependent variations in cutting efficiency (Handel et al., 2009; Lombardo et al., 2011).

於本發明中，將FVIII轉殖基因整合到原代人類幹細胞(體細胞)中作為探索自體FVIII細胞療法可行性的實際模型。非造血的原代人類細胞在體外具有有限的複製能力，這使得衍生與體外擴增以擴大用於臨床治療的單細胞選殖是不切實際的。因此，基因修飾的CD34+造血幹細胞的大量細胞群用於SCID-X1 (Hacein-Bey-Abina等人，2014年)、慢性肉芽腫病(Ott等人，2006年)，以及Wiskott-Aldrich症候群( Hacein-Bey-Abina等人，2015年) 的臨床試驗。嚴格評估大量細胞群中的偏離目標事件是一個必須在邁向臨床細胞療法時必須解決的挑戰。有理由認為，透過幾種不同的分子方法對鋅指核酸酶(ZFN)修飾的臍帶襯裡上皮細胞(CLECs)進行分析比依賴單一的特徵技術更可能識別誘導的致癌事件。為此，鋅指核酸酶(ZFN)修飾的臍帶襯裡上皮細胞(CLECs)透過電腦預測的10個最可能偏離目標位點的目標深度定序進行分析，並透過無偏全基因組定序對其進行補充。這顯示僅在一個預測的偏離目標位點中低頻率(>1.5%)的4-bpu與1-bp缺失。在8q24.3的基因間區域中，這些偏離目標微缺失距離TRAPPC9 (在5'側)38.2 kb，距離CHRAC1 14.6 kb(在3'側)。TRAPPC9 被認為參與NF-κB信號傳導(Mochida等人，2009年)，而CHRAC1 編碼組蛋白折疊DNA結合蛋白(Poot等人，2000年)。這兩種基因的表現在puro-CLEC中都失調。In the present invention, the FVIII transgene is integrated into primary human stem cells (somatic cells) as a practical model for exploring the feasibility of autologous FVIII cell therapy. Non-hematopoietic primary human cells have limited replication capacity in vitro, which makes the derivation and expansion in vitro to expand single cell colonization for clinical treatment impractical. Therefore, a large cell population of genetically modified CD34+ hematopoietic stem cells is used for SCID-X1 (Hacein-Bey-Abina et al., 2014), chronic granulomatous disease (Ott et al., 2006), and Wiskott-Aldrich syndrome (Hacein -Bey-Abina et al., 2015) clinical trials. Strictly assessing off-target events in a large number of cell populations is a challenge that must be addressed when moving toward clinical cell therapy. It is reasonable to believe that analysis of zinc finger nuclease (ZFN)-modified umbilical cord lining epithelial cells (CLECs) through several different molecular methods is more likely to identify induced carcinogenic events than relying on a single signature technique. To this end, zinc finger nuclease (ZFN) modified umbilical cord lining epithelial cells (CLECs) were analyzed by computer prediction of the 10 target depth sequences most likely to deviate from the target site, and unbiased whole genome sequencing was performed on them supplement. This shows that only one predicted deviation from the target site at low frequencies (>1.5%) of 4-bpu and 1-bp deletions. In the intergenic region of 8q24.3, the deviation from the target microdeletion (5 'side) 38.2 kb, CHRAC1 14.6 kb (from 3' side from TRAPPC9). TRAPPC9 is thought to be involved in NF-κB signaling (Mochida et al., 2009), while CHRAC1 encodes histone folding DNA binding protein (Poot et al., 2000). The performance of these two genes is dysregulated in puro-CLEC.

全基因組定序越來越多被用於單核苷酸多態性(single nucleotide polymorphisms, SNP)、***缺失以及重排染色體的無偏鑑定。在24x與28.4x覆蓋深度獲得的全基因組定序(WGS)數據已經確定了***缺失(Shigemizu等人，2013年；Ghoneim等人，2014年)。然而，平均覆蓋深度為14x的全基因組定序(WGS)數據檢測到具有95%靈敏度的單核苷酸多態性(SNP) (Meynert等人，2014年)，***或缺失(indel)檢測靈敏度所需的覆蓋深度則未知。進一步的限制被用於自全基因組定序(WGS)數據推斷***缺失的分析方法不如單核苷酸多態性(SNP)的算法那樣發展(Albers等人，2011年)。本案發明人的結果顯示鋅指核酸酶(ZFN)修飾的臍帶襯裡上皮細胞(CLECs)中***缺失的低發生率反映了最近的報導，即在人類誘導的多能幹細胞中鋅指核酸酶(ZFN)校正SOD1突變沒有誘導單核苷酸多態性(SNP)與***缺失(Kiskinis等人，2014年)。儘管全基因組定序(WGS)似乎是無偏頗地偏離目標評估的理想選擇，但除了成本之外，至少有兩個考慮因素限制了它作為獨立方法的實用性。即使在高覆蓋深度下，應用於全基因組定序(WGS)數據的定序偽像以及各種生物資訊學濾波器也可能產生錯誤的結果。此外，鑑定以10%、1%以及0.1%的頻率存在的具有95%靈敏度的偏離目標事件分別需要定序15、150或1,500個二倍體單細胞選殖(Tsai與Joung，2014年)。檢測1%在500x深度定序的細胞中存在的突變平均僅基於2.5次讀數(Brash，2015年)。考慮到當前的技術及成本狀況，這些考慮因素使得全基因組定序(WGS)作為生物安全評估的唯一或主要技術是不切實際的。Genome-wide sequencing is increasingly used for the unbiased identification of single nucleotide polymorphisms (SNPs), indels, and rearranged chromosomes. Whole genome sequencing (WGS) data obtained at 24x and 28.4x coverage depth have identified indels (Shigemizu et al., 2013; Ghoneim et al., 2014). However, genome-wide sequencing (WGS) data with an average coverage depth of 14x detected single nucleotide polymorphisms (SNPs) with 95% sensitivity (Meynert et al., 2014), and indel detection sensitivity The required depth of coverage is unknown. Further restrictions have been used to deduce the analysis of insertion deletions from genome-wide sequencing (WGS) data as the development of single nucleotide polymorphism (SNP) algorithms (Albers et al., 2011). The results of the present inventors show that the low incidence of insertion deletions in umbilical cord lining epithelial cells (CLECs) modified by zinc finger nucleases (ZFN) reflects recent reports that zinc finger nucleases (ZFN) in human induced pluripotent stem cells ) Correction of SOD1 mutations did not induce single nucleotide polymorphisms (SNPs) and insertion deletions (Kiskinis et al., 2014). Although genome-wide sequencing (WGS) seems to be an ideal choice for unbiased deviation from target evaluation, in addition to cost, at least two considerations limit its usefulness as an independent method. Even at high coverage depths, sequencing artifacts applied to WGS data and various bioinformatics filters can produce erroneous results. In addition, the identification of off-target events with 95% sensitivity at frequencies of 10%, 1%, and 0.1% requires sequencing of 15, 150, or 1,500 diploid single cell colonies, respectively (Tsai and Joung, 2014). Detecting 1% of the mutations present in 500x deep-sequenced cells is based on an average of only 2.5 readings (Brash, 2015). Considering the current technology and cost situation, these considerations make genome-wide sequencing (WGS) as the only or main technology for biosafety assessment unrealistic.

認識到這些局限性，透過定量分析整合連接與轉殖基因複製數擴大了基因組成分析。兩個證據表明在puro-CLEC的AAVS1基因座中準確的鋅指核酸酶(ZFN)調節的FVIII轉殖基因整合。首先，透過定量基因組PCR數據，相對於總(正確目標以及偏離目標)轉殖基因複製數的目標轉殖基因複製數顯示整合連接的複製數以及載體擴增子之間沒有顯著差異(圖10(b))。這證明很少(如果有的話)集成是偏離目標的。其次，定量RT-PCR顯示，與野生型-CLEC相比，puro-CLEC中PPP1R12C mRNA的含量降低了一半(圖10(f))。與複製數的數據合併，這與PPP1R12C 的內含子1中FVIII轉殖基因的單等位基因目標整合一致。Recognizing these limitations, the genetic composition analysis has been expanded by quantitatively analyzing the number of integrated connections and the number of transferred gene copies. Two pieces of evidence indicate precise zinc finger nuclease (ZFN) regulated FVIII transgene integration in the AAVS1 locus of puro-CLEC. First, through quantitative genomic PCR data, the number of target transgenic gene copies relative to the total (correct target and off-target) number of transferred gene showed no significant difference between the number of integrated connections and the vector amplicon (Figure 10( b)). This proves that little (if any) integration is off-target. Second, quantitative RT-PCR showed that the content of PPP1R12C mRNA in puro-CLEC was reduced by half compared to wild-type CLEC (Figure 10(f)). Combined with the data on the number of copies, this is consistent with the integration of the single allele target of the FVIII transgene in PPP1R12C intron 1.

RNA定序產生不可知的全轉錄組數據，其靈敏度與動態範圍比基因陣列更高。鑑於單獨的基因組成分析的局限性，這裡推斷RNA定序將提供互補的全基因組功能讀數，其將發信號通知臍帶襯裡上皮細胞(CLECs)是否在基因及調節元件內持續顯著的鋅指核酸酶(ZFN)誘導的偏離目標命中數。儘管本發明沒有描述非編碼RNA (non-coding RNAs, ncRNAs)的表現，但偏離目標事件也可能改變了短及長的非編碼RNA (ncRNAs)。然而，由於非編碼RNA (ncRNAs)的主要功能是調節轉錄(Morris，2011年；Patil等人，2014年)，非編碼RNA (ncRNAs)表現改變的後果也將反映在RNA定序數據中。在這種情況下，與野生型-CLEC相比，puro-CLEC中僅有0.5%的17,751個表現的轉錄物被錯誤調節2倍或更多(圖12(a)-(c))。注意到失調的基因僅映射到細胞因子-細胞因子受體相互作用途徑。這並不出乎意料，因為PPP1R12C為pro-IL16的已知結合配偶體，其裂解產物參與T細胞免疫反應(Bannert等人，2003年；Cruickshank與Little，2008年)。沒有一個失調的基因定位於KEGG中的任何其他15個經典癌症途徑，儘管有7個是潛在的原癌基因。RNA sequencing produces unknowable whole transcriptome data, with higher sensitivity and dynamic range than gene arrays. Given the limitations of individual gene composition analysis, it is inferred here that RNA sequencing will provide complementary whole-genome functional readings that will signal whether the umbilical cord lining epithelial cells (CLECs) are persistently significant within genes and regulatory elements. (ZFN) Induced off-target hits. Although the present invention does not describe the performance of non-coding RNAs (ncRNAs), off-target events may also change short and long non-coding RNAs (ncRNAs). However, since the main function of non-coding RNAs (ncRNAs) is to regulate transcription (Morris, 2011; Patil et al., 2014), the consequences of changes in the performance of non-coding RNAs (ncRNAs) will also be reflected in RNA sequencing data. In this case, compared with wild-type CLEC, only 0.5% of 17,751 expressed transcripts in puro-CLEC were erroneously regulated by 2 times or more (Figure 12(a)-(c)). Note that the dysregulated genes only map to cytokine-cytokine receptor interaction pathways. This is not unexpected because PPP1R12C is a known binding partner of pro-IL16, and its lysate is involved in T cell immune responses (Bannert et al., 2003; Cruickshank and Little, 2008). None of the dysregulated genes are localized to any other 15 classic cancer pathways in KEGG, although 7 are potentially proto-oncogenes.

PPP1R12C 編碼蛋白磷酸酶1 (protein phosphatase 1, PP1)的調節次單位，參與廣泛的重要生物過程，包括退出有絲***、細胞凋亡、DNA損傷反應、信號以及代謝(Hofman 等人，2000年；Brady與Saltiel，2001年；Bennett，2005年；Kuntziger 等人，2011年；Wurzenberger與Gerlich，2011年；Meadows，2013年；Korrodi-Gregorio 等人，2014年)。PPP1R12C與幾種蛋白質相互作用，包括CAMKK1、CDC42BPB、MPRIP、MYL2、MYL5、MYL7、MYL9、MYL10、MYL12A、MYL12B、MYLPF、PHLPP2 (string-db.org)、PRKG1(Surks等人，1999年)、pro-IL- 16 (Bannert等人，2003年)，以及SRF (Mulder等人，2005年)。其相互作用的配偶體在細胞增殖與分化、細胞存活、細胞遷移、細胞凋亡、轉錄、細胞骨架組織以及訊號傳遞(一氧化氮-環GMP、Akt1、蛋白激酶C及A)中具有作用。鑑於PPP1R12C以及PP1的許多潛在關鍵功能，puro-CLEC的RNA定序數據幾乎沒有證據顯示PPP1R12C 的單倍體不足在功能上是有害的。這與胚胎幹細胞及誘導多能幹細胞對AAVS1 鋅指核酸酶(ZFN)誘導的PPP1R12C 雙等位基因破壞的耐受性相一致(Smith等人，2008年；Hockemeyer等人，2009年；DeKelver等人，2010年)。由於本發明中的FVIII供體DNA構築被設計為不整合外源增強子，因此在此進行的觀察為AAVS1基因座的安全性狀態提供了更多支持。 PPP1R12C encodes a regulatory subunit of protein phosphatase 1, PP1, and participates in a wide range of important biological processes, including withdrawal from mitosis, apoptosis, DNA damage response, signaling, and metabolism (Hofman et al., 2000; Brady and Saltiel, 2001; Bennett, 2005; Kuntziger et al., 2011; Wurzenberger and Gerlich, 2011; Meadows, 2013; Korrodi-Gregorio et al., 2014). PPP1R12C interacts with several proteins, including CAMKK1, CDC42BPB, MPRIP, MYL2, MYL5, MYL7, MYL9, MYL10, MYL12A, MYL12B, MYLPF, PHLPP2 (string-db.org), PRKG1 (Surks et al., 1999), pro-IL-16 (Bannert et al., 2003), and SRF (Mulder et al., 2005). Its interacting partners play a role in cell proliferation and differentiation, cell survival, cell migration, apoptosis, transcription, cytoskeletal organization, and signal transmission (Nitric Oxide-Cyclic GMP, Akt1, Protein Kinase C, and A). Given the many potential key functions of PPP1R12C and PP1, puro-CLEC's RNA sequencing data shows little evidence that PPP1R12C 's insufficient haploidity is functionally harmful. This is consistent with the tolerance of embryonic stem cells and induced pluripotent stem cells to AAPS1 zinc finger nuclease (ZFN)-induced PPP1R12C biallelic damage (Smith et al., 2008; Hockemeyer et al., 2009; DeKelver et al. ,year 2010). Since the FVIII donor DNA construct in the present invention is designed to not integrate foreign enhancers, the observations made here provide more support for the safety status of the AAVS1 locus.

鋅指核酸酶(ZFNs)已被有意用於重建癌症相關的染色體易位(Piganeau等人，2013年)。由於染色體重排也可能由非預期的偏離目標活性引起，本案發明人分析了puro-CLEC全基因組定序(WGS)數據，以了解染色體結構變異的可能跡象。可能出現四種不平衡的染色體重排。 所有假定的斷點都是內含子、基因間或3'UTR (圖11C)。然而，透過定量PCR的實驗驗證未在所檢查的任何可能斷點處顯示不平衡的複製數(圖11(d))且未檢測到預測的融合轉錄物，顯示這些可能是數據分析的偽陽性結果。Zinc finger nucleases (ZFNs) have been intentionally used to reconstruct cancer-related chromosomal translocations (Piganeau et al., 2013). Since chromosomal rearrangements may also be caused by unexpected deviations from target activity, the inventors of this case analyzed puro-CLEC whole genome sequencing (WGS) data to understand possible signs of chromosomal structural variation. There may be four unbalanced chromosome rearrangements. All hypothetical breakpoints are introns, intergenic or 3'UTR (Figure 11C). However, experimental verification through quantitative PCR did not show an unbalanced copy number at any possible breakpoints examined (Figure 11(d)) and no predicted fusion transcripts were detected, showing that these may be false positives for data analysis result.

認識到新鮮的臍帶是臍帶襯裡上皮細胞(CLECs)的來源，但是應該為所有年齡的A型血友病患者開發自體細胞療法，也測試了三種易於獲得的原代人體細胞，即真皮纖維母細胞、骨髓與脂肪組織-來源的基質細胞。使用在臍帶襯裡上皮細胞(CLECs)中整合FVIII轉殖基因的相同AAVS1 鋅指核酸酶(ZFN)構築，所有三種細胞類型都允許相當高含量的DNA切割活性。與puro-CLEC一樣，鋅指核酸酶(ZFN)修飾的原代纖維母細胞以及骨髓衍生的基質細胞也分泌FVIII (圖24)。這表示，對於使用纖維母細胞、骨髓及脂肪組織來源的基質細胞的成年患者，自體細胞療法的方法也可能廣泛應用，其合成並分泌治療性蛋白質的熟練程度是眾所周知的(Naffakh 等人，1995年；Falqui 等人，1999年；Bartholomew 等人，2001年；Schwenter 等人，2003年；Rehman 等人，2004年；Kyriakou 等人，2006年；Suga 等人，2009年；Kakeda 等人，2011年)。Recognizing that fresh umbilical cord is the source of umbilical cord lining epithelial cells (CLECs), but autologous cell therapy should be developed for patients of hemophilia A of all ages, and three readily available primary human cells, dermal fibroblasts, were also tested Cells, bone marrow and adipose tissue-derived stromal cells. Constructed using the same AAVS1 zinc finger nuclease (ZFN) that integrates FVIII transgenes in umbilical cord lining epithelial cells (CLECs), all three cell types allow a fairly high level of DNA cleavage activity. Like puro-CLEC, primary fibroblasts modified with zinc finger nuclease (ZFN) and bone marrow-derived stromal cells also secrete FVIII (Figure 24). This means that for adult patients using stromal cells derived from fibroblasts, bone marrow, and adipose tissue, autologous cell therapy may also be widely used, and their proficiency in synthesizing and secreting therapeutic proteins is well known (Naffakh et al., 1995; Falqui et al., 1999; Bartholomew et al., 2001; Schwenter et al., 2003; Rehman et al., 2004; Kyriakou et al., 2006; Suga et al., 2009; Kakeda et al., 2011 year).

總之，本發明顯示，設計用於高生物安全性的AAVS1 鋅指核酸酶(ZFN)對於使用幾種原代人類細胞類型的自體細胞療法具有廣泛的適用性，並可開發為潛在的FVIII分泌生物植入物，具有低意外致癌作用的風險。In conclusion, the present invention shows that the AAVS1 zinc finger nuclease (ZFN) designed for high biosafety has broad applicability for autologous cell therapy using several primary human cell types and can be developed as a potential FVIII secretor Biological implants have a low risk of accidental carcinogenesis.

材料與方法Materials and Methods

細胞培養Cell culture

K562細胞購自美國典型培養物保藏中心。原代人類細胞(真皮纖維母細胞、脂肪來源的基質細胞，以及骨髓來源的基質細胞)由新加坡CellResearch公司和國立大學衛生系統IRB核准提供並提供。如WO 2006/019357所述分離上皮細胞襯裡幹細胞，然後如上述實施例中所述進行培養。K562 cells were purchased from the American Type Culture Collection. Primary human cells (dermal fibroblasts, adipose-derived stromal cells, and bone marrow-derived stromal cells) were approved and provided by Singapore CellResearch Company and National University Health System IRB. The epithelial cell lining stem cells were isolated as described in WO 2006/019357 and then cultured as described in the above examples.

將K562細胞在補充有10%胎牛血清(Hyclone)的Iscove改良的Eagle培養基(Sigma-Aldrich公司)中培養。除了臍帶上皮細胞外，所有原代人類細胞在補充有10%胎牛血清的Dulbecco改良的Eagle培養基(DMEM)-25 mM葡萄糖(Sigma-Aldrich公司)中培養。臍帶上皮細胞在培養基171 (Cascade Biologicals公司)中培養，培養基中添加50 ng/ml IGF-1、50 ng/ml PDGF-BB、5 ng/ml TGF-β1，以及5 ng/ml胰島素(均來自R&D Systems公司)。K562 cells were cultured in Iscove's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Hyclone). Except for umbilical cord epithelial cells, all primary human cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-25 mM glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum. Umbilical cord epithelial cells were cultured in medium 171 (Cascade Biologicals), supplemented with 50 ng/ml IGF-1, 50 ng/ml PDGF-BB, 5 ng/ml TGF-β1, and 5 ng/ml insulin (all from R&D Systems).

AAVS1AAVS1 鋅指核酸酶Zinc finger nuclease (ZFNs)(ZFNs)

將編碼Fok1 的野生型催化結構域的DNA (pST1374；Addgene公司)誘變用於異二聚化(Miller等人，2007年)。OH (專性異二聚體)鋅指核酸酶(ZFN)在與右AAVS1同源臂(E490K以及I538K；SEQ ID NO. 7)融合的Fok1 單體以及與左同源臂(Q468E與I499L)融合的單體中具有兩個胺基酸變化(SEQ ID NO. 8)。進一步製備專性異二聚體(OH)鋅指核酸酶(ZFN)變體以增強切割活性： a) Sharkey變體在右及左單體中均具有S418P與K441E取代(Guo等人，2010年)(SEQ ID NO: 12與13)。 b) 增強型Sharkey變體具有額外的胺基酸取代：S418P、K441E以及H537R (右Fok1 單體；(SEQ ID NO: 14)以及S418P、K441E以及N496D (左Fok1 單體)(Doyon等人，2011年)(SEQ ID NO: 15)。DNA encoding the wild-type catalytic domain of Fok1 (pST1374; Addgene Corporation) was used for heterodimerization (Miller et al., 2007). OH (obligate heterodimer) zinc finger nuclease (ZFN) in the Fok1 monomer fused to the right AAVS1 homology arm (E490K and I538K; SEQ ID NO. 7) and to the left homology arm (Q468E and I499L) There are two amino acid changes in the fused monomer (SEQ ID NO. 8). Further preparation of obligate heterodimer (OH) zinc finger nuclease (ZFN) variants to enhance cleavage activity: a) Sharkey variants have S418P and K441E substitutions in both the right and left monomers (Guo et al. 2010 ) (SEQ ID NOs: 12 and 13). b) The enhanced Sharkey variant has additional amino acid substitutions: S418P, K441E and H537R (right Fok1 monomer; (SEQ ID NO: 14) and S418P, K441E and N496D (left Fok1 monomer) (Doyon et al., 2011) (SEQ ID NO: 15).

值得注意的是，所示突變的位置對應於野生型FokI 序列的位置，例如，如SEQ ID NO. 6所示。這表示，例如S418P表示SEQ ID NO. 6的野生型FokI 的第418位置的S突變為P。It is worth noting that the position of the mutation shown corresponds to the position of the wild-type FokI sequence, for example, as shown in SEQ ID NO. 6. This means that, for example, S418P indicates that the S at position 418 of the wild-type FokI of SEQ ID NO. 6 is mutated to P.

最終的鋅指核酸酶(ZFN)構築在單個質體中組合了右與左AAVS1-Fok1表現匣(圖14A)。The final zinc finger nuclease (ZFN) construct combines the right and left AAVS1-Fok1 expression cassettes in a single plastid (Figure 14A).

編碼用於AAVS1基因座的一對鋅指胜肽的密碼子優化的DNA (Hockemeyer等人，2009年)是商業合成的(DNA2.0，美國)，並在單獨的質體構築中與其相應的誘變的Fok1 單體連接。The codon-optimized DNA encoding a pair of zinc finger peptides for the AAVS1 locus (Hockemeyer et al., 2009) is commercially synthesized (DNA2.0, USA) and corresponds to it in a separate plastid construct Mutagenic Fok1 monomer linkage.

供體Donor DNADNA

使用具有新黴素抗性標記的三種質體將增加大小的供體DNA整合到PPP1R12C 的內含子1中(圖14B)：Three types of plastids with neomycin resistance markers were used to integrate the increased size donor DNA into intron 1 of PPP1R12C (Figure 14B):

pZDonor (與AAVS1基因座具有1500-bp同源性，由50-bp多選殖位點一分為二；Sigma-Aldrich公司)pZDonor (1500-bp homology with the AAVS1 locus, divided into two by a 50-bp multiple selection site; Sigma-Aldrich)

pZDonor EGFP (從pZDonor中選殖的pEGFP-C1 (Clontech公司)切除的3.75-kb CMV啟動子-GFP)pZDonor EGFP (3.75-kb CMV promoter-GFP excised from pZGFP-C1 (Clontech) selected from pZDonor)

pZDonor雜合FVIII (編碼人類鐵蛋白輕鏈啟動子的9.1-kb供體-選殖在pZDonor中的雜合FVIII cDNA；如下所述)。pZDonor hybrid FVIII (9.1-kb donor encoding human ferritin light chain promoter-hybrid FVIII cDNA cloned in pZDonor; described below).

為了整合到原代人類細胞中，將具有無啟動子的嘌呤黴素抗性選擇基因的供體載體組裝在pAAVS1 SA-2A-嘌呤黴素-pA (Addgene質體編號22075)以及pAAV-CAGGS-EGFP (Addgene質體編號22212)上(圖14C)。For integration into primary human cells, a donor vector with a promoter-free puromycin resistance selection gene was assembled in pAAVS1 SA-2A-puromycin-pA (Addgene plastid number 22075) and pAAV-CAGGS- EGFP (Addgene plastid number 22212) (Figure 14C).

雜合hybrid FVIII cDNAFVIII cDNA

B結構域截短的人-豬FVIII cDNA由豬A1與A3結構域、人類訊息胜肽、A2、殘基B (包含前266個胺基酸以及8個糖基化位點)、C1及C2結構域組成(Sivalingam等人，2014年，也 在SEQ ID NO. 3中描述了重疊PCR。在結構域組裝中使用重疊PCR (參閱圖25)。透過基於參考豬cDNA序列(NM_214167.1)的總豬肝RNA的RT-PCR獲得A1與A3結構域。在pSP64-VIII (美國典型培養物保藏中心)中從完整的人類FVIII cDNA擴增人類結構域。The truncated B-domain human-porcine FVIII cDNA consists of porcine A1 and A3 domains, human signaling peptides, A2, residue B (including the first 266 amino acids and 8 glycosylation sites), C1 and C2 Domain composition (Sivalingam et al., 2014, also described overlapping PCR in SEQ ID NO. 3. Overlapping PCR was used in domain assembly (see FIG. 25). Through the reference pig cDNA sequence (NM_214167.1) RT-PCR of total porcine liver RNA obtained the A1 and A3 domains. The human domain was amplified from the complete human FVIII cDNA in pSP64-VIII (American Type Culture Collection).

限制性片段長度多態性Restriction fragment length polymorphism

限制性片段長度多態性(RFLP)用於量化pZDonor-AAVS1的位點特異性整合。在不存在或存在AAVS1鋅指核酸酶(ZFN)的情況下，在電穿孔10 μg pZDonor-AAVS1後4天從細胞中萃取200 ng基因組DNA。PCR引子擴增跨越AAVSI整合位點的1.9-kb區域。以Hind III消化的擴增子透過在8%聚丙烯醯胺凝膠中電泳解析，以溴化乙錠後染色並成像(BioRad®Gel Doc 2000透照儀)。兩個條帶(1-kb以及0.9-kb)顯示供體整合，而單個1.9-kb條帶表示沒有整合。透過Quantity One軟體(Bio-Rad公司)定量DNA條帶的強度與體積。Restriction fragment length polymorphism (RFLP) was used to quantify the site-specific integration of pZDonor-AAVS1. In the absence or presence of AAVS1 zinc finger nuclease (ZFN), 200 ng of genomic DNA was extracted from cells 4 days after electroporation of 10 μg pZDonor-AAVS1. The PCR primers amplify the 1.9-kb region spanning the AAVSI integration site. Amplicons digested with Hind III were resolved by electrophoresis in 8% polyacrylamide gel, stained with ethidium bromide and imaged (BioRad® Gel Doc 2000 transilluminator). Two bands (1-kb and 0.9-kb) show donor integration, while a single 1.9-kb band indicates no integration. The intensity and volume of DNA bands were quantified by Quantity One software (Bio-Rad).

CEL-1CEL-1 分析analysis

透過使用Surveyor™突變檢測套組(Transgenomic公司)的試劑及說明書檢測CEL-1核酸酶，檢測到的非同源末端連接修復來測定鋅指核酸酶(ZFN)活性。The zinc finger nuclease (ZFN) activity was determined by using the Surveyor™ Mutation Detection Kit (Transgenomic) reagents and instructions to detect the CEL-1 nuclease and the detected non-homologous end junction repair.

流式細胞儀分析Flow cytometry analysis

透過螢光活化細胞分析表現綠色螢光蛋白(GFP)的細胞(BD FACSCalibur™流式細胞儀；488 nm氬激光；530/30帶通濾光器)，在電穿孔後24小時評估轉染效率。Analyze cells expressing green fluorescent protein (GFP) by fluorescent activated cells (BD FACSCalibur™ flow cytometer; 488 nm argon laser; 530/30 bandpass filter) and evaluate transfection efficiency 24 hours after electroporation .

透過組蛋白H2AX磷酸化評估DNA損傷反應。電穿孔後2天，將臍帶襯裡上皮細胞(CLECs)以磷酸鹽緩衝鹽水(phosphate-buffered saline, PBS)/ 90%甲醇中的3.7%甲醛固定、透化(0.5%Triton-X100, 2%牛血清白蛋白) 10分鐘，並與AlexaFluor®647共軛-抗磷酸化合物H2AX抗體(Ser139)(1:40稀釋；Cell Signaling Technology)於25°C下作用1小時。以PBS洗滌細胞兩次，重新懸浮於500 μl PBS中並透過40 μm尼龍網過濾。以633 nm He-Ne激光以及661/16帶通濾波器進行流式細胞儀分析。DNA damage response was assessed by histone H2AX phosphorylation. Two days after electroporation, umbilical cord lining epithelial cells (CLECs) were fixed and permeabilized with phosphate-buffered saline (PBS)/3.7% formaldehyde in 90% methanol (0.5% Triton-X100, 2% bovine Serum albumin) for 10 minutes and reacted with AlexaFluor®647 conjugated anti-phospho compound H2AX antibody (Ser139) (1:40 dilution; Cell Signaling Technology) at 25°C for 1 hour. The cells were washed twice with PBS, resuspended in 500 μl PBS and filtered through a 40 μm nylon mesh. Flow cytometry analysis was performed with 633 nm He-Ne laser and 661/16 bandpass filter.

使用FlowJo版本7.22 (FLOWJO)分析數據。Use FlowJo version 7.22 (FLOWJO) to analyze the data.

基因轉移Gene transfer

將含有10 μg供體DNA質體以及5 μg鋅指核酸酶(ZFN)質體或5 μl AAVS1鋅指核酸酶(ZFN) mRNA(Sigma-Aldrich公司)的100 μl Amaxa®CellLine Nucleofactor Kit V溶液中懸浮的200萬個K562細胞，以Amaxa®NucleofectorI (Lonza公司)中的T016設置進行電穿孔。在指出的情況下，以pEGFP共轉染細胞以評估基因轉移的效率。透過在G418 (0.8 mg/ml)中培養14天來選擇穩定整合的細胞。Place 100 μl of Amaxa® CellLine Nucleofactor Kit V solution containing 10 μg of donor DNA plastid and 5 μg of zinc finger nuclease (ZFN) plasmid or 5 μl of AAVS1 zinc finger nuclease (ZFN) mRNA (Sigma-Aldrich) The suspended 2 million K562 cells were electroporated with the T016 setting in Amaxa® NucleofectorI (Lonza Corporation). Under the indicated conditions, cells were co-transfected with pEGFP to assess the efficiency of gene transfer. Stably integrated cells were selected by culturing in G418 (0.8 mg/ml) for 14 days.

如圖例中所示，以10 μg供體DNA質體以及5 μg鋅指核酸酶(ZFN)質體電穿孔2至1千萬個原代人類細胞(Amaxa®4DNucleofector；設置CM113)。穩定整合的臍帶襯裡上皮細胞對嘌呤黴素具有抗性(0.5 mg/ml，持續7天)(puro-CLECs)。野生型-CLEC未以質體處理。As shown in the legend, 20 to 10 million primary human cells (Amaxa® 4DNucleofector; setting CM113) were electroporated with 10 μg donor DNA plastids and 5 μg zinc finger nuclease (ZFN) plastids. Stably integrated umbilical cord lining epithelial cells are resistant to puromycin (0.5 mg/ml for 7 days) (puro-CLECs). Wild-type CLEC was not treated with plastids.

FVIIIFVIII 分析analysis

使用Coatest SP FVIII套組(Chromogenix公司)以及推薦的方案測定野生型-與puro-CLEC的過夜條件培養基中的FVIII活性。數據表示為mUnits FVIII/百萬細胞/24小時(三次重複的平均值±SD)。Coatest SP FVIII kit (Chromogenix) and the recommended protocol were used to determine the FVIII activity in the overnight conditioned medium of wild-type and puro-CLEC. The data is expressed as mUnits FVIII/million cells/24 hours (mean of three replicates ± SD).

整合連接Integrated connection PCR (JPCR)PCR (JPCR)

使用DyNAzyme EXT DNA聚合酶(Thermo Scientific公司)以及對整合載體特異的引子與緊鄰整合位點的基因組序列對200 ng基因組DNA進行整合連接PCR。陽性對照PCR在距離整合位點2 kb的AAVS1基因座內擴增了單獨的區域。對整合連接PCR產物進行定序以驗證其身份。重疊長PCR與定序證實了完整雜合FVIII轉殖基因的整合。200 ng of genomic DNA was integrated and ligated using DyNAzyme EXT DNA polymerase (Thermo Scientific) and primers specific to the integration vector and the genomic sequence immediately adjacent to the integration site. The positive control PCR amplified a separate region within the AAVS1 locus 2 kb from the integration site. The integrated ligation PCR products are sequenced to verify their identity. Overlapping long PCR and sequencing confirmed the integration of the complete hybrid FVIII transgene.

數位微滴式Digital droplet PCRPCR

將來自野生型-與puro-CLEC的50 ng基因組DNA、1 μM的每種引子以及0.25 μM BHQ1-FAM探針(Sigma-Aldrich公司)加入到QX200™ dd PCR™ supermix (Bio-Rad公司)中，最終反應體積為20 μl並轉移至QX100™液滴發生器，然後使用C1000 Touch™熱循環儀進行40個PCR擴增循環(黏合溫度61°C，延伸時間為每個循環1分鐘)。在QX100™ Droplet讀取器上讀取液滴PCR產物，並使用QuantaSoft™軟體分析數據。對照1 (正向引子：cctgccttaaacccagccag (SEQ ID NO: 17)；反向引子：atgacctcatgctcttggccctcgta (SEQ ID NO: 18)；探針：aaccaccccagcagatactct (SEQ ID NO: 6)以及對照2 (正向引子：tcccctcccagaaagacctgc；SEQ ID NO: 19，反向引子：tcccctcccagaaagacctgc；SEQ ID NO: 20；探針：tacctaacgcactcctgggtga，SEQ ID NO: 21)擴增了人類染色體19q13.42中的基因組基因座。為左側設計的反應(基因組特異性正向引子：ttcgggtcacctctcactcc；SEQ ID NO: 22，載體特異性反性引子：gacgcgcgtgaggaagagttc (SEQ ID NO: 23)；探針：aggcgcaccgtgggcttgt，SEQ ID NO: 24)以及右側(載體特異性正向引子：ctgtggaatgtgtgtcagttag，SEQ ID NO: 25；基因組特異性反向引子：ggctccatcgtaagcaaacc，SEQ ID NO: 26；探針：cgcctctgcctctgagctat，SEQ ID NO: 27)整合連接擴增AAVS1位點特異性整合載體，而所有整合的載體，無論基因組位置如何，都透過載體特異性PCR檢測(正向引子：cgagatgaccgagtacaag；SEQ ID NO: 28；反向引子：gctcgtagaaggggaggttg；SEQ ID NO: 29；探針：tcaccgagctgcaagaact，SEQ ID NO: 30)。每個PCR反應進行四重複。數據為平均值±SD。Add 50 ng genomic DNA from wild-type and puro-CLEC, 1 μM of each primer, and 0.25 μM BHQ1-FAM probe (Sigma-Aldrich) to QX200™ dd PCR™ supermix (Bio-Rad) , The final reaction volume is 20 μl and transferred to the QX100™ droplet generator, and then use the C1000 Touch™ thermal cycler for 40 PCR amplification cycles (adhesion temperature 61°C, extension time is 1 minute per cycle). Read the droplet PCR products on the QX100™ Droplet Reader and analyze the data using QuantaSoft™ software. Control 1 (forward primer: cctgccttaaacccagccag (SEQ ID NO: 17); reverse primer: atgacctcatgctcttggccctcgta (SEQ ID NO: 18); probe: aaccaccccagcagatactct (SEQ ID NO: 6) and control 2 (forward primer: tcccctcccagaaagacctgc; SEQ ID NO: 19, reverse primer: tcccctcccagaaagacctgc; SEQ ID NO: 20; probe: tacctaacgcactcctgggtga, SEQ ID NO: 21) amplified the genomic locus in human chromosome 19q13.42. The reaction designed for the left side (genome Specific forward primer: ttcgggtcacctctcactcc; SEQ ID NO: 22, vector-specific reverse primer: gacgcgcgtgaggaagagttc (SEQ ID NO: 23); probe: aggcgcaccgtgggcttgt, SEQ ID NO: 24) and the right side (vector-specific forward primer : Ctgtggaatgtgtgtcagttag, SEQ ID NO: 25; Genome-specific reverse primer: ggctccatcgtaagcaaacc, SEQ ID NO: 26; Probe: cgcctctgcctctgagctat, SEQ ID NO: 27) Integration Linkage AAVS1 site-specific integration vector, and all integration The vector, regardless of the genomic location, is detected by vector-specific PCR (forward primer: cgagatgaccgagtacaag; SEQ ID NO: 28; reverse primer: gctcgtagaaggggaggttg; SEQ ID NO: 29; probe: tcaccgagctgcaagaact, SEQ ID NO: 30 ). Each PCR reaction was performed in quadruplicate. The data are mean ± SD.

擴增子深度定序Amplicon depth sequencing

以來自AAVS1整合位點以及野生型-與puro-CLECS的OT1-OT10的擴增子的大規模平行配對末端定序(MiSeq，Illumina公司)研究之前針對AAVS1鋅指核酸酶(ZFNs)預測的10個最可能的潛在偏離目標位點(Hockemeyer等人，2009年)(OT1-OT10；圖26)。將商業合成的DNA片段(GenScript公司)與AAVS1基因座序列相似，除了鋅指核酸酶(ZFN)結合半位點之間的5-bp缺失外，以1:10、1:100、1:500以及1:1000的莫爾比摻入野生型AAVS1基因座擴增子中，確定***或缺失(indel)檢測的靈敏度(圖20)。基因庫構建(Nextera® XT DNA樣品製備套組，Illumina公司)且定序由AITbiotech私人有限公司(新加坡)進行。Large-scale parallel paired end sequencing (MiSeq, Illumina) from the AAVS1 integration site and wild-type-OT1-OT10 amplicons with puro-CLECS was used to study the previously predicted 10 for AAVS1 zinc finger nucleases (ZFNs). The most likely potential deviation from the target site (Hockemeyer et al., 2009) (OT1-OT10; Figure 26). The commercially synthesized DNA fragment (GenScript) is similar to the AAVS1 locus sequence, except for the 5-bp deletion between the binding half sites of zinc finger nuclease (ZFN), at 1:10, 1:100, 1:500 And the molar ratio of 1:1000 was incorporated into the wild-type AAVS1 locus amplicon to determine the sensitivity of indel detection (Figure 20). Gene library construction (Nextera ® XT DNA sample preparation kit, Illumina) and sequencing were performed by AITbiotech Sdn Bhd (Singapore).

全基因組定序Genome-wide sequencing

全基因組定序(WGS)由BGI公司(中國深圳)執行。從來自野生型-與puro-CLEC的5 μg隨機片段化的基因組DNA製備具有500-bp***物的基因庫，並在HiSeq 2000 (Illumina公司)上定序。從原始數據中刪除了銜接子序列、重複讀數(使用Picard工具標記；picard.sourceforge.net)、低品質讀數(讀數中超過一半的鹼基品質≤5)以及讀數>10%未知鹼基。使用Burrows-Wheeler Aligner (BWA公司)(Li與Durbin，2009年)將來自兩個樣品的配對末端清潔讀數(90個鹼基)與參考人類基因組(hg19)比對，並以BAM格式文件存儲。兩個樣品的定序深度均為23倍。Whole genome sequencing (WGS) is performed by BGI Corporation (Shenzhen, China). A gene library with a 500-bp insert was prepared from 5 μg of randomly fragmented genomic DNA from wild-type and puro-CLEC, and sequenced on HiSeq 2000 (Illumina Corporation). Adaptor sequences, repeated reads (marked with Picard tool; picard.sourceforge.net), low-quality reads (more than half of the bases in the read are ≤5 in quality), and reads >10% unknown bases were deleted from the original data. Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2009) was used to compare the paired end clean readings (90 bases) from the two samples with the reference human genome (hg19) and store them in a BAM format file. The sequence depth of both samples is 23 times.

RNARNA 定序Sequencing

來自野生型-與puro-CLEC的總RNA透過Bioanalyzer (Agilent公司)定性評估，並透過螢光計(Life Technologies公司)定量。根據標準方案(Illumina TruSeq RNA樣品製備v2套組)，將2 μg高品質RNA (RIN> 9)用於基因庫製備。簡言之，在oligo-dT磁珠上純化poly-A mRNA。純化後，將mRNA片段化(150-250 bp)並使用反轉錄酶及隨機引子轉化為第一鏈cDNA。以DNA聚合酶I以及RNaseH進行第二鏈cDNA合成。接著將cDNA片段平端化，加入單個「A」鹼基以連接到具有互補T-突出端的索引銜接子。將臍帶引產物純化並以PCR富集以產生最終的cDNA基因庫。透過Bioanalyzer驗證索引基因庫的大小及純度，並使用Quant-iT™ PicoGreen® dsDNA測定套組(Life Technologies公司)進行定量。透過即時PCR (iTaq TMUniversalSYBR® GreenSupermix；BioRad公司)將基因庫標準化至10 nM，合併等體積。將合併的基因庫變性並稀釋至20 pM以在cBot上聚類，然後加載到HiSeq 2000上以產生76 bp的配對末端讀數。Total RNA from wild-type and puro-CLEC was qualitatively evaluated by Bioanalyzer (Agilent) and quantified by fluorometer (Life Technologies). According to the standard protocol (Illumina TruSeq RNA sample preparation v2 set), 2 μg high-quality RNA (RIN>9) was used for gene library preparation. Briefly, poly-A mRNA was purified on oligo-dT magnetic beads. After purification, the mRNA is fragmented (150-250 bp) and converted into first-strand cDNA using reverse transcriptase and random primers. DNA polymerase I and RNaseH were used for second-strand cDNA synthesis. The cDNA fragment is then blunted and a single "A" base is added to connect to the index adaptor with complementary T-overhangs. The umbilical cord primer product was purified and enriched by PCR to produce the final cDNA gene library. The size and purity of the index gene library were verified by Bioanalyzer, and quantified using the Quant-iT™ PicoGreen® dsDNA assay kit (Life Technologies). The gene library was standardized to 10 nM by real-time PCR (iTaq TM Universal SYBR® Green Supermix; BioRad), and equal volumes were pooled. The pooled gene pool was denatured and diluted to 20 pM to cluster on cBot, and then loaded onto HiSeq 2000 to generate 76 bp paired end reads.

生物資訊資料分析Bioinformatics data analysis

檢測目標***缺失Detect target insertion deletion

將puro-CLEC樣品的所有讀數與含有預期鋅指核酸酶(ZFN)切割位點的980-bp序列比對，e值截止值為0.00001。使用SMALT (www.sanger.ac.uk/resources/software/smalt/)將所有比對的讀數映射回參考人類基因組(hg19)以產生序列比對文件。VarScan v2.3.6用於使用命令「pileup2indel」檢測***缺失(Koboldt等人，2012年)。All reads of the puro-CLEC sample were aligned with the 980-bp sequence containing the expected zinc finger nuclease (ZFN) cleavage site, and the e-value cutoff was 0.00001. Use SMALT (www.sanger.ac.uk/resources/software/smalt/) to map all aligned reads back to the reference human genome (hg19) to generate sequence alignment files. VarScan v2.3.6 is used to detect insertion deletions using the command "pileup2indel" (Koboldt et al., 2012).

檢測非目標***缺失Detect non-target indels

使用VarScan v2.3.6分析與hg19比對的所有野生型-與puro-CLEC讀數的體細胞變體。使用SAMtools將二元比對格式的序列比對轉化為SAMtools堆積格式(Li等人，2009年)。產生的堆積文件交由VarScan使用命令「somatic」進行體細胞變體調用，其中野生型-CLEC為「normal」，puro-CLEC為「tumor」 (java –jar VarScan.jar somatic >pileup file of WT sample> >pileup file of PURO sample> output)。使用命令「processSomatic」 (java -jar VarScan.jar processSomatic output.indel)將VarScan識別的體細胞變體進一步分類為高置信度(.hc)或低置信度(.lc)。進一步分析高可信度的體細胞變體以產生用於實驗驗證的候選***或缺失(indel)的最終列表。VarScan v2.3.6 was used to analyze all somatic variants of wild-type and puro-CLEC readings aligned with hg19. SAMtools was used to convert the sequence alignment of the binary alignment format into the SAMtools stacked format (Li et al., 2009). The generated stack file is transferred to VarScan using the command "somatic" for somatic cell variant calls, where wild-type CLEC is "normal" and puro-CLEC is "tumor" (java –jar VarScan.jar somatic >pileup file of WT sample > >pileup file of PURO sample> output). Use the command "processSomatic" (java -jar VarScan.jar processSomatic output.indel) to further classify the somatic variants identified by VarScan as high confidence (.hc) or low confidence (.lc). The high-confidence somatic cell variants are further analyzed to produce a final list of candidate insertions or deletions (indel) for experimental verification.

使用鋅指核酸酶(ZFN)-位點在電腦中鑑定了hg19中的推定的偏離目標位點(Cradick等人，2011年)。左及右鋅指核酸酶(ZFN)半位點分別為ATCCTGTCCCTA (SEQ ID NO: 31)以及ACCCCACAGTGG (SEQ ID NO: 32)。允許的間距為5或6 bp，每個半位點的最大錯配數設定為2。高信度體細胞***與電腦預測的AAVS1-鋅指核酸酶(ZFN)偏離目標位點之間沒有重疊。The zinc finger nuclease (ZFN)-site was used to identify putative off-target sites in hg19 in a computer (Cradick et al., 2011). The left and right zinc finger nuclease (ZFN) half sites are ATCCTGTCCCTA (SEQ ID NO: 31) and ACCCCACAGTGG (SEQ ID NO: 32), respectively. The allowed spacing is 5 or 6 bp, and the maximum number of mismatches per half-site is set to 2. There is no overlap between the high-confidence somatic cell insertion and the computer predicted AAVS1-zinc finger nuclease (ZFN) deviation from the target site.

接下來，採用另一種方法來識別可能的偏離目標效應。使用內部Perl腳本，掃描讀數的熱點基因組區域，其中映射多個重疊的高置信度體細胞***或缺失(indel)變體。透過PCR-Sanger定序對這些基因組基因座進行實驗驗證。Next, use another method to identify possible off-target effects. Using an internal Perl script, the hot-spot genomic region of the reading is scanned, where multiple overlapping high-confidence somatic cell insertion or deletion (indel) variants are mapped. These genomic loci were experimentally verified by PCR-Sanger sequencing.

鑑定AAVS1 鋅指核酸酶(ZFN)調節的FVIII供體DNA***Identify AAVS1 zinc finger nuclease (ZFN) regulated FVIII donor DNA insertion

將所有puro-CLEC讀數與透過整合FVIII供體DNA和參考人類基因組(hg19)修飾的AAVS1序列比對。這在載體中鑑定了對FVIII供體DNA整合特異的原核序列。在野生型-CLEC樣品讀數中缺少這些原核序列並且它們僅存在於puro-CLEC樣品中證實了FVIII供體DNA的整合。為了鑑定偏離目標整合，從puro-CLEC鑑定讀數，該puro-CLEC為人類與非人類序列的組合(下文稱為混合讀數)。混合讀數被映射到參考人類基因組。這定義了推定的偏離目標整合的位置，同時證實了hg19中不存在非人類序列。只有與修飾的AAVS1區域對齊的讀段才被認為是AAVS1基因座中目標鋅指核酸酶(ZFN)調節的FVIII供體DNA***。All puro-CLEC reads were aligned with the AAVS1 sequence modified by integrating FVIII donor DNA and the reference human genome (hg19). This identified a prokaryotic sequence specific for FVIII donor DNA integration in the vector. The lack of these prokaryotic sequences in the wild-type-CLEC sample reads and their presence only in puro-CLEC samples confirmed the integration of FVIII donor DNA. To identify off-target integrations, reads were identified from puro-CLEC, which is a combination of human and non-human sequences (hereinafter referred to as mixed reads). The mixed readings are mapped to the reference human genome. This defines the putative deviation from target integration and confirms the absence of non-human sequences in hg19. Only reads aligned with the modified AAVS1 region are considered to be FVIII donor DNA insertions regulated by the target zinc finger nuclease (ZFN) in the AAVS1 locus.

檢測結構變體Detect structural variants

作為SVDetect (版本r0.08；http://svdetect.sourceforge.net) (Zeitouni等人，2010年)以及BreakDancer (版本1.4.4；http://breakdancer.sourceforge.net/) (陳等人，2009年) 只需要異常映射的讀取，原始讀取首先使用SAMtools (版本0.1.19；http://samtools.sourceforge.net/) 以及Picard進行預處理。用於SAMtools的命令行為： samtools view -b -h -F 10 -q 22 input.bam> output.bam。As SVDetect (version r0.08; http://svdetect.sourceforge.net) (Zeitouni et al., 2010) and BreakDancer (version 1.4.4; http://breakdancer.sourceforge.net/) (Chen et al., 2009) Only the reading of the exception map is required. The original reading is first preprocessed using SAMtools (version 0.1.19; http://samtools.sourceforge.net/) and Picard. The command line for SAMtools is: samtools view -b -h -F 10 -q 22 input.bam> output.bam.

保留在output.bam中的此命令僅讀取異常映射的(映射到不同的染色體以及不同的鏈)且其最小映射品質為22。生成的bam文件進一步過濾，以使用Picard工具中的以下命令丟棄重複讀取： java -jar /opt/picard-1.111/MarkDuplicates.jar INPUT=output.bam OUTPUT=output.nodup.bam REMOVE_DUPLICATES=true ASSUME_SORTED=true METRICS_FILE=metrics.output.txt.This command retained in output.bam only reads abnormal mappings (mapped to different chromosomes and different chains) and its minimum mapping quality is 22. The generated bam file is further filtered to discard the repeated reading using the following command in the Picard tool: java -jar /opt/picard-1.111/MarkDuplicates.jar INPUT=output.bam OUTPUT=output.nodup.bam REMOVE_DUPLICATES=true ASSUME_SORTED=true METRICS_FILE=metrics.output.txt.

生成的bam文件交給SVDetect以及BreakDancer以調用結構變體(SV)。SVDetect與BreakDancer都配置為使用8倍標準偏差作為閾值來檢測具有2個或更多支持讀取對的重排。在進行SVDetect的第一步之後，包含所有被調用的SV的所得「.links」文件被過濾為「不完美的重複」(由Mijuskovic等人，2012定義)，具有內部Perl腳本。僅刪除了由不完美重複的群集支持的鏈接。但是，在刪除支持不完美的副本之後，保留了僅具有一些不完美副本的鏈接。在此之後，文件被SVDetect自己的過濾過程進一步過濾。下一步是比較為野生型-與puro-CLEC調用的過濾SV。僅關閉僅比較相同SV類型的選項。沒有為BreakDancer輸出過濾不完美的重複項，因為如果錨定區域設置為3並且BreakDancer中的默認值為7，則不需要這樣做。The generated bam file is handed over to SVDetect and BreakDancer to call the structural variant (SV). Both SVDetect and BreakDancer are configured to use 8 times the standard deviation as a threshold to detect rearrangements with 2 or more supported read pairs. After the first step of SVDetect, the resulting ".links" file containing all called SVs is filtered into "imperfect repetitions" (defined by Mijuskovic et al., 2012), with internal Perl scripts. Only links backed by imperfectly duplicated clusters have been removed. However, after deleting the copy that supports imperfections, links that only have some imperfect copies remain. After this, the file is further filtered by SVDetect's own filtering process. The next step is to compare the filtered SV called for wild-type and puro-CLEC. Only turn off the option to compare only the same SV type. There are no imperfect duplicates filtered for BreakDancer output, because if the anchor area is set to 3 and the default value in BreakDancer is 7, you do not need to do this.

對BreakDancer與SVDetect的結果進行最終過濾，以鑑定重複DNA以及低可映射區域的重疊(Mijuskovic等人，2012年)。僅刪除了與重疊到任何這些區域的讀取支持的SV。使用內部Perl腳本與BEDtools (版本2.17.0；http://bedtools.readthedocs.org/en/latest/)完成過濾(Quinlan與Hall，2010年)。 如所述(Mijuskovic等人，2012年)從UCSC基因組瀏覽器(http://genome.ucsc.edu/)中萃取過濾所需的這些區域的BED文件。在最後一步中，檢查來自BreakDancer以及SVDetect的輸出的基因組位點，其具有與puro-CLEC特異的染色體間或染色體內SV一致的讀數簇。這是透過內部Perl腳本以及不同的聚類自由度(DF)來完成的。最大DF設定為3，即最多999個鹼基的基因組坐標仍被認為是聚類的證據。The results of BreakDancer and SVDetect were finally filtered to identify the overlap of duplicate DNA and low mappable regions (Mijuskovic et al., 2012). Only SVs with read support that overlapped into any of these areas were removed. Use internal Perl scripts and BEDtools (version 2.17.0; http://bedtools.readthedocs.org/en/latest/) to complete filtering (Quinlan and Hall, 2010). As described (Mijuskovic et al., 2012), the BED files of these regions required for filtering are extracted from the UCSC genome browser (http://genome.ucsc.edu/). In the last step, the genomic loci from the output of BreakDancer and SVDetect are examined, which have clusters of reads consistent with puro-CLEC-specific inter- or intra-chromosomal SVs. This is done through internal Perl scripts and different degrees of freedom in clustering (DF). The maximum DF is set to 3, that is, genomic coordinates of up to 999 bases are still considered as evidence of clustering.

RNA定序RNA sequencing

使用TopHat (tophat.cbcb.umd.edu)將序列讀數映射到參考人類基因組(hg19)。 使用Cufflinks (cufflinks.cbcb.umd.edu)計算差異表現。相較於野生型-CLEC，puro-CLEC中表現含量相差≥2倍的轉錄物被認為是顯著改變的。DAVID (用於註釋、可視化以及集成發現的數據庫) 2.1功能註釋工具(http://david.abcc.ncifcrf.gov)(Huang等人，2009年)用於註釋顯著改變的轉錄物及用於途徑作圖。改變的轉錄物亦參考了癌基因與腫瘤抑制基因的匯總彙編(http://www.bushmanlab.org/links/genelists)。TopHat (tophat.cbcb.umd.edu) was used to map sequence reads to the reference human genome (hg19). Use Cufflinks (cufflinks.cbcb.umd.edu) to calculate differential performance. Compared to wild-type CLEC, transcripts with a difference of ≥ 2 times the content of puro-CLEC were considered to have changed significantly. DAVID (database for annotation, visualization, and integrated discovery) 2.1 Functional annotation tool (http://david.abcc.ncifcrf.gov) (Huang et al., 2009) for annotating significantly changed transcripts and for pathways Drawing. The modified transcripts also refer to the summary compilation of oncogenes and tumor suppressor genes (http://www.bushmanlab.org/links/genelists).

驗證潛在的***或缺失Verify potential insertions or deletions (indel)(indel) 與結構變體With structural variants

以phi29聚合酶(REPLI-g套組；Qiagen公司)擴增來自野生型-與puro-CLEC的基因組DNA。透過PCR-Sanger定序研究了高置信度***或缺失(indel)。透過由SVDetect以及BreakDancer鑑定的基因組斷裂區域的定量PCR研究預測的不平衡結構變體。每個15 μl反應三重複進行，在iTaq™ Universal SYBR® Green Supermix (Bio-Rad公司)中包含30 ng基因組DNA以及0.3 μM每種引子。β-肌動蛋白擴增在每個實驗中作為內部對照。在CFX96 Touch™即時檢測系統(Bio-Rad公司)上運行50個擴增循環，之後確定熔解曲線確定產物特異性和閾值循環(C T )值(CFX Manager™軟體，Bio-Rad公司)。將每個測試基因座的平均C T 值標準化為其自身的肌動蛋白C T 值。結果表示為puro-CLEC基因組DNA的標準化C T 值的比率：在每個推定的斷裂點基因座處的野生型-CLEC基因組DNA的標準化C T 值。Genomic DNA from wild-type and puro-CLEC was amplified with phi29 polymerase (REPLI-g kit; Qiagen). High-confidence indels were studied by PCR-Sanger sequencing. Unbalanced structural variants predicted by quantitative PCR studies of genomic break regions identified by SVDetect and BreakDancer. Each 15 μl reaction was performed in triplicate, and included 30 ng of genomic DNA and 0.3 μM of each primer in iTaq™ Universal SYBR® Green Supermix (Bio-Rad). β-actin amplification was used as an internal control in each experiment. 50 amplification cycles were run on the CFX96 Touch™ real-time detection system (Bio-Rad), after which the melting curve was determined to determine product specificity and threshold cycle ( C T ) values (CFX Manager™ software, Bio-Rad). The mean C T value for each test locus normalized for actin own C T value. Results are expressed as normalized ratios C T value puro-CLEC genomic DNA: normalized C T value for each putative wild type genomic DNA breaks -CLEC locus point.

***或缺失Insertion or deletion (indels)(indels) 的of SangerSanger 定序Sequencing

使用BigDye®化學在3730xI定序儀(Life Technologies公司)中對***或缺失(indel)的PCR擴增子進行定序。由於在indel 1-3中高度重複的DNA序列，甜菜鹼被添加到定序反應中。indel 2擴增子不能獲得作為連續序列。Sequences of indel PCR amplicons were sequenced in a 3730xI sequencer (Life Technologies) using BigDye® chemistry. Due to the highly repetitive DNA sequence in indel 1-3, betaine is added to the sequencing reaction. The indel 2 amplicon cannot be obtained as a continuous sequence.

RT-PCRRT-PCR

進行定量RT-PCR。圖10(a)-(f)驗證了puro-CLEC中PPP1R12C 與所選轉錄物含量的變化。在沒有質體DNA的情況下電穿孔的臍帶襯裡上皮細胞(CLECs)與相同數量的群體倍增作為對照。使用內含子外顯子引子擴增內源性PPP1R12C 轉錄物(外顯子4-6)，以AAVS1整合位點為中心的1-Mb區間內的相鄰基因(LILRB4 、ISOC2 、PPP6R1 、NAT14 、ZNF579 、FIZ1 以及RDH13 )，由Gene Network Central™ (http://www.sabiosciences.com)與人類蛋白質-蛋白質相互作用預測(http://www.compbio.dundee.ac.uk)所預測的PPP1R12C 的潛在相互作用配偶體，其被RNA定序分析顯著改變(DUSP1 、DUSP6 、CDC6 以及DUSP16 )以及管家基因GAPDH。將轉錄物含量標準化為GAPDH表現，並使用'delta-deltaC (T) 方法(Livak與Schmittgen，2001年)相對於野生型-CLEC表現puro-CLEC中轉錄物含量的倍數變化。Perform quantitative RT-PCR. Figures 10(a)-(f) verify the changes in the content of PPP1R12C and selected transcripts in puro-CLEC. In the absence of plastid DNA, electroporated umbilical cord lining epithelial cells (CLECs) were doubled with the same number of populations as a control. Use intron exon primers to amplify endogenous PPP1R12C transcripts (exons 4-6), adjacent genes in the 1-Mb interval centered on the AAVS1 integration site ( LILRB4 , ISOC2 , PPP6R1 , NAT14 , ZNF579 , FIZ1 and RDH13 ), predicted by Gene Network Central™ (http://www.sabiosciences.com) and human protein-protein interaction prediction (http://www.compbio.dundee.ac.uk) The potential interaction partner of PPP1R12C was significantly changed by RNA sequencing analysis ( DUSP1 , DUSP6 , CDC6 and DUSP16 ) and housekeeping gene GAPDH. The transcript content was normalized to GAPDH performance, and the delta-delta C (T) method (Livak and Schmittgen, 2001) was used to express the fold change in transcript content in puro-CLEC relative to wild-type-CLEC.

細胞增殖分析Cell proliferation analysis

將100 μl培養基中的100個野生型-或puro-CLEC接種到平底96孔組織培養盤中，一樣品四重複。 根據推薦的方案，在7天後進行MTS測定(CellTiter 96® AQueous One Solution Cell Proliferation Assay；Promega公司)。使用MRX II 96孔板讀取器(Dynex公司)定量490 nm處的吸光度。100 wild-type or puro-CLECs in 100 μl of medium were inoculated into a flat-bottom 96-well tissue culture dish, and one sample was repeated four times. According to the recommended protocol, MTS measurement (CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay; Promega) was performed after 7 days. The absorbance at 490 nm was quantified using an MRX II 96-well plate reader (Dynex Corporation).

統計分析Statistical Analysis

使用GraphPad Prism (GraphPad軟體公司)進行統計學分析。ANOVA與Tukey-Kramer檢驗用於比較三組或更多組的平均值。學生氏的非配對t-檢驗用於兩組之間具有相等方差的比較，並且當假設方差不相等時使用Mann-Whitney檢驗。P值>0.05被認為是顯著的。Statistical analysis was performed using GraphPad Prism (GraphPad Software Company). ANOVA and Tukey-Kramer test are used to compare the average of three or more groups. Student's unpaired t-test is used for comparisons between groups with equal variances, and the Mann-Whitney test is used when the variances are assumed to be unequal. P-value>0.05 is considered significant.

AAVS1 鋅指核酸酶(ZFN)用於在原代人類臍帶襯裡上皮細胞(CLECs)中整合FVIII轉殖基因。組合幾個胺基酸取代以令人驚訝地增強鋅指核酸酶(ZFN)準確度與活性(即專性異二聚化與增強的DNA切割活性)，並且瞬間次低溫能夠提高轉殖基因整合的效率。使用基因捕獲策略以及嘌呤黴素抗性的篩選來促進FVIII轉殖基因的目標整合，FVIII轉殖基因為B結構域截短的人-豬雜交體，其分泌比人類FVIII更高的FVIII活性。在AAVS1 鋅指核酸酶(ZFN)處理的優化條件下，將完整的FVIII轉殖基因整合到分泌有效量的FVIII的臍帶襯裡上皮細胞(CLECs)中。總FVIII供體DNA的定量基因組複製數分析，AAVS1基因座中供體DNA的左及右整合連接與單等位基因轉殖基因整合與不存在顯著的偏離目標整合一致。這得到定量RT-PCR的支持，其顯示PPP1R12C 表現減少一半，該基因被FVIII轉殖基因整合破壞。有針對性的電腦深度定序預測十個最可能的偏離目標位點僅在8q24基因間位點顯示出低頻率的微缺失。全基因組序列數據分析提出了三種潛在的***缺失。這三個都是基因間的；兩個實驗定序並顯示為單鹼基替換或微衛星DNA擴增。透過全基因組序列分析顯示的四種不平衡的染色體重排未透過實驗基因組複製數分析與預測的異常染色體連接的缺失來進行驗證。RNA定序用於提供偏離目標基因組事件的功能讀數。這揭示了在AAVS1基因座中鋅指核酸酶(ZFN)調節的FVIII整合的轉錄組中的非常小的足跡。在17,751個總轉錄本中，只有90個(0.5%)的含量被改變了2倍或更多。途徑分析沒有將失調的轉錄物映射到任何規範的致癌途徑，而只映射趨化因子-趨化因子受體相互作用，這與PPP1R12C 的已知功能一致。用於探測PPP1R12C 單倍體不足的可能後果的RNA定序數據的更廣泛的分析是均勻消極的，除了僅增加DUSP6的表現，其已知的作用是抗增殖以及抗癌。此由鋅指核酸酶(ZFN)修飾的臍帶襯裡上皮細胞(CLECs)的增殖率略低獲得支持。AAVS1 鋅指核酸酶(ZFN)在原代成人真皮纖維母細胞、骨髓以及脂肪來源的基質細胞中也具有活性。AAVS1 zinc finger nuclease (ZFN) is used to integrate the FVIII transgene in primary human umbilical cord lining epithelial cells (CLECs). Combining several amino acid substitutions to surprisingly enhance the accuracy and activity of zinc finger nucleases (ZFN) (ie obligate heterodimerization and enhanced DNA cleavage activity), and instant sub-low temperature can improve the integration of transgenic genes s efficiency. A gene capture strategy and selection of puromycin resistance were used to promote target integration of FVIII transgenes, which are human-porcine hybrids with truncated B domains that secrete higher FVIII activity than human FVIII. Under optimized conditions of AAVS1 zinc finger nuclease (ZFN) treatment, the complete FVIII transgene was integrated into umbilical cord lining epithelial cells (CLECs) that secrete an effective amount of FVIII. Quantitative genomic copy number analysis of total FVIII donor DNA, the left and right integration junctions of the donor DNA in the AAVS1 locus and single allele transfer gene integration are consistent with no significant deviation from target integration. This is supported by quantitative RT-PCR, which shows that the performance of PPP1R12C is reduced by half, and the gene is integrated and destroyed by the FVIII transgene gene. Targeted computerized deep sequencing predicts that the ten most likely off-target loci show low-frequency microdeletions only at the 8q24 intergenic locus. Whole genome sequence data analysis proposed three potential insertion deletions. All three are intergenic; the two experiments are sequenced and shown as single base substitution or microsatellite DNA amplification. The four unbalanced chromosomal rearrangements revealed through genome-wide sequence analysis were not verified by experimental genome copy number analysis and the predicted loss of abnormal chromosome connections. RNA sequencing is used to provide functional readings of events that deviate from the target genome. This revealed a very small footprint in the transcriptome of FVIII integration regulated by zinc finger nuclease (ZFN) in the AAVS1 locus. Of 17,751 total transcripts, only 90 (0.5%) were changed by 2 times or more. Pathway analysis did not map dysregulated transcripts to any canonical oncogenic pathway, but only mapped chemokine-chemokine receptor interactions, which is consistent with the known function of PPP1R12C . The more extensive analysis of RNA sequencing data used to detect the possible consequences of PPP1R12C haploid deficiency is uniformly negative, except that it only increases the performance of DUSP6, whose known effects are anti-proliferation and anti-cancer. This is supported by the slightly lower proliferation rate of umbilical cord lining epithelial cells (CLECs) modified by zinc finger nuclease (ZFN). AAVS1 zinc finger nuclease (ZFN) is also active in primary adult dermal fibroblasts, bone marrow, and adipose-derived stromal cells.

雜合hybrid FVIIIFVIII 胺基酸序列之比較Comparison of amino acid sequences

(頂部序列) Hybrid FVIII_KON lab = 1709個胺基酸(保留266個胺基酸的B結構域，具有8個糖基化位點，SEQ ID NO: 4)(Top sequence) Hybrid FVIII_KON lab = 1709 amino acids (retains B domain of 266 amino acids, with 8 glycosylation sites, SEQ ID NO: 4)

(底部序列) Hybrid FVIII_Doering lab = 1467個胺基酸(SEQ ID NO: 5，描述於Doerin等人，Molecular Therapy，第17卷第7期第1145-1154頁，2009年7月。)(Bottom sequence) Hybrid FVIII_Doering lab = 1467 amino acids (SEQ ID NO: 5, described in Doerin et al., Molecular Therapy, Vol. 17, No. 7, pages 1145-1154, July 2009.)

查詢：./ wwwwtmp /lalign29380.1.seq 1>>>Hybrid FVIII_KON lab 1709 bp-1709 aa Library: ./wwwtmp/lalign29380.2.seq 1個序列中有1467個殘基Enquiry: ./wwwwtmp/lalign29380.1.seq 1>>>Hybrid FVIII_KON lab 1709 bp-1709 aa Library: ./wwwtmp/lalign29380.2.seq 1467 residues in 1 sequence

統計: (洗牌[500]) MLE統計：Lambda = 0.1781；K =0.02504 從1(1)到500個序列採樣的統計數據Statistics: (shuffle [500]) MLE statistics: Lambda = 0.1781; K = 0.02504 Statistics from 1 (1) to 500 sequence samples

閾值：E() > 10得分：49Threshold: E()> 10 Score: 49

演算法：Smith-Waterman (SSE2，Michael Farrar 2006年)(7.2，2010年11月)Algorithm: Smith-Waterman (SSE2, Michael Farrar 2006) (7.2, November 2010)

參數：BL50 matrix (15:-5), open/ext: -12/-2Parameters: BL50 matrix (15:-5), open/ext: -12/-2

掃描時間：0.180Scan time: 0.180

>>Hybrid FVIII_Doering lab 1467 bp (1467個胺基酸)>>Hybrid FVIII_Doering lab 1467 bp (1467 amino acids)

Waterman-Eggert得分: 9322; 2400.4 bits; E(1) > 0 在1709個胺基酸重疊中有84.1%的同一性(85.2%相似)(1-1709:1-1467) 10 20 30 40 50 60 雜合 MQIELSTCFFLCLLRFCFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPL ::.::::: ::::: . ::::::::::::::::::::::::::::::::::::::::::: 雜合 MQLELSTCVFLCLLPLGFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPL 10 20 30 40 50 60 70 80 90 100 110 120 雜合 GPSVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPTIQAEVYDTVVVTLKNMASHPVSLHA :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 GPSVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPTIQAEVYDTVVVTLKNMASHPVSLHA 70 80 90 100 110 120 130 140 150 160 170 180 雜合 VGVSFWKSSEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLS :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 VGVSFWKSSEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLS 130 140 150 160 170 180 190 200 210 220 230 240 雜合 HVDLVKDLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAM :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 HVDLVKDLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAM 190 200 210 220 230 240 250 260 270 280 290 300 雜合 DPAPARAQPAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHSIFLEGHTFLVRH :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 DPAPARAQPAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHSIFLEGHTFLVRH 250 260 270 280 290 300 310 320 330 340 350 360 雜合 HRQASLEISPLTFLTAQTFLMDLGQFLLFCHISSHHHGGMEAHVRVESCAEEPQLRRKAD :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 HRQASLEISPLTFLTAQTFLMDLGQFLLFCHISSHHHGGMEAHVRVESCAEEPQLRRKAD 310 320 330 340 350 360 370 380 390 400 410 420 雜合 EEEDYDDNLYDSDMDVVRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLA :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 EEEDYDDNLYDSDMDVVRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLA 370 380 390 400 410 420 430 440 450 460 470 480 雜合 PDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTL :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 PDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTL 430 440 450 460 470 480 490 500 510 520 530 540 雜合 LIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGP :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 LIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGP 490 500 510 520 530 540 550 560 570 580 590 600 雜合 TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE 550 560 570 580 590 600 610 620 630 640 650 660 雜合 NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS 610 620 630 640 650 660 670 680 690 700 710 720 雜合 IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG 670 680 690 700 710 720 730 740 750 760 770 780 雜合 MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTI :::::::::::::::::::::::::::::::::::::::::.:::: ::. 雜合 MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFAQNSRPPSA---------- 730 740 750 760 770 790 800 810 820 830 840 雜合 PENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPS 雜合 ------------------------------------------------------------ 850 860 870 880 890 900 雜合 PGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST 雜合 ------------------------------------------------------------ 910 920 930 940 950 960 雜合 SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE :.: 雜合 --------------------------------------------SAP------------- 970 980 990 1000 1010 1020 雜合 NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDGRTERLCSQNPPVL .:::: 雜合 -------------------------------------------------------KPPVL 1030 1040 1050 1060 1070 1080 雜合 KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKRTRHYFIAAV .::::.:. :.: .....:::: .:.: : :::::: :::::.:::::::::::::::: 雜合 RRHQRDISLPTFQPEEDKMDYDDIFSTETKGEDFDIYGEDENQDPRSFQKRTRHYFIAAV 780 790 800 810 820 830 1090 1100 1110 1120 1130 1140 雜合 EQLWDYGMSESPRALRNRAQNGEVPRFKKVVFREFADGSFTQPSYRGELNKHLGLLGPYI :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 EQLWDYGMSESPRALRNRAQNGEVPRFKKVVFREFADGSFTQPSYRGELNKHLGLLGPYI 840 850 860 870 880 890 1150 1160 1170 1180 1190 1200 雜合 RAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAEPRHNFVQPNETRTYFWKVQHHM :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 RAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAEPRHNFVQPNETRTYFWKVQHHM 900 910 920 930 940 950 1210 1220 1230 1240 1250 1260 雜合 APTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTLNAAHGRQVTVQEFALFFTIF :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 APTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTLNAAHGRQVTVQEFALFFTIF 960 970 980 990 1000 1010 1270 1280 1290 1300 1310 1320 雜合 DETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYVMDTLPGLVMAQNQRIRWY :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 DETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYVMDTLPGLVMAQNQRIRWY 1020 1030 1040 1050 1060 1070 1330 1340 1350 1360 1370 1380 雜合 LLSMGSNENIHSIHFSGHVFSVRKKEEYKMAVYNLYPGVFETVEMLPSKVGIWRIECLIG :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 LLSMGSNENIHSIHFSGHVFSVRKKEEYKMAVYNLYPGVFETVEMLPSKVGIWRIECLIG 1080 1090 1100 1110 1120 1130 1390 1400 1410 1420 1430 1440 雜合 EHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWS :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 EHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWS 1140 1150 1160 1170 1180 1190 1450 1460 1470 1480 1490 1500 雜合 TKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTL :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 TKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTL 1200 1210 1220 1230 1240 1250 1510 1520 1530 1540 1550 1560 雜合 MVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESK :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 MVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESK 1260 1270 1280 1290 1300 1310 1570 1580 1590 1600 1610 1620 雜合 AISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGV :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 AISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGV 1320 1330 1340 1350 1360 1370 1630 1640 1650 1660 1670 1680 雜合 TTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 雜合 TTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT 1380 1390 1400 1410 1420 1430 1690 1700 雜合 RYLRIHPQSWVHQIALRMEVLGCEAQDLY ::::::::::::::::::::::::::::: 雜合 RYLRIHPQSWVHQIALRMEVLGCEAQDLY 1440 1450 1460Waterman-Eggert score: 9322; 2400.4 bits; E(1)> 0 84.1% identity (15.2% similarity) among 1709 amino acid overlaps (1-1709:1-1467) 10 20 30 40 50 60 Hybrid MQIELSTCFFLCLLRFCFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPL ::.::::: :::::. :::::::::::::::::::::::::::::::::::: ::::::: Hybrid MQLELSTCVFLCLLPLGFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPL 10 20 30 40 50 60 70 80 90 100 110 120 Hybrid GPSVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPTIQAEVYDTVVVTLKNMASHPVSLHA :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid GPSVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPTIQAEVYDTVVVTLKNMASHPVSLHA 70 80 90 100 110 120 130 140 150 160 170 180 Hybrid VGVSFWKSSEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLS :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid VGVSFWKSSEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLS 130 140 150 160 170 180 190 200 210 220 230 240 Hybrid HVDLVKDLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAM :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid HVDLVKDLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAM 190 200 210 220 230 240 250 260 270 280 290 300 Hybrid DPAPARAQPAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHSIFLEGHTFLVRH :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid DPAPARAQPAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHSIFLEGHTFLVRH 250 260 270 280 290 300 310 320 330 340 350 360 Hybrid HRQASLEISPLTFLTAQTFLMDLGQFLLFCHISSHHHGGMEAHVRVESCAEEPQLRRKAD :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid HRQASLEISPLTFLTAQTFLMDLGQFLLFCHISSHHHGGMEAHVRVESCAEEPQLRRKAD 310 320 330 340 350 360 370 380 390 400 410 420 Hybrid EEEDYDDNLYDSDMDVVRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLA :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid EEEDYDDNLYDSDMDVVRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLA 370 380 390 400 410 420 430 440 450 460 470 480 Hybrid PDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTL :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid PDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTL 430 440 450 460 470 480 490 500 510 520 530 540 Hybrid LIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGP :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid LIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGP 490 500 510 520 530 540 550 560 570 580 590 600 Hybrid TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE 550 560 570 580 590 600 610 620 630 640 650 660 Hybrid NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS 610 620 630 640 650 660 670 680 690 700 710 720 Hybrid IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG 670 680 690 700 710 720 730 740 750 760 770 780 Hybrid MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTI :::::::::::::::::::::::::::::::::::::::::.::::::. Hybrid MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFAQNSRPPSA---------- 730 740 750 760 770 790 800 810 820 830 840 Hybrid PENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPS hybrid------------------------------------------------ ------------ 850 860 870 880 890 900 Hybrid PGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST hybrid------------------------------------------------ ------------ 910 920 930 940 950 960 Hybrid SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE :.: Hybrid --------------------SAP--- ---------- 970 980 990 1000 1010 1020 Hybrid NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDGRTERLCSQNPPVL .:::: hybrid------------------------------------------------ -------KPPVL 1030 1040 1050 1060 1070 1080 Hybrid KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKRTRHYFIAAV .::::.:. :.: .....:::: .:.:: :::::: :::::.:::::::::::::: ::: Hybrid RRHQRDISLPTFQPEEDKMDYDDIFSTETKGEDFDIYGEDENQDPRSFQKRTRHYFIAAV 780 790 800 810 820 830 1090 1100 1110 1120 1130 1140 Hybrid EQLWDYGMSESPRALRNRAQNGEVPRFKKVVFREFADGSFTQPSYRGELNKHLGLLGPYI :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid EQLWDYGMSESPRALRNRAQNGEVPRFKKVVFREFADGSFTQPSYRGELNKHLGLLGPYI 840 850 860 870 880 890 1150 1160 1170 1180 1190 1200 Hybrid RAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAEPRHNFVQPNETRTYFWKVQHHM :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid RAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAEPRHNFVQPNETRTYFWKVQHHM 900 910 920 930 940 950 1210 1220 1230 1240 1250 1260 Hybrid APTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTLNAAHGRQVTVQEFALFFTIF :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid APTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTLNAAHGRQVTVQEFALFFTIF 960 970 980 990 1000 1010 1270 1280 1290 1300 1310 1320 Hybrid DETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYVMDTLPGLVMAQNQRIRWY :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid DETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYVMDTLPGLVMAQNQRIRWY 1020 1030 1040 1050 1060 1070 1330 1340 1350 1360 1370 1380 Hybrid LLSMGSNENIHSIHFSGHVFSVRKKEEYKMAVYNLYPGVFETVEMLPSKVGIWRIECLIG :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid LLSMGSNENIHSIHFSGHVFSVRKKEEYKMAVYNLYPGVFETVEMLPSKVGIWRIECLIG 1080 1090 1100 1110 1120 1130 1390 1400 1410 1420 1430 1440 Hybrid EHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWS :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid EHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWS 1140 1150 1160 1170 1180 1190 1450 1460 1470 1480 1490 1500 Hybrid TKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTL :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid TKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTL 1200 1210 1220 1230 1240 1250 1510 1520 1530 1540 1550 1560 Hybrid MVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESK :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid MVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESK 1260 1270 1280 1290 1300 1310 1570 1580 1590 1600 1610 1620 Hybrid AISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGV :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid AISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGV 1320 1330 1340 1350 1360 1370 1630 1640 1650 1660 1670 1680 Hybrid TTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT :::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::: Hybrid TTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT 1380 1390 1400 1410 1420 1430 1690 1700 Hybrid RYLRIHPQSWVHQIALRMEVLGCEAQDLY ::::::::::::::::::::::::::::: Hybrid RYLRIHPQSWVHQIALRMEVLGCEAQDLY 1440 1450 1460

實施例2.2：使用CRISPR-Cas9將轉殖基因整合到AAVS1基因座中Example 2.2: Integration of transgenic genes into the AAVS1 locus using CRISPR-Cas9

使用CRISPR-Cas9將轉殖基因整合到本發明之轉殖基因間質幹細胞群中(例如，一幹細胞群，其中至少約97%或更多細胞表現以下標記中的每一種：CD73、CD90以及CD105並且缺乏表現CD34、CD45以及HLA-DR)可使用各自的市售套組進行。如上所述，合適的商業套組的實例包括OriGene的套組(Technologies公司，Rockville，馬里蘭州，美國)，其具有用於AAVS1目標的一體化CRISPR載體(pCas-Guide-AAVS1)，「Genome-CRISPR™ Human AAVS1 Safe Harbor Gene Knock-in Kit」型號SH004，可從GeneCopoeia公司，馬里蘭州購得，或System Biosciences (SBI)公司(Palo Alto，加州，美國)的AAVS1安全港目標通用型HR供體載體2.0無啟動子(AAVS1-SA-puro-MCS)套組(GE620A-KIT)。Use CRISPR-Cas9 to integrate transgenic genes into a population of transgenic mesenchymal stem cells of the present invention (eg, a stem cell population in which at least about 97% or more cells exhibit each of the following markers: CD73, CD90, and CD105 And the lack of performance CD34, CD45 and HLA-DR) can be carried out using their respective commercial kits. As mentioned above, examples of suitable commercial kits include OriGene's kit (Technologies, Rockville, Maryland, USA), which has an integrated CRISPR vector (pCas-Guide-AAVS1) for the AAVS1 target, "Genome- CRISPR™ Human AAVS1 Safe Harbor Gene Knock-in Kit” model SH004, available from GeneCopoeia, Maryland, or AAVS1 Safe Harbor Target Universal HR Donor from System Biosciences (SBI) (Palo Alto, California, United States) Vector 2.0 promoterless (AAVS1-SA-puro-MCS) kit (GE620A-KIT).

使用AAVS1安全港目標通用HR供體載體載體2.0無啟動子(AAVS1-SA-puro-MCS)套組(GE620A-KIT)，用於整合編碼例如SEQ ID NO: 4的雜合FVIII的轉殖基因。本發明之間質幹細胞群的AAVS1基因座將使偏離目標整合最小化。於此，使用合適的限制酶與連接反應，將具有啟動子序列以及poly A位點的雜合FVIII cDNA匣選殖到多選殖位點的供體載體中。供體載體將與All-in-one Cas9 SmartNuclease & AAVS1 gRNA載體共轉染，用於將DSB作為目標引導到AAVS1位點，通常具有約10-15 μg供體載體以及5-7 μg Zloer-Cas9核酸酶載體可用於轉染約1-5百萬個細胞。Using AAVS1 Safe Harbor Target Universal HR Donor Vector 2.0 Promoter-Free (AAVS1-SA-puro-MCS) Kit (GE620A-KIT) for integration of transgenic genes encoding hybrid FVIII such as SEQ ID NO: 4 . The AAVS1 locus of the mesenchymal stem cell population of the present invention will minimize off-target integration. Here, a suitable restriction enzyme and a ligation reaction are used to clone a hybrid FVIII cDNA cassette having a promoter sequence and a poly A site into a donor vector with multiple selection sites. The donor vector will be co-transfected with the All-in-one Cas9 SmartNuclease & AAVS1 gRNA vector for directing DSB to the AAVS1 site, usually with about 10-15 μg donor vector and 5-7 μg Zloer-Cas9 Nuclease vectors can be used to transfect approximately 1-5 million cells.

如果需要，可以優化基因轉移/轉染方案以提供允許最大基因表現效率與最低細胞死亡的條件。可以使用報告基因質體優化轉染，例如使用製造商的說明書表現綠色螢光蛋白(GFP)以及Lonza細胞株優化96孔Nucleofector™套組。或者，可以基於脂質的轉染試劑/製劑轉染本發明之間質幹細胞群，其允許有效且無毒的轉染。通常在轉染後24-48小時內透過流式細胞儀分析評估轉染效率。If necessary, the gene transfer/transfection protocol can be optimized to provide conditions that allow maximum gene expression efficiency and minimum cell death. Reporter gene plastids can be used to optimize transfection, for example using the manufacturer's instructions to express green fluorescent protein (GFP) and the Lonza cell line to optimize the 96-well Nucleofector™ kit. Alternatively, a lipid-based transfection reagent/formulation can be used to transfect the mesenchymal stem cell population of the present invention, which allows efficient and non-toxic transfection. The transfection efficiency is usually evaluated by flow cytometry analysis within 24-48 hours after transfection.

具有目標轉殖基因整合的細胞將透過嘌呤黴素篩選在嘌呤黴素的劑量/濃度下篩選1週，該嘌呤黴素已被確定用於殺死對嘌呤黴素無抗性的間質幹細胞(通常在0.1-1 µg/ml的範圍內)。選擇後出現的嘌呤黴素抗性細胞可能在AAVS1基因座處具有轉殖基因整合。AAVS1基因座的位點特異性整合可透過使用套組中提供的連接PCR引子混合物(GE640PR-1)與從嘌呤黴素抗性細胞中萃取的基因組DNA建立PCR反應來驗證。一旦鑑定了本發明之轉殖基因間質幹細胞群，其已證實在AAVS1基因座處編碼SEQ ID NO: 4的雜合體FVIII的基因的整合，該細胞群將被擴展並冷凍保存以具有可從其衍生工作細胞庫的主細胞庫。隨後，透過使用Chromogenix Coamatic® FactorVIII套組(Chromogenix公司)測試條件培養基的FVIII活性，並透過Visulize™ FVIII抗原ELISA套組(Affinity Biologicals公司)，詳見製造商說明書，檢測FVIII蛋白的存在，可以測試具有整合到AAVS1基因座中的轉殖基因之轉殖基因間質幹細胞群分泌因子VIII。如果需要，可以對從轉殖基因幹細胞群萃取的基因組DNA進行進一步測試，以評估基因組中的偏離目標效應(Cel-1核酸酶測定，Amplicon Deep Sequenceing公司)。額外的遺傳毒性評估可能潛在地包括轉錄組研究以比較具有與不具有基因組修飾的轉殖基因幹細胞群，如上文實施例2.1中詳述的全基因組定序比較。Cells with target transgene integration will be screened by puromycin at the dose/concentration of puromycin for 1 week. The puromycin has been determined to kill mesenchymal stem cells that are not resistant to puromycin ( Usually in the range of 0.1-1 µg/ml). The puromycin-resistant cells that appeared after selection may have a transgene integration at the AAVS1 locus. The site-specific integration of the AAVS1 locus can be verified by establishing a PCR reaction with the genomic DNA extracted from puromycin resistant cells using the ligated PCR primer mix (GE640PR-1) provided in the kit. Once the transgenic mesenchymal stem cell population of the present invention has been identified, which has confirmed the integration of the gene encoding the hybrid FVIII of SEQ ID NO: 4 at the AAVS1 locus, the cell population will be expanded and cryopreserved to have It derives the main cell bank of the working cell bank. Subsequently, by using the Chromogenix Coamatic® FactorVIII kit (Chromogenix) to test the FVIII activity of the conditioned medium, and through the Visulize™ FVIII antigen ELISA kit (Affinity Biologicals), see the manufacturer’s instructions for the presence of the FVIII protein. A population of transgenic mesenchymal stem cells with a transgene integrated into the AAVS1 locus secretes factor VIII. If necessary, the genomic DNA extracted from the transgenic stem cell population can be further tested to assess the off-target effect in the genome (Cel-1 nuclease assay, Amplicon Deep Sequenceing). Additional genotoxicity assessments may potentially include transcriptome studies to compare populations of transgenic gene stem cells with and without genomic modifications, such as genome-wide sequencing comparisons detailed in Example 2.1 above.

對於本領域技術人員顯而易見的是，在不脫離本發明之範圍及精神的情況下，可以對本文公開的發明進行各種替換及修改。It is obvious to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

說明書中提及的所有專利及出版物指示了本發明所屬領域的普通技術人員之水準。所有的專利及出版物都透過引用方式併入本文，其程度如同每個單獨的出版物被具體地且單獨地指出透過引用併入。All patents and publications mentioned in the specification indicate the level of those of ordinary skill in the art to which this invention belongs. All patents and publications are incorporated herein by reference to the same extent as each individual publication is specifically and individually indicated to be incorporated by reference.

這裡說明性描述的發明可適當地在沒有任何一個或多個要素，一個或多個限制的情況下實施，這裡沒有具體公開。因此，例如，術語「包括」，「包含」，「含有」等應該被廣義地解讀而沒有限制。此外，這裡使用的術語及表現已經被作為描述性的術語而非限制性的，且不意圖使用這樣的術語及表現來排除所示出及描述的特徵或者其部分的任何等同物，但其認識到在要求保護的本發明之範圍內可以進行各種修改。因此，應該理解的是，儘管透過較佳實施例及可選特徵具體公開了本發明，但是本領域技術人員可以採取本文公開之其中體現本發明之修改及變化，且這樣的修改及變化被認為在本發明之範圍內。這裡已經廣泛地且一般性地描述了本發明。落入一般性公開內容中的每個較窄的物種及亞屬群也構成本發明之一部分。這包括對本發明之一般性描述，附帶條件或負面限制從該屬中移除任何主題，而不管在本文中是否具體列舉了所摘取的材料。另外，在根據馬庫西群組描述本發明之特徵或方面的情況下，本領域的技術人員將認識到，本發明也由此根據馬庫西群組的任何單個成員或亞組成員進行描述。從下面的申請專利範圍中，本發明之其它實施例將變得明顯。 本發明之特徵還在於以下項目： 1. 一種產生轉殖基因間質幹細胞群之方法，該方法包括透過酶調節的整合將轉殖基因***該間質幹細胞群的基因組中，其中該間質幹細胞群為一臍帶羊膜的分離的間質幹細胞群，其中至少約90%或更多的間質幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105。 2. 如第1項之方法，其中該酶調節的整合將該轉殖基因***該間質幹細胞群的該基因組中是透過酵素所進行的，該酵素選自由重組酶、類轉錄活化劑效應子核酸酶以及核酸酶所組成之群組。 3. 如第2項之方法，其中該重組酶選自由酪胺酸重組酶(tyrosine recombinase, YR)以及絲胺酸重組酶(serine recombinase, SR)及其混合物所組成之群組。 4. 如第3項之方法，其中該酪胺酸重組酶選自由Cre (Cre重組酶調節的整合)、Dre (Dre重組酶調節的整合)，以及Flp所組成之群組或其中該絲胺酸重組酶選自由重組酶phiC31 (phiC31整合酶調節的整合)、Bxb1，以及R4重組酶調節的整合所組成之群組。 5. 如第2項之方法，其中該核酸酶選自由鋅指核酸酶(zinc finger nuclease, ZFN)(鋅指核酸酶調節的整合)或Cas9 (CRISPR/Cas9調節的整合)所組成之群組。 6. 如第1至5項任一項之方法，其中至少約90%或更多的分離的間質幹細胞群的細胞缺乏下列標記之表現：CD34、CD45以及HLA-DR。 7. 如第1至6項任一項之方法，其中至少約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多該分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，並且缺乏CD34、CD45以及HLA-DR各自的表現。 8. 如第1至7項任一項之方法，其中該間質幹細胞群選自由人類幹細胞群、犬幹細胞群、貓幹細胞群、鼠幹細胞群、鼠幹細胞群、豬幹細胞群、馬幹細胞群、猿幹細胞群，或獼猴幹細胞群所組成之群組。 9. 如第1至8項任一項之方法，其中該轉殖基因整合到人類間質幹細胞群的AAVS1基因座(存在於人類染色體19 q13.3-qter上)或基因座8p22 (存在於人類染色體8上)的DLC1的內含子7中。 10. 如第5至9項任一項之方法，其中該方法包括透過突變的鋅指核酸酶***該轉殖基因。 11. 如第10項之方法，其中該鋅指核酸酶在FokI 切割結構域FokI 中包含至少一個突變，以提供具有增強的切割活性之變體。 12. 如第10或11項之方法，其中該突變的鋅指核酸酶在右、左單體中於對應於SEQ ID NO. 6的序列的野生型FokI 的第418及441個位置處包含S418P及K441E的取代。 13. 如第5至12項任一項之方法，其中該鋅指核酸酶為異二聚體。 14. 如第10至13項任一項之方法，其中該鋅指核酸酶具有Sharkey變體的催化結構域，其具有如SEQ ID NOs. 9、12或13所示任一之胺基酸序列。 15. 如第10至14項任一項之方法，其中該突變的鋅指核酸酶包含在該右Fok1 單體中的胺基酸取代S418P、K441E以及H537R，以及左Fok1 單體中的胺基酸取代S418P、K441E以及N496D，且其中該位置對應於SEQ ID NO. 6的各個位置。 16. 如第10至15項任一項之方法，其中該突變的鋅指核酸酶(專性異二聚體)包含與該右AAVS1同源臂融合的Fok1 單體中的二個胺基酸取代E490K以及I538K，以及與該左同源臂融合的該單體中的二個胺基酸取代Q468E以及I499L，且其中該位置對應於SEQ ID NO. 6的各個位置。 17. 如第5至16項任一項之方法，其中該鋅指核酸酶包含一鋅指蛋白，該鋅指蛋白選自由具有胺基酸序列基序X2 -Cys-X2,4 -Cys-X12 -His-X3,4,5 -His的類Cys2 His2 鋅指蛋白、Gag-knuckle鋅指蛋白高音譜號、鋅帶鋅指蛋白，以及Zn2 /Cys6 鋅指蛋白所組成之群組。 18. 如第5至17項任一項之方法，其中該鋅指蛋白選自由P3鋅指蛋白、E2C (E6)鋅指蛋白、E5鋅指蛋白、E4鋅指蛋白，以及E3鋅指蛋白所組成之群組。 19. 如第5至18項任一項之方法，其中該整合反應在約25°C至約32°C的溫度範圍內進行。 20. 如第19項之方法，其中該整合至該哺乳動物幹細胞群的基因組中是透過在一約30°C的溫度下的整合反應所進行的。 21. 如第5至20項任一項之方法，其中該轉殖基因之整合透過轉染作用所進行。 22. 如第21項之方法，其中該轉染作用使用單一質體所進行，該質體遞送該鋅指核酸酶的兩種單體，較佳為該Sharkey或強化的Sharkey AAVS1鋅指核酸酶單體。 23. 如第21或22項之方法，其中該轉染作用使用瞬間低溫所進行。 24. 如前述任一項之方法，其中該轉殖基因選自由編碼凝血因子的核酸分子(基因)以及編碼內分泌腺分泌的蛋白激素的核酸分子(基因)所組成之群組。 25. 如第24項之方法，其中該凝血因子選自由因子VII、因子VIII，以及因子IX所組成之群組。 26. 如第24項之方法，其中該由內分泌腺分泌的蛋白激素的表現或分泌之缺乏與內分泌缺乏有關。 27. 如第26項之方法，其中該蛋白激素的缺乏與內分泌缺乏有關，該內分泌缺乏選自由胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常所組成之群組。 28. 如第25或26項之方法，其中該轉殖基因為編碼嵌合因子VIII多胜肽的核酸分子。 29. 如第28項之方法，其中該轉殖基因編碼包含人類及豬結構域的嵌合因子VIII多胜肽。 30. 如第29項之方法，其中該轉殖基因編碼包含豬A1與A3結構域、人類訊息胜肽、人類A2訊號胜肽、殘留的人類B結構域，以及人類C1及C2結構域或由其組成的嵌合因子VIII多胜肽，及/或其中該轉殖基因包含一如SEQ ID NO. 3所示的序列。 31. 如第30項之方法，其中該殘留的人類B結構域包含前266個胺基酸以及8個糖基化位點。 32. 如第30或31項之方法，其中該轉殖基因編碼SEQ ID NO: 4的嵌合因子VIII多胜肽，或與SEQ ID NO. 4的多胜肽序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的序列同一性的多胜肽。 33. 一種轉殖基因間質幹細胞群，其攜帶透過如第1至32項任一項所述之方法而可獲得或獲得之轉殖基因。 34. 如第33項之轉殖基因間質幹細胞群，其中該間質幹細胞群選自由人類幹細胞群、犬幹細胞群、貓幹細胞群、馬幹細胞群、鼠幹細胞群、鼠幹細胞群、豬幹細胞群、猿幹細胞群，或獼猴幹細胞群所組成之群組。 35. 如第33或34項之轉殖基因間質幹細胞，其中該轉殖基因被整合到人類臍帶襯裡幹細胞的AAVS1基因座(存在於人類染色體19 q13.3-qter上)或整合到基因座8p22 (存在於人類染色體8上)的DLC1的內含子7中。 36. 如第33至35項任一項之轉殖基因間質幹細胞，其中該轉殖基因為編碼嵌合因子VIII多胜肽的基因。 37. 如第36項之轉殖基因間質幹細胞，其中該轉殖基因編碼包含人類與豬結構域的嵌合因子VIII多胜肽。 38. 如第36或37項任一項之轉殖基因間質幹細胞，其中該轉殖基因編碼一包含或由豬A1與A3結構域、人類訊息胜肽、人類A2結構域、一殘留的人類B結構域，以及人類C1及C2結構域所組成之嵌合因子VIII多胜肽，及/或其中該轉殖基因包含如SEQ ID NO. 3所示之序列。 39. 如第38項之轉殖基因間質幹細胞，其中該殘留的人類B結構域包含前266個胺基酸以及8個糖基化位點。 40. 如第36至39項任一項之轉殖基因間質幹細胞，其中該轉殖基因編碼SEQ ID NO: 4的嵌合因子VIII多胜肽，或與SEQ ID NO. 4的多胜肽序列具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的序列同一性的多胜肽。 41. 如第33至37項任一項所定義之轉殖基因間質幹細胞用於治療疾病之用途。 42. 如第41項之用途，其中該疾病為與基因缺陷或該基因表現缺陷相關的疾病，其中該基因選自由編碼凝血因子的基因以及編碼由內分泌腺分泌的蛋白激素的基因所組成之群組。 43. 如第42項之用途，其中該凝血因子選自因子VII、因子VIII，以及因子IX。 44. 如第41至43項任一項之用途，其中該疾病為血友病。 45. 如第44項之用途，其中該血友病選自由A型血友病、B型血友病，以及C型血友病所組成之群組。 46. 如第41項之用途，其中該疾病與內分泌缺乏有關。 47. 如第41或46項之用途，其中該疾病與由內分泌腺分泌的該蛋白激素之表現或分泌缺乏有關。 48. 如第47項之用途，其中該蛋白激素的缺乏與內分泌缺乏有關，該內分泌缺乏選自由胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常所組成之群組。 49. 一種治療患有疾病的患者之方法，該方法包括對該患者施用如第33至40項任一項所定義之轉殖基因間質幹細胞群。 50. 如第49項之治療患者之方法，其中該疾病為與基因缺陷或該基因表現缺陷相關的疾病，其中該基因選自由編碼凝血因子的基因以及編碼由內分泌腺分泌的蛋白激素的基因所組成之群組。 51. 如第50項之方法，其中該凝血因子選自因子VII、因子VIII，以及因子IX。 52. 如第49至51項任一項之方法，其中該疾病為血友病。 53. 如第52項之方法，其中該血友病選自由A型血友病、B型血友病，以及C型血友病所組成之群組。 54. 如第49至53項任一項之方法，其中該疾病與內分泌缺乏有關。 55. 如第49至52項任一項或第54項之方法，其中該其中該疾病與由內分泌腺分泌的該蛋白激素之表現或分泌缺乏有關。 56. 如第55項之方法，其中該蛋白激素的缺乏與內分泌缺乏有關，該內分泌缺乏選自由胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常所組成之群組。 57. 如第49至56項任一項之方法，其中該轉殖基因間質幹細胞群透過植入或注射給藥。 58. 如第57項之方法，其中該轉殖基因間質幹細胞群將以皮下植入。 59. 一種含有如第33至40項任一項所定義之轉殖基因間質幹細胞群之醫藥組合物。 60. 如第59項之醫藥組合物，其適於植入或注射。 61. 如第59或60項之醫藥組合物，其適於皮下植入。 62. 一種如第33至40項任一項所定義之轉殖基因間質幹細胞群用於基因療法之用途。 63. 臍帶羊膜的一間質幹細胞群之用途，其中至少約90%或更多的該間質幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105，以用於產生用於基因療法之如第33至40項任一項所定義之一轉殖基因間質幹細胞群。 64. 如第63項之用途，其中至少約90%或更多的間質幹細胞群的細胞缺乏以下標記之表現：CD34、CD45以及HLA-DR。 65. 如第62及63項任一項之用途，其中至少約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多該分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，並且缺乏CD34、CD45以及HLA-DR各自的表現。引用的科學文獻 The invention illustratively described herein can be suitably implemented without any one or more elements, one or more limitations, and is not specifically disclosed herein. Therefore, for example, the terms "include", "include", "include", etc. should be interpreted broadly without limitation. In addition, the terms and expressions used herein have been taken as descriptive terms rather than limiting, and it is not intended to use such terms and expressions to exclude the illustrated and described features or any equivalents of parts thereof, but their recognition Various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through the preferred embodiments and optional features, those skilled in the art may adopt the modifications and changes disclosed herein which reflect the present invention, and such modifications and changes are considered Within the scope of the present invention. The invention has been described broadly and generally here. Each narrower species and subgenus group falling within the general disclosure also forms part of the present invention. This includes a general description of the present invention, with conditions or negative restrictions to remove any subject from the genus, regardless of whether the extracted material is specifically listed in this article. In addition, where the features or aspects of the present invention are described in terms of the Makusi group, those skilled in the art will recognize that the invention is thus described in terms of any single member or subgroup member of the Makusi group . Other embodiments of the present invention will become apparent from the following patent application scope. The present invention is also characterized by the following items: 1. A method of generating a population of transgenic mesenchymal stem cells, the method comprising inserting a transgenic gene into the genome of the population of mesenchymal stem cells through enzyme-mediated integration, wherein the mesenchymal stem cells The population is an isolated mesenchymal stem cell population of umbilical cord amniotic membrane, wherein at least about 90% or more of the cells of the mesenchymal stem cell population exhibit each of the following markers: CD73, CD90, and CD105. 2. The method according to item 1, wherein the integration regulated by the enzyme inserts the transferred gene into the genome of the mesenchymal stem cell group through an enzyme selected from the group consisting of a recombinase and a transcription activator-like effector Nucleases and nucleases. 3. The method according to item 2, wherein the recombinase is selected from the group consisting of tyrosine recombinase (YR) and serine recombinase (SR) and mixtures thereof. 4. The method according to item 3, wherein the tyrosine recombinase is selected from the group consisting of Cre (Cre recombinase-regulated integration), Dre (Dre recombinase-regulated integration), and Flp or the serine The acid recombinase is selected from the group consisting of recombinase phiC31 (phiC31 integrase-mediated integration), Bxb1, and R4 recombinase-mediated integration. 5. The method according to item 2, wherein the nuclease is selected from the group consisting of zinc finger nuclease (ZFN) (zinc finger nuclease-mediated integration) or Cas9 (CRISPR/Cas9-mediated integration) . 6. The method according to any one of items 1 to 5, wherein at least about 90% or more of the cells of the isolated mesenchymal stem cell population lack the performance of the following markers: CD34, CD45, and HLA-DR. 7. The method according to any one of items 1 to 6, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more , About 96% or more, about 97% or more, about 98% or more, about 99% or more, the cells of the isolated mesenchymal stem cell population exhibit each of CD73, CD90, and CD105, and lack The performance of CD34, CD45 and HLA-DR. 8. The method according to any one of items 1 to 7, wherein the mesenchymal stem cell population is selected from the group consisting of human stem cell population, canine stem cell population, cat stem cell population, murine stem cell population, murine stem cell population, porcine stem cell population, equine stem cell population, Ape stem cell population, or a group of macaque stem cell populations. 9. The method according to any one of items 1 to 8, wherein the transferred gene is integrated into the AAVS1 locus of human mesenchymal stem cell population (present on human chromosome 19 q13.3-qter) or the locus 8p22 (present on Intron 7 of DLC1 on human chromosome 8). 10. The method according to any one of items 5 to 9, wherein the method comprises inserting the transgene through a mutated zinc finger nuclease. 11. The method of item 10, wherein the zinc finger nuclease contains at least one mutation in the FokI cleavage domain FokI to provide a variant with enhanced cleavage activity. 12. The method according to item 10 or 11, wherein the mutated zinc finger nuclease comprises S418P in the right and left monomers at positions 418 and 441 of the wild-type FokI corresponding to the sequence of SEQ ID NO. 6. And the replacement of K441E. 13. The method according to any one of items 5 to 12, wherein the zinc finger nuclease is a heterodimer. 14. The method according to any one of items 10 to 13, wherein the zinc finger nuclease has the catalytic domain of the Sharkey variant, which has an amino acid sequence as shown in SEQ ID NOs. 9, 12, or 13. . 15. The method according to any one of items 10 to 14, wherein the mutated zinc finger nuclease contains amino acid substitutions S418P, K441E, and H537R in the right Fok1 monomer, and amino groups in the left Fok1 monomer The acid substitutes S418P, K441E and N496D, and this position corresponds to each position of SEQ ID NO. 16. The method according to any one of items 10 to 15, wherein the mutated zinc finger nuclease (obligate heterodimer) comprises two amino acids in the Fok1 monomer fused to the right AAVS1 homology arm 6. Substitute E490K and I538K, and two amino acids in the monomer fused to the left homology arm replace Q468E and I499L, and this position corresponds to each position of SEQ ID NO. 6. 17. The method according to any one of items 5 to 16, wherein the zinc finger nuclease comprises a zinc finger protein selected from the group consisting of amino acid sequence motifs X 2 -Cys-X 2,4- Cys -X 12 -His-X 3,4,5 -His Cys 2 His 2 zinc finger protein, Gag-knuckle zinc finger protein treble clef, zinc band zinc finger protein, and Zn 2 /Cys 6 zinc finger protein Formed into groups. 18. The method according to any one of items 5 to 17, wherein the zinc finger protein is selected from the group consisting of P3 zinc finger protein, E2C (E6) zinc finger protein, E5 zinc finger protein, E4 zinc finger protein, and E3 zinc finger protein Formed into groups. 19. The method according to any one of items 5 to 18, wherein the integration reaction is performed at a temperature ranging from about 25°C to about 32°C. 20. The method according to item 19, wherein the integration into the genome of the mammalian stem cell population is performed by an integration reaction at a temperature of about 30°C. 21. The method according to any one of items 5 to 20, wherein the integration of the transgene is performed by transfection. 22. The method of item 21, wherein the transfection is performed using a single plastid, which delivers two monomers of the zinc finger nuclease, preferably the Sharkey or enhanced Sharkey AAVS1 zinc finger nuclease monomer. 23. The method according to item 21 or 22, wherein the transfection is performed using an instant low temperature. 24. The method according to any one of the preceding items, wherein the transgene is selected from the group consisting of nucleic acid molecules (genes) encoding coagulation factors and nucleic acid molecules (genes) encoding protein hormones secreted by endocrine glands. 25. The method according to item 24, wherein the coagulation factor is selected from the group consisting of factor VII, factor VIII, and factor IX. 26. The method according to item 24, wherein the lack of performance or secretion of the protein hormone secreted by the endocrine glands is related to endocrine deficiency. 27. The method according to item 26, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, Adrenal deficiency, and thyroid abnormalities. 28. The method according to item 25 or 26, wherein the transgene is a nucleic acid molecule encoding a chimeric factor VIII polypeptide. 29. The method according to item 28, wherein the transferred gene encodes a chimeric factor VIII polypeptide comprising human and porcine domains. 30. The method according to item 29, wherein the transgenic gene encoding comprises porcine A1 and A3 domains, human signal peptides, human A2 signal peptides, residual human B domains, and human C1 and C2 domains or The chimeric factor VIII polypeptide composed thereof, and/or wherein the transgene contains a sequence shown in SEQ ID NO.3. 31. The method according to item 30, wherein the residual human B domain contains the first 266 amino acids and 8 glycosylation sites. 32. The method according to item 30 or 31, wherein the transgenic gene encodes the chimeric factor VIII polypeptide of SEQ ID NO: 4, or has at least 85%, 86% of the polypeptide sequence of SEQ ID NO. 4. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the peptides of sequence identity. 33. A population of transgenic mesenchymal stem cells carrying a transgenic gene obtainable or obtained by the method according to any one of items 1 to 32. 34. The transgenic gene mesenchymal stem cell population according to item 33, wherein the mesenchymal stem cell population is selected from the group consisting of human stem cell population, canine stem cell population, cat stem cell population, horse stem cell population, mouse stem cell population, mouse stem cell population, and pig stem cell population , Simian stem cell population, or cynomolgus monkey stem cell population. 35. The transgenic gene mesenchymal stem cell as described in item 33 or 34, wherein the transgenic gene is integrated into the AAVS1 locus of human umbilical cord lining stem cells (present on human chromosome 19 q13.3-qter) or integrated into the locus 8p22 (present on human chromosome 8) is in intron 7 of DLC1. 36. The transgenic gene mesenchymal stem cell according to any one of items 33 to 35, wherein the transgenic gene is a gene encoding a chimeric factor VIII polypeptide. 37. The transgenic mesenchymal stem cell according to item 36, wherein the transgenic gene encodes a chimeric factor VIII polypeptide comprising human and porcine domains. 38. The transgenic mesenchymal stem cell according to any one of items 36 or 37, wherein the transgenic gene encodes a gene comprising or consisting of porcine A1 and A3 domains, human message peptides, human A2 domains, and a residual human The B domain and the chimeric factor VIII polypeptide composed of human C1 and C2 domains, and/or the transgenic gene thereof include the sequence shown in SEQ ID NO.3. 39. The transgenic mesenchymal stem cell according to item 38, wherein the residual human B domain contains the first 266 amino acids and 8 glycosylation sites. 40. The transgenic mesenchymal stem cell according to any one of items 36 to 39, wherein the transgenic gene encodes the chimeric factor VIII polypeptide of SEQ ID NO: 4, or the polypeptide of SEQ ID NO. 4. Sequences have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity Sexual polypeptide. 41. The use of transgenic mesenchymal stem cells as defined in any one of items 33 to 37 for the treatment of diseases. 42. The use according to item 41, wherein the disease is a disease related to a genetic defect or a defect in the expression of the gene, wherein the gene is selected from the group consisting of a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland group. 43. The use according to item 42, wherein the coagulation factor is selected from factor VII, factor VIII, and factor IX. 44. The use according to any one of items 41 to 43, wherein the disease is hemophilia. 45. The use according to item 44, wherein the hemophilia is selected from the group consisting of hemophilia A, hemophilia B, and hemophilia C. 46. The use according to item 41, wherein the disease is related to endocrine deficiency. 47. The use according to item 41 or 46, wherein the disease is related to the lack of expression or secretion of the protein hormone secreted by the endocrine glands. 48. The use according to item 47, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, Adrenal deficiency, and thyroid abnormalities. 49. A method of treating a patient suffering from a disease, the method comprising administering to the patient a population of transgenic mesenchymal stem cells as defined in any one of items 33 to 40. 50. The method for treating a patient according to item 49, wherein the disease is a disease related to a gene defect or a defect in the gene expression, wherein the gene is selected from a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland Formed into groups. 51. The method according to item 50, wherein the coagulation factor is selected from factor VII, factor VIII, and factor IX. 52. The method according to any one of items 49 to 51, wherein the disease is hemophilia. 53. The method according to item 52, wherein the hemophilia is selected from the group consisting of hemophilia A, hemophilia B, and hemophilia C. 54. The method according to any one of items 49 to 53, wherein the disease is related to endocrine deficiency. 55. The method according to any one of items 49 to 52 or item 54, wherein the disease is related to a lack of performance or secretion of the protein hormone secreted by the endocrine glands. 56. The method according to item 55, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, Adrenal deficiency, and thyroid abnormalities. 57. The method according to any one of items 49 to 56, wherein the population of transgenic mesenchymal stem cells is administered by implantation or injection. 58. The method according to item 57, wherein the population of transgenic mesenchymal stem cells will be implanted subcutaneously. 59. A pharmaceutical composition containing a population of transgenic mesenchymal stem cells as defined in any one of items 33 to 40. 60. The pharmaceutical composition according to item 59, which is suitable for implantation or injection. 61. The pharmaceutical composition according to item 59 or 60, which is suitable for subcutaneous implantation. 62. The use of a population of transgenic mesenchymal stem cells as defined in any one of items 33 to 40 for gene therapy. 63. The use of a mesenchymal stem cell group of umbilical cord amniotic membrane, wherein at least about 90% or more of the cells of the mesenchymal stem cell group exhibit each of the following markers: CD73, CD90, and CD105 for use in gene generation The therapy is as described in any one of items 33 to 40, as one of the transgenic gene mesenchymal stem cell populations. 64. The use according to item 63, wherein at least about 90% or more of the cells of the mesenchymal stem cell population lack the performance of the following markers: CD34, CD45 and HLA-DR. 65. The use according to any one of items 62 and 63, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more , About 96% or more, about 97% or more, about 98% or more, about 99% or more, the cells of the isolated mesenchymal stem cell population exhibit each of CD73, CD90, and CD105, and lack The performance of CD34, CD45 and HLA-DR. Cited scientific literature

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無no

附圖將進一步說明本發明，其中：The drawings will further illustrate the invention, in which:

圖 1 所示為針對達爾伯克改良伊格爾培養基(DMEM)之Lonza公司的技術資訊表，包括在實驗部分中用於製造本發明之培養基(PTT-6)的說明性實施例的DMEM之目錄型號； FIG. 1 shows a technical information sheet of Lonza Corporation for Dulbecco’s Modified Eagle Medium (DMEM), including the DMEM of the illustrative embodiment used to manufacture the medium (PTT-6) of the present invention in the experimental section. Catalog model

圖 2 所示為針對Ham's F12培養基之Lonza公司的技術資訊表； Figure 2 shows Lonza's technical information table for Ham's F12 medium;

圖 3 所示為針對DMEM：F12 (1：1)培養基之Lonza公司的技術資訊表，包括在實驗部分中用於製造本發明之培養基(PTT-6)的說明性實施例的DMEM：F12 (1：1)培養基之目錄型號； Fig. 3 shows a technical information sheet of Lonza Corporation for DMEM: F12 (1:1) medium, including DMEM: F12 (Illustrative Example of the illustrative embodiment used to manufacture the medium (PTT-6) of the present invention in the experimental part. 1:1) the catalog model of the culture medium;

圖 4 所示為針對M171培養基之Life Technologies公司的技術資訊表，包括在實驗部分中用於製造本發明之培養基(PTT-6)的說明性實施例的M171培養基之目錄型號； Figure 4 shows the M171 culture medium for the Life Technologies Corporation technical information table, comprising means for producing an illustrative media (PTT-6) of the present invention in the experimental section of the embodiment in M171 medium directory type embodiment;

圖 5 所示為成分表，包括其商業供應商與在實驗部分中用於製造該培養基PTT-6的目錄型號。 Figure 5 shows the ingredient list, including its commercial suppliers and catalog models used in the experimental part to manufacture the medium PTT-6.

圖 6A-C 所示為流式細胞儀實驗之結果，其中已分析了從臍帶分離的間質幹細胞關於間質幹細胞標記CD73、CD90以及CD105的表現。針對這些實驗，自臍帶組織分離間質幹細胞接著在三種不同的培養基中培養。在這些實驗中使用以下三種培養基：a) 90% (v/v)補充有10% FBS (v/v)的DMEM，b) 在美國專利申請2006/0078993與相應之國際專利申請WO2006/019357中描述的培養基PTT-4，其由90% (v/v) CMRL1066與10% (v/v) FBS所組成(參閱WO2006/019357的第[0183]段)，以及c)培養基PTT-6其組成係在本文中描述。在該流式細胞儀分析中，針對這三種所用之培養基中的每一種，以二種不同的臍帶襯裡間質幹細胞(CLMC)群之樣品進行分析。 Figures 6A-C show the results of flow cytometry experiments in which the performance of mesenchymal stem cells isolated from the umbilical cord with respect to mesenchymal stem cell markers CD73, CD90 and CD105 has been analyzed. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue and then cultured in three different media. The following three media were used in these experiments: a) 90% (v/v) DMEM supplemented with 10% FBS (v/v), b) in US patent application 2006/0078993 and the corresponding international patent application WO2006/019357 The described medium PTT-4 consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO2006/019357), and c) medium PTT-6 its composition It is described in this article. In this flow cytometry analysis, samples of two different umbilical cord lined mesenchymal stem cell (CLMC) populations were analyzed for each of the three media used.

結果如圖 6A-C 所示。更詳細地，圖 6A 所示為從臍帶組織分離後並在DMEM/10% FBS中培養的分離的間質臍帶襯裡幹細胞表現幹細胞標記CD73、CD90以及CD105之百分比，圖 6B 所示為從臍帶組織分離後並在PTT-4中培養的分離的間質臍帶襯裡幹細胞表現幹細胞標記CD73、CD90以及CD105之百分比，以及圖 6C 所示為從臍帶組織分離後並在PTT-6中培養的分離的間質臍帶襯裡幹細胞表現幹細胞標記CD73、CD90以及CD105之百分比。The results are shown in FIG. 6A-C. In more detail, FIG. 6A shows the percentage of isolated interstitial umbilical cord lining stem cells isolated from umbilical cord tissue and cultured in DMEM/10% FBS. The percentage of stem cell markers CD73, CD90, and CD105 is shown in FIG. 6B . The isolated interstitial umbilical cord lining stem cells after isolation and cultured in PTT-4 showed the percentage of stem cell markers CD73, CD90, and CD105, and Figure 6C shows the separation time after separation from the umbilical cord tissue and cultured in PTT-6 The umbilical cord lining stem cells expressed the percentage of stem cell markers CD73, CD90 and CD105.

從圖 6A 中可以看出，使用DMEM/10% FBS作為培養基培養物分離的群體具有約75%的CD73+細胞，78%的90+細胞以及80%的CD105+細胞(兩次實驗的平均值)，而在分離後/使用PTT-4培養基培養臍帶組織(參閱圖 6B )，CD73陽性、CD90陽性以及CD105陽性的間質幹細胞的數量為約87% (CD73+細胞)、93%/ CD90+細胞)以及86% (CD105+細胞)平均兩次實驗。透過在本發明之PTT-6培養基中培養獲得的間質幹細胞群的純度相對於所有三種標記(CD73、CD90，CD105)至少為99.0%，這表示該細胞群的純度顯著高於使用PTT-4培養基或DMEM/10%FBS培養的種群。此外，甚至更重要的是，透過在PTT-6中培養獲得的間質幹細胞群基本上是100%純的且確定的幹細胞群。這使得本發明之幹細胞群是基於幹細胞的療法的理想候選者。因此，這種間質臍帶襯裡幹細胞群可能成為這種基於幹細胞的治療方法的黃金標準。As can be seen from FIG. 6A , the population isolated using DMEM/10% FBS as the culture medium had approximately 75% CD73+ cells, 78% 90+ cells, and 80% CD105+ cells (average of two experiments), After separation/cultivation of umbilical cord tissue using PTT-4 medium (see Figure 6B ), the number of CD73-positive, CD90-positive, and CD105-positive mesenchymal stem cells was approximately 87% (CD73+ cells), 93%/CD90+ cells), and 86 % (CD105+ cells) averaged two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0% relative to all three markers (CD73, CD90, CD105), which means that the purity of the cell population is significantly higher than that using PTT-4 Medium or DMEM/10% FBS cultured population. Moreover, even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is basically a 100% pure and definite stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell-based therapy. Therefore, this interstitial umbilical cord lining stem cell population may become the gold standard for this stem cell-based treatment.

圖 7A-B 所示為流式細胞儀實驗之結果，其中分析了從臍帶分離之間質幹細胞關於幹細胞標記(CD73、CD90以及CD105、CD34、CD45以及HLA-DR (人類白血球抗原-抗原D相關))之表現，其用於確定多能人類間質幹細胞用於細胞治療之適用性，以及與骨髓間質幹細胞表現的這些標記進行比較。本實驗中，臍帶羊膜的間質幹細胞係透過在本發明之培養基PTT-6中培養臍帶組織以自臍帶組織中分離，而使用標準方法自人類骨髓中分離骨髓間質幹細胞。 Figures 7A-B shows the results of flow cytometry experiments, which analyzes from the umbilical cord between the isolated mesenchymal stem cells on stem cell markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen - Related Antigen D )) performance, which is used to determine the applicability of pluripotent human mesenchymal stem cells for cell therapy and to compare these markers with bone marrow mesenchymal stem cell performance. In this experiment, the umbilical cord amniotic mesenchymal stem cell line was isolated from umbilical cord tissue by culturing the umbilical cord tissue in the culture medium PTT-6 of the present invention, and bone marrow mesenchymal stem cells were isolated from human bone marrow using standard methods.

圖 7A 所示為自臍帶組織分離並培養於PTT-6培養基後，表現幹細胞標記CD73、CD90與CD105以及缺乏CD34、CD45與HLA-DR之分離的間質臍帶襯裡幹細胞的百分比，而圖 7B 所示為表現CD73、CD90與CD105以及缺乏CD34、CD45與HLA-DR之分離的骨髓間質幹細胞的百分比。 FIG. 7A isolated and cultured, expression of stem cell markers CD73, CD90 and CD105 and the absence of CD34 percentage of mesenchymal umbilical cord lining stem cells after the PTT-6 medium, the isolated CD45 and HLA-DR of self umbilical cord tissue, while FIG. 7B It is shown as the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking CD34, CD45 and HLA-DR.

如圖 7A 所示，間質幹細胞群含有97.5%的活細胞，其中100%表現CD73、CD90以及CD105的每一種(參閱「CD73+ CD90+」以及「CD73+ CD105+」行)，而99.2%的幹細胞群不表現CD45，且100%的幹細胞群不表現CD34以及HLA-DR (參閱「CD34-CD45-」 以及「CD34-HLA-DR-」行)。因此，透過在PTT-6培養基中培養獲得的間質幹細胞群基本上是100%純的且確定的幹細胞群，其滿足間質幹細胞將用於細胞療法的標準(95%或更多的幹細胞群表現CD73、CD90以及CD105，而98%或更多的幹細胞群缺乏CD34、CD45以及HLA-DR的表現，參閱Sensebe等人 「Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review」，同上)。於此應注意的是，羊膜的間質幹細胞在標準培養條件下黏附於塑料並在體外分化為成骨細胞、脂肪細胞以及成軟骨細胞，參閱美國專利9,085,755、美國專利8,287,854或WO2007/046775，因此符合通常接受在細胞療法中使用間質幹細胞的標準。As shown in Figure 7A, mesenchymal stem cells contained 97.5% viable cells, wherein 100% performance of CD73, CD90 and CD105 in each (see "CD73 + CD90 +" and "CD73 + CD105 +" line), while 99.2% of the population of stem cells not Express CD45, and 100% of the stem cell population does not express CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-" rows). Therefore, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is basically a 100% pure and definite stem cell population, which meets the criteria that the mesenchymal stem cells will be used for cell therapy (95% or more stem cell population Performance of CD73, CD90 and CD105, and 98% or more of the stem cell population lacks performance of CD34, CD45 and HLA-DR, see Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", ibid. ). It should be noted here that the mesenchymal stem cells of amniotic membrane adhere to plastic under standard culture conditions and differentiate into osteoblasts, adipocytes and chondrocytes in vitro, see US Patent 9,085,755, US Patent 8,287,854 or WO2007/046775, so It meets the standards that are generally accepted for the use of mesenchymal stem cells in cell therapy.

圖 7B 所示為表現CD73、CD90以及CD105並且缺乏CD34、CD45以及HLA-DR表現的分離的骨髓間質幹細胞的百分比。如圖 7B 所示，骨髓間質幹細胞群含有94.3%的活細胞，其中100%表現CD73、CD90以及CD105 (參閱 「CD73+ CD90+」以及「CD73+ CD105+」行)，而僅62.8%的骨髓幹細胞群缺乏CD45的表現，且99.9%的幹細胞群缺乏CD34與HLA-DR的表現(參閱「CD34-CD45-」以及「CD34-HLA-DR-」行)。因此，被認為是間質幹細胞的黃金標準的骨髓間質幹細胞就幹細胞標記而言，遠比間質幹細胞群(臍帶的羊膜)更不均質/純。該發現還顯示，本發明之幹細胞群可為基於幹細胞的療法的理想候選者，並且可以成為基於幹細胞的治療方法的黃金標準。 Figure 7B shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking the expression of CD34, CD45 and HLA-DR. As shown in Figure 7B, mesenchymal stem cell population contained 94.3% viable cells, wherein 100% performance of CD73, CD90 and of CD105 (see "CD73 + CD90 +" and "CD73 + CD105 +" line), and the lack of 62.8% of the bone marrow stem cells only CD45 performance, and 99.9% of stem cell populations lack CD34 and HLA-DR performance (see "CD34-CD45-" and "CD34-HLA-DR-" rows). Therefore, bone marrow mesenchymal stem cells, considered to be the gold standard for mesenchymal stem cells, are far more heterogeneous/pure than the mesenchymal stem cell population (umbilical cord amniotic membrane) in terms of stem cell labeling. This finding also shows that the stem cell population of the present invention can be an ideal candidate for stem cell-based therapy and can become the gold standard for stem cell-based therapy.

圖 8(a)-(b) 所示為鋅指核酸酶(ZFN)構築的位點特異性切割活性之比較。 Figures 8(a)-(b) show a comparison of the site-specific cleavage activity constructed by zinc finger nuclease (ZFN).

圖 8(a) 所示為AAVS1 鋅指核酸酶(ZFN)變體與瞬間低溫對切割效率的比較。來自K562細胞的基因組DNA與pZDonor共同進行(參閱圖14(a)-(c))以及以下AAVS1 鋅指核酸酶(ZFN)變體：專性異二聚體(obligate heterodimer，OH)，Sharkey或增強型Sharkey。OH (專性異二聚體)鋅指核酸酶(ZFN)在與右AAVS1同源臂(E490K與I538K；包含SEQ ID NO. 7)融合的Fok1 單體中以及在與左同源臂(Q468E與I499L；包含SEQ ID NO. 8)融合的單體中有兩個胺基酸變化。Sharkey變體在右及左單體中都具有額外的S418P與K441E取代(分別包含SEQ ID NOs.12與13)。增強型Sharkey變體具有額外的胺基酸取代：右Fok1 單體中的H537R；(包含SEQ ID NO: 14)以及左Fok1 單體(包含SEQ ID NO: 15)中的N496D。所示突變的位置對應於野生型FokI 序列的位置，例如，如SEQ ID NO. 6所示。這表示，例如S418P表示SEQ ID NO. 6的野生型FokI的第418位置上的S突變為P。攜帶鋅指核酸酶(ZFN)變體的載體也在圖14(a)-(c)中進一步詳細描述。不同的鋅指核酸酶(ZFN)變體在37°C或30°C下培養。每次電穿孔中的pEGFP作為轉染效率的指標。透過限制性片段長度多態性評估位點特異性切割。顯示的結果為AAVS1修飾基因座的三重複密度測量值的平均值±標準差(SD)，表示為組合的未修飾與修飾基因座的百分比。透過限制性片段長度多態性(RFLP)評估，Sharkey以及增強型Sharkey 鋅指核酸酶(ZFNs)在30°C誘導供體DNA整合的最高效率(分別為44.5與47.9%)。 Figure 8(a) shows the comparison of the cutting efficiency between the AAVS1 zinc finger nuclease (ZFN) variant and instant low temperature. Genomic DNA from K562 cells was performed with pZDonor (see Figure 14(a)-(c)) and the following AAVS1 zinc finger nuclease (ZFN) variants: obligate heterodimer (OH), Sharkey or Enhanced Sharkey. OH (obligate heterodimer) zinc finger nuclease (ZFN) in the Fok1 monomer fused to the right AAVS1 homology arm (E490K and I538K; contains SEQ ID NO. 7) and in the left homology arm (Q468E There are two amino acid changes in the monomer fused with I499L; comprising SEQ ID NO. 8). Sharkey variants have additional S418P and K441E substitutions in both the right and left monomers (including SEQ ID NOs. 12 and 13 respectively). The enhanced Sharkey variant has additional amino acid substitutions: H537R in the right Fok1 monomer; (including SEQ ID NO: 14) and N496D in the left Fok1 monomer (including SEQ ID NO: 15). The position of the mutation shown corresponds to the position of the wild-type FokI sequence, for example, as shown in SEQ ID NO. This means, for example, S418P means that the S at position 418 of the wild-type FokI of SEQ ID NO. 6 is mutated to P. Vectors carrying zinc finger nuclease (ZFN) variants are also described in further detail in Figures 14(a)-(c). Different zinc finger nuclease (ZFN) variants were cultured at 37°C or 30°C. The pEGFP in each electroporation serves as an indicator of transfection efficiency. Site-specific cleavage was assessed by restriction fragment length polymorphism. The results shown are the mean ± standard deviation (SD) of the triple repeat density measurements of the AAVS1 modified locus, expressed as a percentage of the combined unmodified and modified loci. Through the evaluation of restriction fragment length polymorphism (RFLP), Sharkey and enhanced Sharkey zinc finger nucleases (ZFNs) induced the highest efficiency of donor DNA integration at 30°C (44.5 and 47.9%, respectively).

圖 8(b) 所示為小圖數據的圖形表示。 對於每種鋅指核酸酶(ZFN)變體(由小圖a確定)(左軸)獲得50-bp供體DNA的AAVS1特異性整合的K562基因組DNA的不同百分比針對透過電穿孔(右軸)的pEGFP基因轉移顯示相當效率。數據為平均值±SD (n = 3)。相較於37℃所有鋅指核酸酶(ZFN)變體，對於30°C培養的細胞，整合顯著更高(P >0.05)。於37°C時，使用增強型Sharkey (P = 0.0024)以及Sharkey (P = 0.03)與OH相比，實現了顯著更高的積分。於30°C時，增強型Sharkey以及Sharkey具有相當的活性，前者比OH顯著更為活躍(P = 0.02)。 Figure 8(b) shows a graphical representation of the thumbnail data. For each zinc finger nuclease (ZFN) variant (as determined by panel a) (left axis) a different percentage of K562 genomic DNA with 50-bp donor DNA-specific integration of AAVS1 is obtained for electroporation (right axis) The pEGFP gene transfer showed considerable efficiency. The data is the mean ± SD (n = 3). Compared to all zinc finger nuclease (ZFN) variants at 37°C, the integration was significantly higher for cells cultured at 30°C (P>0.05). At 37°C, the use of enhanced Sharkey (P = 0.0024) and Sharkey (P = 0.03) achieved significantly higher points compared to OH. At 30°C, enhanced Sharkey and Sharkey are quite active, the former is significantly more active than OH (P = 0.02).

圖 9(a)-(c) 所示為不同大小供體DNA的AAVS1基因座特異性整合。 Figures 9(a)-(c) show the specific integration of the AAVS1 locus of donor DNA of different sizes.

圖 9(a) 供體DNA的鋅指核酸酶(ZFN)依賴性整合。將K562細胞與pEGFP (用於轉染效率的報告基因)與具有或不具有AAVS1 ZFN mRNA的pZDonor共同電穿孔(亦參閱圖14(a)-(c))。 (左)：轉染細胞的明場與螢光圖像。比例尺=100 μm (右)：限制性片段長度多態性(Restriction fragment length polymorphism， RFLP)用於量化pZDonor-AAVS1的位點特異性整合(亦參閱圖7(B))。 在不存在或存在AAVS1鋅指核酸酶(ZFN)的情況下，在電穿孔10 μg pZDonor-AAVS1後4天從細胞中萃取200 ng的基因組DNA。PCR引子擴增跨越AAVSI整合位點的1.9-kb區域。在僅以pZdonor處理後4天，對來自細胞的基因組DNA進行PCR；在以pZdonor與AAVS1 鋅指核酸酶(ZFN) mRNA處理後4天(第4天)以及16天(第16天)，提供50-bp供體DNA位點特異性整合的證據。對照PCR擴增AAVS1基因座的900 bp區域。兩個條帶(1-與0.9-kb)表示供體整合，而單個1.9-kb條帶表示沒有整合。 Figure 9(a) Zinc finger nuclease (ZFN) dependent integration of donor DNA. K562 cells were co-electroporated with pEGFP (reporter gene for transfection efficiency) and pZDonor with or without AAVS1 ZFN mRNA (see also Figure 14(a)-(c)). (Left): Brightfield and fluorescent images of transfected cells. Scale bar=100 μm (right): Restriction fragment length polymorphism (RFLP) is used to quantify the site-specific integration of pZDonor-AAVS1 (see also Figure 7(B)). In the absence or presence of AAVS1 zinc finger nuclease (ZFN), 200 ng of genomic DNA was extracted from cells 4 days after electroporation of 10 μg pZDonor-AAVS1. The PCR primers amplify the 1.9-kb region spanning the AAVSI integration site. PCR was performed on genomic DNA from cells 4 days after treatment with pZdonor only; 4 days (day 4) and 16 days (day 16) after treatment with pZdonor and AAVS1 zinc finger nuclease (ZFN) mRNA Evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified the 900 bp region of the AAVS1 locus. Two bands (1- and 0.9-kb) indicate donor integration, while a single 1.9-kb band indicates no integration.

圖 9(b) 所示為增強型Sharkey AAVS1 鋅指核酸酶(ZFN)調節的pZdonor EGFP整合的準確性(亦參閱圖14(b))。 左圖：在具有或不具有G418篩選的pZDonor EGFP以及增強型Sharkey 鋅指核酸酶(ZFN)共同電穿孔的K562細胞的基因組DNA上進行的左與右整合連接的PCR擴增。值得注意的是，透過在G418 (0.8 mg/ml)中培養14天來篩選穩定整合的細胞。透過Quantity One軟體(Bio-Rad公司)定量DNA條帶的強度與體積。由穩定轉染的細胞實現最高的整合，因為在G418選擇的細胞中DNA條帶的強度傾向於最高。 右圖：左(上)以及右(下)連接PCR擴增子的DNA序列色層分析圖。矢量序列以灰色加底線；增強型Sharkey AAVS1 鋅指核酸酶(ZFN)識別半位點以淺灰色加底線。對照PCR擴增了AAVS1基因座的900 bp區域。野生型K562表示未轉染的對照K562細胞。 Figure 9(b) shows the accuracy of pZdonor EGFP integration regulated by enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) (see also Figure 14(b)). Left: PCR amplification of left and right integrated connections on the genomic DNA of K562 cells electroporated with or without G418 screened pZDonor EGFP and enhanced Sharkey zinc finger nuclease (ZFN). It is worth noting that stable integration cells were selected by culturing in G418 (0.8 mg/ml) for 14 days. The intensity and volume of DNA bands were quantified by Quantity One software (Bio-Rad). The highest integration is achieved by stably transfected cells, because the DNA bands tend to have the highest intensity in G418-selected cells. Right: Chromatographic analysis of the DNA sequence of the PCR amplicon connected to the left (top) and right (bottom). Vector sequences are underlined in gray; enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) recognition half-sites are underlined in light gray. Control PCR amplified the 900 bp region of the AAVS1 locus. Wild-type K562 represents untransfected control K562 cells.

圖 9(c) 顯示了增強型Sharkey AAVS1 鋅指核酸酶(ZFN)調節的pZDonor Hybrid FVIII整合的準確性(亦參閱圖14(b))。 左圖：在僅以pZDonor Hybrid FVIII電穿孔的K562細胞的基因組DNA上進行的左以及右整合連接的PCR擴增，或者以增強型Sharkey鋅指核酸酶(ZFN)共同電穿孔，然後進行G418篩選。對照PCR擴增了AAVS1基因座的900 bp區域。野生型K562表示未轉染的對照K562細胞。凝膠圖像中的白色垂直線劃分了為了清晰而合併的泳道。僅透過穩定轉染的細胞實現整合。 右圖：左(上)以及右(下)連接PCR擴增子的DNA序列色層分析圖。矢量序列以灰色加底線；增強型Sharkey AAVS1 鋅指核酸酶(ZFN)識別半位點以淺灰色加底線。 Figure 9(c) shows the accuracy of pZDonor Hybrid FVIII integration regulated by enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) (see also Figure 14(b)). Left: PCR amplification of left and right integration junctions on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only, or co-electroporation with enhanced Sharkey zinc finger nuclease (ZFN), followed by G418 screening . Control PCR amplified the 900 bp region of the AAVS1 locus. Wild-type K562 represents untransfected control K562 cells. The white vertical lines in the gel image divide the lanes merged for clarity. Integration is achieved only through stably transfected cells. Right: Chromatographic analysis of the DNA sequence of the PCR amplicon connected to the left (top) and right (bottom). Vector sequences are underlined in gray; enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) recognition half-sites are underlined in light gray.

圖 10(a)-(f) 所示為FVIII供體DNA在臍帶襯裡上皮細胞(CLECs)中的AAVS1基因座特異性整合。 Figures 10(a)-(f) show the specific integration of the FVIII donor DNA at the AAVS1 locus in umbilical cord lined epithelial cells (CLECs).

圖 10(a) 為透過同源定向整合(未按比例繪製)整合在AAVS1基因座中的供體DNA的示意圖。從人類鐵蛋白輕鏈啟動子(hFER L)表現FVIII轉殖基因(SEQ ID NO. 3的5.1 kb雜合FVIII)。為了消除鐵蛋白啟動子的鐵調節，FerL的5'UTR已經被黑猩猩延伸因子1 (elongation factor 1， EF1)基因的5'UTR (Chipanzee EF1 5'UTR)取代。***物還包含剪接受體(splice acceptor，SA)、嘌呤黴素匣(2A與嘌呤黴素)、聚腺苷酸化(poly A)序列，人類細胞巨大病毒(CMV增強子)的主要立即早期增強子的強增強子，以及一牛生長激素多腺苷酸化(BGH poly A)。重淺灰色條表示同源臂。虛線淺灰色線表示同源臂側翼的AAVS1基因座基因組DNA。箭頭表示用於整合連接與重疊PCR的引子，以記錄完整FVIII轉殖基因DNA的整合。穩定整合的臍帶襯裡上皮細胞對嘌呤黴素具有抗性(0.5 mg/ml，持續7天)(puro-CLECs)。野生型臍帶襯裡上皮細胞(Wt-CLECs)未以質體處理。 Figure 10(a) is a schematic diagram of donor DNA integrated in the AAVS1 locus through homologous directed integration (not drawn to scale). The FVIII transfer gene (5.1 kb hybrid FVIII of SEQ ID NO. 3) was expressed from the human ferritin light chain promoter (hFER L). In order to eliminate the iron regulation of the ferritin promoter, the 5'UTR of FerL has been replaced by the 5'UTR (Chipanzee EF1 5'UTR) of the chimpanzee elongation factor 1 (EF1) gene. The insert also contains splice acceptor (SA), puromycin cassette (2A and puromycin), polyadenylation (poly A) sequence, the main immediate early enhancement of human cytomegalovirus (CMV enhancer) The strong enhancer of the seed, and a bovine growth hormone polyadenylation (BGH poly A). Heavy gray bars indicate homology arms. The dashed light gray line indicates the genomic DNA of the AAVS1 locus flanking the homology arm. Arrows indicate primers used for integration ligation and overlapping PCR to record the integration of complete FVIII transgenic DNA. Stably integrated umbilical cord lining epithelial cells are resistant to puromycin (0.5 mg/ml for 7 days) (puro-CLECs). Wild-type umbilical cord lining epithelial cells (Wt-CLECs) were not treated with plastids.

圖 10(b) 所示為增強型Sharkey AAVS1 鋅指核酸酶(ZFN)調節的pSA-2A-Puro雜合體FVIII整合的準確性(亦參閱圖14(c))。已經進行了整合連接PCR (junction PCR，JPCR)，其檢查供體整合的正確性。JPCR顯示供體DNA整合到PPP1R12C的內含子1中。左及右JPCR分別擴增診斷性5'與3'基因組連接，透過供體整合使用對供體DNA特異的引子以及超出同源臂的相鄰基因組區域來產生。對照陽性PCR在遠離整合位點2 kb的AAVS1基因座中擴增900 bp序列。DNA大小標記為10、3以及1 kb。右圖所示為兩個不同的長PCR，包括以供體以及基因座特異性引子進行的全長轉殖基因。只有puro-CLEC基因組DNA對預測的6.9以及4.2 kb擴增子呈陽性，對其進行定序以證實完整FVIII轉殖基因cDNA的整合。標明了10、5以及3 kb的DNA大小標記。凝膠圖像中的白色垂直線劃分了為清晰起見而合併的泳道。 Figure 10(b) shows the accuracy of pSA-2A-Puro hybrid FVIII integration regulated by enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) (see also Figure 14(c)). Junction PCR (JPCR) has been performed, which checks the correctness of donor integration. JPCR showed that the donor DNA integrated into intron 1 of PPP1R12C. The left and right JPCRs amplify the diagnostic 5'and 3'genomic junctions respectively, and are generated by donor integration using donor DNA-specific primers and adjacent genomic regions beyond the homology arms. Control positive PCR amplifies a 900 bp sequence in the AAVS1 locus 2 kb away from the integration site. The DNA size markers are 10, 3, and 1 kb. The picture on the right shows two different long PCRs, including full-length transgenes with donor and locus-specific primers. Only puro-CLEC genomic DNA was positive for the predicted 6.9 and 4.2 kb amplicons, which were sequenced to confirm the integration of the complete FVIII transgene cDNA. DNA size markers of 10, 5, and 3 kb are indicated. The white vertical lines in the gel image divide the lanes merged for clarity.

圖 10(c) 所示為定序的JPCR擴增子，以確認位點特異性的左及右積分連接。供體特異性序列以灰色加底線且增強型Sharkey AAVS1 鋅指核酸酶(ZFN)結合位點以淺灰色加底線。 Figure 10(c) shows sequenced JPCR amplicons to confirm site-specific left and right integral ligation. Donor-specific sequences are underlined in gray and the enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) binding site is underlined in light gray.

圖 10(d) 所示為如上所述之人類臍帶上皮幹細胞，其以供體DNA質體以及鋅指核酸酶(ZFN)質體進行電穿孔。僅以FVIII供體質體(C1-CLEC)轉染對照臍帶襯裡上皮細胞(CLECs)或以FVIII供體質體以及不含嘌呤黴素篩選的鋅指核酸酶(ZFN)(C2-CLEC)共轉染對照臍帶襯裡的上皮細胞(CLECS )。將Puro-CLEC轉染像C2-CLEC，並進行嘌呤黴素篩選 (電穿孔後4天開始，0.5 mg/ml，連續7天)。穩定整合的臍帶襯裡上皮幹細胞對嘌呤黴素具有抗性(0.5 mg/ml，持續7天)(puro-CLECs)。野生型臍帶襯裡的上皮細胞(Wt-CLECs)未以質體處理。然後在電穿孔後1天以及37天在野生型以及puro-CLEC的條件培養基中測量FVIII活性。大多數FVIII在電穿孔後1天從puro-CLEC分泌。puro-CLEC的分泌於第37天比於第1天更高，而於第37天在C1-CLC以及C2-CLC中未檢測到FVIII的分泌。這表示只有puro-CLEC提供連續的FVIII分泌，而其他轉染的臍帶襯裡的上皮細胞(CLECs)隨時間沒有顯示出這種分泌。數據為平均值±SEM；n = 3。 Figure 10(d) shows the human umbilical cord epithelial stem cells as described above, which are electroporated with donor DNA plastids and zinc finger nuclease (ZFN) plastids. Co-transfection of control umbilical cord lining epithelial cells (CLECs) with FVIII donor plastids (C1-CLEC) or zinc finger nuclease (ZFN) (C2-CLEC) screened with FVIII donor plastids and puromycin-free screening Control epithelial cells lined with umbilical cord (CLEC S ). Puro-CLEC was transfected like C2-CLEC, and puromycin screening was performed (starting 4 days after electroporation, 0.5 mg/ml for 7 consecutive days). Stably integrated umbilical cord lining epithelial stem cells are resistant to puromycin (0.5 mg/ml for 7 days) (puro-CLECs). Wild-type umbilical cord-lined epithelial cells (Wt-CLECs) were not treated with plastids. FVIII activity was then measured in wild-type and puro-CLEC conditioned media 1 day and 37 days after electroporation. Most FVIII is secreted from puro-CLEC 1 day after electroporation. Puro-CLEC secretion was higher on day 37 than on day 1, and FVIII secretion was not detected in C1-CLC and C2-CLC on day 37. This means that only puro-CLEC provides continuous FVIII secretion, while other transfected umbilical cord lined epithelial cells (CLECs) have not shown this secretion over time. Data are mean ± SEM; n = 3.

圖 10(e) 所示為在AAVS1基因座上的總整合供體DNA與整合連接的複製數相對於染色體19q13.42上對照基因座的複製數。為了測量整合供體DNA的複製數，已經進行了數位微滴式PCR (digital droplet PCR，dPCR)。該技術將樣品分離成大量分區，然後分別在每個分區中進行反應。這種分離允許更可靠的收集與核酸量的靈敏測量。具體而言，在圖10(e)中，進行puro-CLEC基因組DNA的dPCR以確定整合載體、左整合連接、右整合連接，以及染色體19q13.42中的兩個對照基因座的複製。Puro-CLEC係指嘌呤黴素選擇的細胞，在電穿孔後1個月具有FVIII轉殖基因的穩定整合。沒有模板DNA進行的反應為陰性對照。只有隨著時間的推移才能檢測到整合到puro-CLEC中的載體。每μl整合約300-600個複製數。此外，透過定量基因組PCR數據相對於總(在目標上以及偏離目標)轉殖基因複製數的目標轉殖基因複製數顯示整合連接的複製數以及載體擴增子之間沒有顯著差異。數據為平均值±SD；n = 4。 Fig. 10(e) shows the number of copies of the total integration donor DNA and integration junction at the AAVS1 locus relative to the number of copies of the control locus on chromosome 19q13.42. In order to measure the number of copies of the integrated donor DNA, digital droplet PCR (dPCR) has been performed. This technique separates the sample into a large number of partitions, and then reacts in each partition separately. This separation allows more reliable collection and sensitive measurement of the amount of nucleic acid. Specifically, in FIG. 10(e), dPCR of puro-CLEC genomic DNA was performed to determine the integration vector, left integration junction, right integration junction, and replication of two control loci in chromosome 19q13.42. Puro-CLEC refers to cells selected by puromycin, which has stable integration of FVIII transgenes 1 month after electroporation. Reactions without template DNA are negative controls. Only over time can the vector integrated into puro-CLEC be detected. Each μl integrates about 300-600 copies. In addition, the quantitative genomic PCR data showed no significant differences between the integrated ligated copy number and the vector amplicon relative to the total (on target and off target) copy number of the transferred gene. Data are mean ± SD; n = 4.

圖 10(f) 所示為野生型CLEC與puro-CLEC中PPP1R12C轉錄物含量(外顯子4-6)的定量RT-PCR。RT-PCR檢測RNA表現，而定量RT-PCR用於定量RNA。數據為平均值±SEM；n = 3。定量RT-PCR顯示，相較於野生型CLEC，puro-CLEC中PPP1R12C mRNA的含量降低了一半(圖10(f))。與複製數的數據合併，這與PPP1R12C 的內含子1中FVIII轉殖基因的單等位正確目標基因整合是一致的。 Figure 10(f) shows the quantitative RT-PCR of PPP1R12C transcript content (exons 4-6) in wild-type CLEC and puro-CLEC. RT-PCR measures RNA performance, while quantitative RT-PCR is used to quantify RNA. Data are mean ± SEM; n = 3. Quantitative RT-PCR showed that compared to wild-type CLEC, the content of PPP1R12C mRNA in puro-CLEC was reduced by half (Figure 10(f)). Combined with the data on the number of copies, this is consistent with the integration of the single allelic target gene of the FVIII transgene in PPP1R12C intron 1.

圖 11(a)-(d) 所示為與增強型Sharkey AAVS1 鋅指核酸酶(ZFN)調節的FVIII轉殖基因整合相關的偏離目標基因組變化。來自野生型與puro-CLEC的基因組DNA如圖10(a)-(f)中所述。以phi29聚合酶(REPLI-g套組；Qiagen公司)擴增。透過PCR-Sanger定序研究了高置信度***或缺失(indel)。***或缺失(indel)通常是用於***或缺失DNA中鹼基的分子生物學術語。因此，術語***或缺失(indel)描述了導致核苷酸***及/或核苷酸缺失的突變。在該位置存在兩個單鹼基取代而沒有***或缺失。已發現3個***或缺失。(由於高度重複的序列基序，indel 2無法連續定序。)indel 2擴增子不能獲得作為連續序列。 Figures 11(a)-(d) show the off-target genomic changes associated with the integration of enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) regulated FVIII transgene integration. Genomic DNA from wild-type and puro-CLEC is described in Figure 10(a)-(f). Amplified with phi29 polymerase (REPLI-g kit; Qiagen). High-confidence indels were studied by PCR-Sanger sequencing. Insertion or deletion (indel) is usually a molecular biology term used to insert or delete bases in DNA. Therefore, the term indel describes a mutation that results in nucleotide insertion and/or nucleotide deletion. There are two single base substitutions at this position without insertions or deletions. 3 insertions or deletions have been found. (Because of highly repetitive sequence motifs, indel 2 cannot be sequenced continuously.) The indel 2 amplicon cannot be obtained as a continuous sequence.

圖 11(a) 描繪了indel 1的DNA序列。indel 1為偽陽性***或缺失(indel)，因為序列數據僅顯示兩個單鹼基取代。 Figure 11 (a) depicts the DNA sequence of indel 1. indel 1 is a false positive insertion or deletion (indel) because the sequence data shows only two single base substitutions.

圖 11(b) 描繪了indel 3的序列。indel 3包括兩個不同衛星DNA的擴展(weblogo.berkeley.edu)。每個淺灰色以及灰色矩形代表相應衛星DNA的單個複製。indel 3包含兩個不同衛星DNA的***。較大的***(1,489 bp)為衛星DNA的15倍擴展，而較小的***(186 bp)為不同衛星的兩倍擴展。 Figure 11(b) depicts the sequence of indel 3. indel 3 includes two different satellite DNA extensions (weblogo.berkeley.edu). Each light gray and gray rectangle represents a single copy of the corresponding satellite DNA. indel 3 contains the insertion of two different satellite DNAs. The larger insertion (1,489 bp) is a 15-fold expansion of satellite DNA, while the smaller insertion (186 bp) is a two-fold expansion of different satellites.

圖 11(c) 所示為全基因組定序(whole genome sequencing，WGS)分析顯示四種染色體重排，均具有不平衡的基因組複製數。描述了來自全基因組定序(WGS)數據的生物資訊學分析的puro-CLEC中不平衡結構變體(structural variants，SVs)的假設斷點。所有染色體位置均指hg19。 Figure 11(c) shows a whole genome sequencing (WGS) analysis showing that the four chromosome rearrangements all have unbalanced genome replication numbers. Describes the hypothetical breakpoints of unbalanced structural variants (SVs) in puro-CLEC from bioinformatics analysis of genome-wide sequencing (WGS) data. All chromosome positions refer to hg19.

圖 11(d) 描繪了定量PCR。由於預測四種結構變體(SV)中的所有斷裂位點具有不平衡的基因組複製數，已經進行了定量PCR以確定puro-CLEC的基因組DNA中的每個斷裂點基因座的複製數相對於野生型臍帶襯裡的上皮細胞(CLECs)(wt-CLECs)中的相同斷裂點基因座。每個斷裂點基因座處的相對複製數表示為puro-以及野生型CLEC的標準化CT值的比率。標準化是必要的，因為SV1-SV4斷裂點基因座在不同的黏合溫度下被擴增以實現擴增的特異性。使用肌動蛋白基因座擴增的CT值來標準化相同實驗中每個斷裂點基因座的CT值。不能擴增SV1的斷裂點2基因座。SV4的候選斷裂點位點間隔8bp並作為單個基因座擴增。在所有分析的候選斷裂點，puro-CLEC與野生型CLEC基因組複製數的比率為0.97-1.07與不平衡結構改變的實質頻率不一致(圖11(d))。基於透過PCR擴增不存在異常染色體連接並且透過實驗驗證在斷裂點基因座處沒有異常基因組複製數，鋅指核酸酶(ZFN)處理不太可能誘導具有生物學意義的染色體重排。因此，圖11(d)顯示了透過定量PCR在推定斷裂點處的相對基因組複製數。將每個斷裂點基因座處的平均CT值標準化為其自身的肌動蛋白CT值。對於SV2以及SV3，顯示了每個假定斷裂點處puro-CLEC：野生型-CLEC的標準化複製數的比率。基因組複製數可以僅在SV1與SV4的一個假設斷裂點處量化。數據為三重複反應的平均值±SD。 Figure 11(d) depicts quantitative PCR. Since all breakpoints in the four structural variants (SV) are predicted to have an unbalanced genomic copy number, quantitative PCR has been performed to determine the copy number of each breakpoint locus in the genomic DNA of puro-CLEC relative to The same breakpoint locus in wild-type umbilical cord lined epithelial cells (CLECs) (wt-CLECs). The relative number of copies at each breakpoint locus is expressed as the ratio of puro- and normalized CT values of wild-type CLEC. Standardization is necessary because the SV1-SV4 breakpoint loci are amplified at different bonding temperatures to achieve specificity of amplification. The CT value of the actin locus amplification was used to normalize the CT value of each breakpoint locus in the same experiment. The SV1 breakpoint 2 locus cannot be amplified. The candidate breakpoints of SV4 are separated by 8 bp and amplified as a single locus. At all candidate breakpoints analyzed, the ratio of puro-CLEC to wild-type CLEC genome replication number was 0.97-1.07, which was inconsistent with the substantial frequency of unbalanced structural changes (Figure 11(d)). Based on the absence of abnormal chromosomal junctions through PCR amplification and the experimental verification that there is no abnormal genomic copy number at the breakpoint locus, zinc finger nuclease (ZFN) treatment is unlikely to induce biologically significant chromosomal rearrangements. Therefore, FIG. 11(d) shows the relative genomic copy number at the predicted break point by quantitative PCR. The average CT value at each breakpoint locus is normalized to its own actin CT value. For SV2 and SV3, the ratio of normalized copy number of puro-CLEC: wild-type-CLEC at each assumed break point is shown. The number of genome copies can be quantified only at a hypothetical breakpoint of SV1 and SV4. The data is the mean ± SD of triplicate reactions.

圖 12(a)-(c) 所示為puro-CLEC中過度表現以及表現不足基因的基因本體分類以及所選轉錄物的定量RT-PCR。如圖3中所述之野生型-CLEC與puro-CLEC的RNA-seq共鑑定了17,751個轉錄物，其中僅如圖 12(a) 所示57個過度表現並且如圖 12(b) 所示33個在puro-CLEC中，至少有兩倍的表現不足。FVIII為過度表現的轉錄物之一。途徑分析顯示10個失調的轉錄物透過DAVID分析28(Benjamini校正的P = 0.011)定位於細胞因子-細胞因子受體相互作用，這與PPP1R12C在發炎反應中的作用一致(亦參閱圖21)。雖然7個失調的基因是在超過1,600個致癌基因的綜合目錄中潛在的原癌基因(http://www.bushmanlab.org/links/genelists)，但沒有一個定位於KEGG的任何經典癌症途徑。 Figures 12(a)-(c) show the gene ontology classification of over- and under-expressed genes in puro-CLEC and quantitative RT-PCR of selected transcripts. Figure 3 of the wild type and puro-CLEC -CLEC of RNA-seq were identified 17,751 transcripts, which only in Figure 12 (a) and FIG 57 as over-represented in FIG 12 (b) shown in FIG. Of the 33 puro-CLECs, at least two times underperformed. FVIII is one of the overexpressed transcripts. Pathway analysis showed that 10 dysregulated transcripts were localized to cytokine-cytokine receptor interaction through DAVID analysis 28 (Benjamini corrected P = 0.011), which is consistent with the role of PPP1R12C in the inflammatory response (see also Figure 21). Although the 7 dysregulated genes are potential proto-oncogenes in the comprehensive catalog of more than 1,600 oncogenes (http://www.bushmanlab.org/links/genelists), none of them is located in any classic cancer pathway of KEGG.

圖 12(c) 所示為進行定量RT-PCR以驗證puro-CLEC中PPP1R12C 以及所選轉錄物含量的變化，如圖10(a)-(F)所述。在沒有質體DNA的情況下電穿孔的CLEC與相同數量的群體倍增作為對照。使用跨內含子外顯子引子擴增內源性PPP1R12C 轉錄物(外顯子4-6)，以AAVS1整合位點為中心的1-Mb區間內的相鄰基因(LILRB4 、ISOC2 、PPP6R1 、NAT14 、ZNF579 、FIZ1 ，以及RDH13 )，由Gene Network Central™ (http://www.sabiosciences.com)與人類蛋白質-蛋白質相互作用預測(http://www.compbio.dundee.ac.uk)預測的PPP1R12C 的潛在相互作用配偶體，其被RNA-Seq分析顯著改變(DUSP1 、DUSP6 、CDC6 ，以及DUSP16 )，以及一管家基因GAPDH 。將轉錄物含量標準化為GAPDH 表現，並使用「delta-deltaC (T) 方法」 (Livak與Schmittgen，2001年)相對於野生型-CLEC表現puro-CLEC中轉錄物含量的倍數變化。該圖顯示僅DUSP6的失調，DUSP6為一PPP1R12C相互作用配偶體，其表現在puro-CLEC中增加4.2倍。DUSP6負調節ERK1/230，高表現損害上皮-間質轉化以及致瘤性。數據為平均值±SEM；每組實驗n = 2，每個樣品重複3次。 Figure 12(c) shows quantitative RT-PCR to verify the changes in the content of PPP1R12C and selected transcripts in puro-CLEC, as described in Figure 10(a)-(F). In the absence of plastid DNA, electroporated CLEC doubled with the same number of population as a control. Amplify endogenous PPP1R12C transcripts (exons 4-6) using intron exon primers, adjacent genes within the 1-Mb interval centered on the AAVS1 integration site ( LILRB4 , ISOC2 , PPP6R1 , NAT14 , ZNF579 , FIZ1 , and RDH13 ), predicted by Gene Network Central™ (http://www.sabiosciences.com) and human protein-protein interaction prediction (http://www.compbio.dundee.ac.uk) The potential interaction partner of PPP1R12C was significantly changed by RNA-Seq analysis ( DUSP1 , DUSP6 , CDC6 , and DUSP16 ), as well as a housekeeping gene GAPDH . The transcript content was normalized to GAPDH performance, and the delta-delta C (T) method (Livak and Schmittgen, 2001) was used to compare the wild-type-CLEC performance to the fold change in transcript content in puro-CLEC. The figure shows the dysregulation of only DUSP6, which is a PPP1R12C interacting partner, whose performance is increased by 4.2 times in puro-CLEC. DUSP6 negatively regulates ERK1/230, high expression impairs epithelial-mesenchymal transition and tumorigenicity. The data is the mean ± SEM; n = 2 for each group of experiments, each sample was repeated 3 times.

圖 13(a)-(b) 所示為在其他原代人類細胞類型中增強型Sharkey AAVS1 鋅指核酸酶(ZFN)活性以及FVIII轉殖基因分泌。 Figures 13(a)-(b) show enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) activity and FVIII transgene secretion in other primary human cell types.

圖 13(a) 所示為在成人原代細胞中AAVS1 鋅指核酸酶(ZFN)調節的FVIII轉殖基因整合。來自嘌呤黴素抗性人類皮膚纖維母細胞(纖維母細胞FVIII穩定物)以及骨髓基質細胞(BMSC FVIII穩定物)的基因組DNA的PCR與FVIII供體質體共同電穿孔(pSA-2A-Puro Hybrid FVIII；見圖10(a)-(f)以及14(c))以及增強型Sharkey AAVS1 鋅指核酸酶(ZFN)(增強型Sharkey專性異二聚體鋅指核酸酶(ZFNs)；見圖10(a)-(f)以及14(a))顯示存在整合載體(521 bp)，左(602 bp)以及右(551 bp)整合連接。對照PCR擴增了AAVS1基因座的510-bp區域。減去模板泳道顯示在沒有基因組DNA的情況下進行的陰性對照反應。凝膠圖像中的白色垂直線劃分了為清晰起見而合併的泳道。 Fig. 13(a) shows the integration of FVIII transgene regulated by AAVS1 zinc finger nuclease (ZFN) in adult primary cells. Co-electroporation of genomic DNA from puromycin-resistant human skin fibroblasts (fibroblast FVIII stabilizer) and bone marrow stromal cells (BMSC FVIII stabilizer) with FVIII donor plastids (pSA-2A-Puro Hybrid FVIII ; See Figures 10(a)-(f) and 14(c)) and enhanced Sharkey AAVS1 zinc finger nucleases (ZFN) (enhanced Sharkey specific heterodimer zinc finger nucleases (ZFNs); see Figure 10 (a)-(f) and 14(a)) show the presence of integration vector (521 bp), left (602 bp) and right (551 bp) integration connections. Control PCR amplified the 510-bp region of the AAVS1 locus. Subtracting the template lane shows the negative control reaction performed in the absence of genomic DNA. The white vertical lines in the gel image divide the lanes merged for clarity.

圖 13(b) 所示為鋅指核酸酶(ZFN)修飾的細胞分泌FVIII。在電穿孔後6週，野生型與嘌呤黴素抗性人類皮膚纖維母細胞(152±14 mU/106 細胞/24小時)以及骨髓基質細胞(253.3±6.4 mU/106 細胞/24小時)的條件培養基中的FVIII活性。 與puro-CLEC一樣，鋅指核酸酶(ZFN)修飾的原代纖維母細胞以及骨髓衍生的基質細胞也分泌FVIII。數據為平均值±SEM；n = 3。 Fig. 13(b) shows that the cells modified by zinc finger nuclease (ZFN) secrete FVIII. Six weeks after electroporation, wild-type and puromycin-resistant human skin fibroblasts (152±14 mU/10 6 cells/24 hours) and bone marrow stromal cells (253.3±6.4 mU/10 6 cells/24 hours) FVIII activity in conditioned medium. Like puro-CLEC, primary fibroblasts modified by zinc finger nuclease (ZFN) and bone marrow-derived stromal cells also secrete FVIII. Data are mean ± SEM; n = 3.

圖 14(a)-(c) 所示為鋅指核酸酶(ZFN)變體構築。 Figures 14(a)-(c) show the construction of zinc finger nuclease (ZFN) variants.

圖 14(a) 所示為AAVS1 鋅指核酸酶(ZFN)變體構築。三種變體是專性異二聚體(OH)鋅指核酸酶(ZFN)(根據Miller, J.C.等人(2007年) NatBiotechnol 25：778-785所修飾)，其包含SEQ ID NO. 7的催化序列(右同源臂)以及SEQ ID NO. 8的催化序列(左同源臂)；Sharkey專性異二聚體鋅指核酸酶(ZFN)(根據Guo, J.等人(2010年) J Mol Biol 400: 96107進一步修飾的專性異二聚體)，其包含SEQ ID NO. 12的催化序列(右同源臂)以及SEQ ID NO. 13的催化序列(左同源臂)；增強型Sharkey專性異二聚體鋅指核酸酶(ZFN)(根據Doyon, Y等人(2011年) Nat Methods 8: 7479進一步修飾的Sharkey專性異二聚體鋅指核酸酶(ZFN)變體)，其包含SEQ ID NO. 14的催化序列(右同源臂)以及SEQ ID NO. 15的催化序列(左同源臂)。 Figure 14(a) shows the construction of the AAVS1 zinc finger nuclease (ZFN) variant. The three variants are obligate heterodimer (OH) zinc finger nucleases (ZFN) (modified according to Miller, JC et al. (2007) NatBiotechnol 25: 778-785), which contains the catalysis of SEQ ID NO. 7 Sequence (right homology arm) and the catalytic sequence of SEQ ID NO. 8 (left homology arm); Sharkey specific heterodimer zinc finger nuclease (ZFN) (according to Guo, J. et al. (2010) J Mol Biol 400: 96107 further modified obligate heterodimer), which contains the catalytic sequence of SEQ ID NO. 12 (right homology arm) and the catalytic sequence of SEQ ID NO. 13 (left homology arm); enhanced Sharkey obligate heterodimer zinc finger nuclease (ZFN) (Sharkey obligate heterodimer zinc finger nuclease (ZFN) variant further modified according to Doyon, Y et al. (2011) Nat Methods 8: 7479) , Which contains the catalytic sequence of SEQ ID NO. 14 (right homology arm) and the catalytic sequence of SEQ ID NO. 15 (left homology arm).

圖 14(b) 所示為具有AAVS1同源臂的供體構築。三個供體為pZDonor (50 bp***以及多選殖位點)；pZDonor EGFP (編碼3-kb EGFP)；pZDonor雜合FVIII (編碼SEQ ID NO. 3的9-kb人-豬FVIII cDNA)。 Figure 14(b) shows the donor architecture with the AAVS1 homology arm. The three donors were pZDonor (50 bp insertion and multiple selection site); pZDonor EGFP (encoding 3-kb EGFP); pZDonor hybrid FVIII (9-kb human-porcine FVIII cDNA encoding SEQ ID NO. 3).

圖 14(c) 所示為用於基因捕獲策略的供體構築。具有AAVS1同源臂以及剪接受體序列的供體構築在AAVS1基因座整合後表現來自內源性PPP1R12C啟動子的無啟動子嘌呤黴素抗性基因為(左) AAVS1 SA-2A-puro-pA供體(Addgene質體編號22075)，其整合 AAVS1位點的1-kb嘌呤黴素抗性基因；(中間) AAV-CAGGS-EGFP (Addgene質體編號22212)，其整合了由嘌呤黴素抗性基因組成的4.2kb片段以及由CAGGS啟動子表現的EGFP報告基因；(右) pSA-2A-Puro雜合體FVIII，其整合了SEQ ID NO. 3的9-kb片段，包含嘌呤黴素抗性基因以及由人類鐵蛋白輕鏈啟動子表現的人-豬FVIII cDNA。 Figure 14(c) shows the donor construction for the gene capture strategy. The donor construct with the AAVS1 homology arm and the splice acceptor sequence after integration of the AAVS1 locus shows that the promoter-free puromycin resistance gene from the endogenous PPP1R12C promoter is (left) AAVS1 SA-2A-puro-pA Donor (Addgene plastid number 22075), which integrates the 1-kb puromycin resistance gene at the AAVS1 site; (middle) AAV-CAGGS-EGFP (Addgene plastid number 22212), which integrates the puromycin resistance 4.2kb fragment composed of sex genes and the EGFP reporter gene expressed by the CAGGS promoter; (right) pSA-2A-Puro hybrid FVIII, which integrates the 9-kb fragment of SEQ ID NO. 3, including puromycin resistance Gene and human-porcine FVIII cDNA expressed by human ferritin light chain promoter.

圖 15(a)-(b) 所示為瞬間電穿孔的CLEC中鋅指核酸酶(ZFN)轉錄的時間過程以及次低溫對鋅指核酸酶(ZFN)蛋白含量的影響。使用兩個單獨的鋅指核酸酶(ZFN)質體進行圖15(a)與(b)中所示的實驗，該質體具有增強型Sharkey專性異二聚體突變(AAVS1 right Sharkey E490K；I538K；S418P；K441E；包含SEQ ID NO. 14的H537RfokI 以及pSCB AAVS1 left Sharkey Q468E；I499L；S418P；K441E；包含SEQ ID NO. 15的N496DfokI )。來自這2個質體的關鍵片段用於衍生增強型Sharkey專性異二聚體鋅指核酸酶(ZFN)構築(亦參閱圖14(a)-(c))。 Figures 15(a)-(b) show the time course of zinc finger nuclease (ZFN) transcription in CLEC with transient electroporation and the effect of sub-low temperature on the protein content of zinc finger nuclease (ZFN). The experiments shown in Figures 15(a) and (b) were performed using two separate zinc finger nuclease (ZFN) plastids with enhanced Sharkey obligate heterodimer mutations (AAVS1 right Sharkey E490K; I538K; S418P; K441E;. H537R fokI comprising SEQ ID NO 14 and pSCB AAVS1 left Sharkey Q468E; I499L; S418P; K441E; comprising SEQ ID NO N496D fokI 15 a). The key fragments from these two plastids were used to derive enhanced Sharkey's obligate heterodimeric zinc finger nuclease (ZFN) construct (see also Figure 14(a)-(c)).

圖 15(a) 所示為鋅指核酸酶(ZFN)構築的轉錄時程。以編碼左鋅指核酸酶(ZFN)(頂部)或右鋅指核酸酶(ZFN)(底部)的質體DNA進行電穿孔後，在指定的時間使用同源臂特異性引子，對來自轉染的CLEC的總RNA進行RT-PCR。陰性對照是在沒有反轉錄的情況下進行的反應(Minus RT PCR)。γ-肌動蛋白mRNA的RT-PCR為陽性對照。將鋅指核酸酶(ZFN)轉錄物條帶的光密度測量值標準化為它們各自的肌動蛋白含量，並表示為8小時時鋅指核酸酶(ZFN) mRNA轉錄物含量的百分比(在兩種凝膠中針對每個泳道指示)。在以AAVS1鋅指核酸酶(ZFN)質體電穿孔臍帶襯裡上皮細胞(CLECs)後8-48小時，RT-PCR顯示最高含量的鋅指核酸酶(ZFN)表現。 Fig. 15(a) shows the transcription time course constructed by zinc finger nuclease (ZFN). After electroporation of plastid DNA encoding left zinc finger nuclease (ZFN) (top) or right zinc finger nuclease (ZFN) (bottom), use homology arm-specific primers at designated times to Perform RT-PCR on the total RNA of CLEC. The negative control is a reaction performed without reverse transcription (Minus RT PCR). RT-PCR of γ-actin mRNA is a positive control. The optical density measurements of zinc finger nuclease (ZFN) transcript bands are normalized to their respective actin content and expressed as the percentage of zinc finger nuclease (ZFN) mRNA transcript content at 8 hours (in both (Indicated for each lane in the gel). 8-48 hours after electroporation of umbilical cord lined epithelial cells (CLECs) with AAVS1 zinc finger nuclease (ZFN) plastids, RT-PCR showed the highest content of zinc finger nuclease (ZFN) performance.

圖 15(b) 所示為鋅指核酸酶(ZFN)蛋白表現的時間過程。上圖：以編碼左右鋅指核酸酶(ZFN)的單個質體轉染的臍帶襯裡上皮細胞(CLECs)中FLAG標記的鋅指核酸酶(ZFN)蛋白的西方墨點分析法，並在37°C或30°C培養指定的天數，顯示當細胞暴露於次低溫時，鋅指核酸酶(ZFN)蛋白的豐度更高。未轉染的臍帶襯裡上皮細胞(CLECs) (野生型)對FLAG標記的鋅指核酸酶(ZFN)蛋白的表現呈現陰性的。下圖：ß-肌動蛋白作為注入樣品的對照。 Figure 15(b) shows the time course of zinc finger nuclease (ZFN) protein expression. Above: Western blot analysis of FLAG-labeled zinc finger nuclease (ZFN) protein in umbilical cord lining epithelial cells (CLECs) transfected with a single plastid encoding left and right zinc finger nucleases (ZFN) at 37° Incubation at C or 30°C for a specified number of days shows that the zinc finger nuclease (ZFN) protein is more abundant when cells are exposed to sub-low temperatures. Untransfected umbilical cord lining epithelial cells (CLECs) (wild type) were negative for FLAG-labeled zinc finger nuclease (ZFN) protein. Bottom: ß-actin serves as a control for injected samples.

圖 16(a)-(b) 所示為原代人類臍帶襯裡上皮細胞(CLECs)中位點特異性雙鏈DNA切割以及同源定向修復。透過酶促突變檢測測定(CEL-1)測定評估鋅指核酸酶(ZFN)活性(透過非同源末端連接報告DNA斷裂修復)。CEL I是一種從芹菜中分離的內切核酸酶，為一種核酸酶，對雙鏈DNA的錯配、***以及缺失具有高度特異性。該酶已經純化，可用於突變檢測試驗。 Figures 16(a)-(b) show site-specific double-stranded DNA cleavage and homologous directed repair in primary human umbilical cord lining epithelial cells (CLECs). The zinc finger nuclease (ZFN) activity (report DNA break repair through non-homologous end ligation) was assessed by the Enzymatic Mutation Assay (CEL-1) assay. CEL I is an endonuclease isolated from celery. It is a nuclease with high specificity for double-stranded DNA mismatch, insertion and deletion. The enzyme has been purified and can be used for mutation detection tests.

圖 16(a) 所示為AAVS1鋅指核酸酶(ZFN)單體遞送構築的比較。兩個單獨的鋅指核酸酶(ZFN)質體，其具有增強型Sharkey專用異二聚體突變(AAVS1 right Sharkey E490K；I538K；S418P；K441E；包含SEQ ID NO. 14的H537RfokI 以及pSCB AAVS1 left Sharkey Q468E；I499L；S418P；K441E；包含SEQ ID NO. 15的N496DfokI )共同電穿孔。使用增強型Sharkey專性異二聚體鋅指核酸酶(ZFN)構築進行具有雙鋅指核酸酶(ZFN)的標題(於圖14中)。以編碼左或右AAVS1 鋅指核酸酶(ZFN)(2個單一鋅指核酸酶(ZFN))的兩個單獨質體電穿孔的臍帶襯裡上皮細胞(CLECs)或編碼左和右AAVS1 鋅指核酸酶(ZFN)(雙鋅指核酸酶(ZFN))的單個質體在37°C下培養3天(37°C)或37°C持續1天，然後於30°C持續2天(30°C)。跨越AAVS1 鋅指核酸酶(ZFN)目標位點的基因組區域被擴增並以CEL-1核酸酶(+)消化或未消化(-)。透過10%聚丙烯醯胺凝膠電泳解析PCR擴增子，使用BioRad®GelDoc2000透射儀以及QuantityOne軟體成像並定量。來自Surveyor™突變檢測套組(Transgenomic公司)的PCR擴增子為CEL-1核酸酶消化的陽性對照。對於每種處理條件，在凝膠圖像中的相應泳道下方報告了基於光密度測定法在大量處理群體中修飾的基因組DNA (以***或缺失(indel)切割並修復的鋅指核酸酶(ZFN))的比例的估算值。當右及左增強型Sharkey AAVS1 鋅指核酸酶(ZFN)單體作為單一構築遞送時，CEL-1測定結果顯示顯著更高的鋅指核酸酶(ZFN)活性(與作為兩種構築遞送的鋅指核酸酶(ZFN)單體相比，43±1.9% (35±1.8%，P = 0.045))。 Figure 16(a) shows a comparison of the AAVS1 zinc finger nuclease (ZFN) monomer delivery architecture. Two separate zinc finger nucleases (the ZFNs) plastids, having enhanced dedicated Sharkey mutant heterodimer (AAVS1 right Sharkey E490K; I538K; S418P; K441E; comprising SEQ ID NO H537R fokI 14 and pSCB AAVS1 left. sharkey Q468E; I499L; S418P; K441E ;. comprising SEQ ID NO N496D fokI 15) is co-electroporation. The enhanced Sharkey obligate heterodimer zinc finger nuclease (ZFN) construction was used to carry out the title with double zinc finger nuclease (ZFN) (in Figure 14). Electroporated umbilical cord lining epithelial cells (CLECs) with two separate plastids encoding left or right AAVS1 zinc finger nuclease (ZFN) (2 single zinc finger nucleases (ZFN)) or encoding left and right AAVS1 zinc finger nucleic acids Individual plastids of enzyme (ZFN) (double zinc finger nuclease (ZFN)) were incubated at 37°C for 3 days (37°C) or 37°C for 1 day, then at 30°C for 2 days (30° C). The genomic region spanning the AAVS1 zinc finger nuclease (ZFN) target site is amplified and digested with CEL-1 nuclease (+) or undigested (-). The PCR amplicons were resolved by 10% polyacrylamide gel electrophoresis, and imaged and quantified using BioRad® GelDoc2000 transmission instrument and QuantityOne software. The PCR amplicon from Surveyor™ Mutation Detection Kit (Transgenomic Corporation) is a positive control for CEL-1 nuclease digestion. For each processing condition, genomic DNA modified in a large number of processing populations based on densitometry (zinc finger nucleases cut and repaired with indels (ZFN) is reported below the corresponding lanes in the gel image )) The estimated value of the ratio. When the right and left enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) monomers were delivered as a single construct, CEL-1 assay results showed significantly higher zinc finger nuclease (ZFN) activity (compared with zinc delivered as two constructs) Refers to nuclease (ZFN) monomer compared with 43±1.9% (35±1.8%, P = 0.045)).

圖 16(b) 所示為依賴AAVS1 鋅指核酸酶(ZFN)的供體DNA整合。左：以pZDonor單獨電穿孔或以雙AAVS1 鋅指核酸酶(ZFN)質體電穿孔的臍帶襯裡上皮細胞(CLECs)，均在pEGFP存在下進行，透過螢光顯微鏡評估基因轉移效率(原始放大倍數×100)。比例尺=100 μm。 中心：透過整合連接PCR分析這些細胞的pZDonor的位點特異性整合。以載體特異性以及基因組特異性引子進行整合連接PCR，以擴增跨越整合連接的1-kb區域。對照基因組PCR擴增了AAVS1基因座的900 bp區域。右：透過以Hind III消化跨越整合位點的PCR擴增子然後進行5%聚丙烯醯胺凝膠電泳來進行RFLP測定。使用提供增強型Sharkey AAVS1 鋅指核酸酶(ZFN)單體以及瞬間低溫的單一質體。在這些條件下，整合連接PCR與RFLP分析顯示在僅用pZDonor電穿孔的臍帶襯裡上皮細胞(CLECs)中沒有供體DNA整合。 Fig. 16(b) shows the integration of donor DNA dependent on AAVS1 zinc finger nuclease (ZFN). Left: Umbilical cord lined epithelial cells (CLECs) electroporated with pZDonor alone or with double AAVS1 zinc finger nuclease (ZFN) plastids, all performed in the presence of pEGFP, and the gene transfer efficiency was evaluated by fluorescence microscopy (original magnification) ×100). Scale bar=100 μm. Center: The site-specific integration of pZDonor in these cells was analyzed by integration ligation PCR. Vector-specific and genome-specific primers were used to perform integrated ligation PCR to amplify the 1-kb region spanning the integrated ligation. The control genome PCR amplified the 900 bp region of the AAVS1 locus. Right: RFLP assay was performed by digesting the PCR amplicon across the integration site with Hind III and then performing 5% polyacrylamide gel electrophoresis. Use a single plastid that provides enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) monomer and instant low temperature. Under these conditions, integrated ligation PCR and RFLP analysis revealed no donor DNA integration in umbilical cord lined epithelial cells (CLECs) electroporated with pZDonor alone.

圖 17(a)-(e) 所示為供體DNA或AAVS1 鋅指核酸酶(ZFN)誘導的細胞毒性及遺傳毒性。 Figures 17(a)-(e) show the cytotoxicity and genotoxicity induced by donor DNA or AAVS1 zinc finger nuclease (ZFN).

圖 17(a) 所示為供體DNA對遺傳毒性的劑量滴定。供體DNA劑量增加對誘導DNA雙鏈斷裂的影響。以2 μg pEGFP (報告基因)，固定劑量的AAVS1鋅指核酸酶(ZFN)(5 μg)以及增加劑量的pZDonor電穿孔200萬個臍帶襯裡上皮細胞(CLECs)，如圖所示。 臍帶襯裡上皮細胞(CLECs)野生型表示未經電穿孔的未修飾細胞。透過電穿孔後第4天的磷酸化H2AX陽性細胞的百分比來評估遺傳毒性。通常，H2AX表現的分析可用於檢測不同有毒物質的遺傳毒性作用。如圖17(a)所示，供體的量越高，雙鏈斷裂的量越高。*表示與供體(30 μg) EP相比P >0.01，且＃表示與供體(10 μg) + 鋅指核酸酶(ZFN)以及供體(20 μg) + 鋅指核酸酶(ZFN)相比， P >0.01。數據為平均值±SEM；n = 3。 Figure 17(a) shows the dose titration of donor DNA for genotoxicity. The effect of donor DNA dose increase on the induction of DNA double-strand breaks. Two million umbilical cord lining epithelial cells (CLECs) were electroporated with 2 μg pEGFP (reporter gene), a fixed dose of AAVS1 zinc finger nuclease (ZFN) (5 μg) and an increased dose of pZDonor. Umbilical cord lining epithelial cells (CLECs) wild type means unmodified cells without electroporation. Genotoxicity was assessed by the percentage of phosphorylated H2AX positive cells on day 4 after electroporation. Generally, the analysis of H2AX performance can be used to detect the genotoxic effects of different toxic substances. As shown in FIG. 17(a), the higher the amount of donor, the higher the amount of double-strand breaks. * Indicates P> 0.01 compared to donor (30 μg) EP, and # indicates phase compared to donor (10 μg) + zinc finger nuclease (ZFN) and donor (20 μg) + zinc finger nuclease (ZFN) Ratio, P>0.01. Data are mean ± SEM; n = 3.

圖 17(b) 所示為供體DNA的細胞毒性劑量滴定。透過比較在與上述(a)相同的實驗條件下電穿孔後第4天相對於電穿孔後第1天的GFP陽性細胞百分比的下降以評估細胞毒性。從(b)可以看出，於第4天，對於所有供體濃度，檢測到的GFP比第1天少。這表示第4天的細胞毒性高於第1天。當使用30 μg的供體濃度時，還存在細胞毒性最高的趨勢。數據表示為相對於第1天，於第4天的表現GFP的細胞的百分比。*表示與供體(10μg) + 鋅指核酸酶(ZFN)相比P >0.01，且＃表示與供體(20μg) + 鋅指核酸酶(ZFN)相比， P >0.01。數據為平均值±SEM；n = 3。 Figure 17(b) shows the cytotoxic dose titration of donor DNA. Cytotoxicity was evaluated by comparing the percentage decrease of GFP-positive cells on day 4 after electroporation relative to day 1 after electroporation under the same experimental conditions as in (a) above. It can be seen from (b) that on day 4, for all donor concentrations, less GFP was detected than on day 1. This means that the cytotoxicity on day 4 is higher than on day 1. When a donor concentration of 30 μg is used, there is also a trend for the highest cytotoxicity. The data is expressed as the percentage of cells expressing GFP on day 4 relative to day 1. * Indicates P>0.01 compared to donor (10 μg) + zinc finger nuclease (ZFN), and # indicates P >0.01 compared to donor (20 μg) + zinc finger nuclease (ZFN). Data are mean ± SEM; n = 3.

圖 17(c) 所示為AAVS1 鋅指核酸酶(ZFN)對遺傳毒性的劑量滴定。增加鋅指核酸酶(ZFN) DNA用量對誘導DNA雙鏈斷裂的影響。以2 μg pEGFP (報告基因)，固定劑量的pZDonor (10 μg)以及增加劑量的AAVS1 鋅指核酸酶(ZFN)電穿孔200萬個臍帶襯裡上皮細胞(CLECs)，如圖所示。透過在電穿孔後第4天磷酸化的H2AX陽性細胞的百分比來評估遺傳毒性。EP僅表示在沒有任何添加的質體構築的情況下進行電穿孔的臍帶襯裡上皮細胞(CLECs)。在測試的不同供體濃度下，細胞毒性非常相似。*表示與供體(10 μg) EP相比P >0.05，且＃表示與供體(10 μg) + 鋅指核酸酶(ZFN)(5 μg)，供體(10 μg) + 鋅指核酸酶(ZFN)(10 μg)以及供體(10 μg) + 鋅指核酸酶(ZFN)(30 µg)相比P >0.05。數據為平均值±SEM；n = 3。 Figure 17(c) shows the dose titration of AAVS1 zinc finger nuclease (ZFN) for genotoxicity. The effect of increasing the amount of zinc finger nuclease (ZFN) DNA on inducing DNA double-strand breaks. Two million umbilical cord lined epithelial cells (CLECs) were electroporated with 2 μg pEGFP (reporter gene), fixed dose pZDonor (10 μg) and increasing dose of AAVS1 zinc finger nuclease (ZFN), as shown. Genotoxicity was assessed by the percentage of H2AX-positive cells phosphorylated on day 4 after electroporation. EP only represents umbilical cord lined epithelial cells (CLECs) electroporated without any added plastid architecture. The cytotoxicity was very similar at the different donor concentrations tested. * Indicates P> 0.05 compared to donor (10 μg) EP, and # indicates compared to donor (10 μg) + zinc finger nuclease (ZFN) (5 μg), donor (10 μg) + zinc finger nuclease (ZFN) (10 μg) and donor (10 μg) + zinc finger nuclease (ZFN) (30 μg) compared to P>0.05. Data are mean ± SEM; n = 3.

圖 17(d) 所示為AAVS1 鋅指核酸酶(ZFN)對細胞毒性的劑量滴定。透過比較在與上述(c)相同的實驗條件下電穿孔後第4天相對於電穿孔後第1天的GFP陽性細胞百分比的下降以評估細胞毒性。數據表示為相對於第1天，於第4天的表現GFP的細胞的百分比。檢測所用的所有不同實驗裝置的細胞毒性。細胞毒性在電穿孔後4天比在電穿孔後1天更高。此外，當使用20 μg鋅指核酸酶(ZFN)時，細胞毒性最高。*表示與供體(10 μg) EP相比P >0.05，且＃表示與供體(10 μg) + 鋅指核酸酶(ZFN)(10 μg)相比， P >0.01。數據為平均值±SEM；n = 3。因此，(a)-(d)中描述的總體結果顯示，測試了不同劑量的增強型Sharkey AAVS1 鋅指核酸酶(ZFN)與供體DNA，以確定誘導由細胞活力及磷酸化組蛋白H2AX定量的最小細胞毒性的條件。總體結果顯示5-10 μg增強型Sharkey AAVS1 鋅指核酸酶(ZFN)與10 μg供體DNA的共電穿孔誘導最小的細胞毒性。 Figure 17(d) shows the dose titration of AAVS1 zinc finger nuclease (ZFN) for cytotoxicity. Cytotoxicity was evaluated by comparing the percentage of GFP-positive cells on day 4 after electroporation relative to day 1 after electroporation under the same experimental conditions as in (c) above. The data is expressed as the percentage of cells expressing GFP on day 4 relative to day 1. Test the cytotoxicity of all different experimental devices used. Cytotoxicity was higher at 4 days after electroporation than at 1 day after electroporation. In addition, when 20 μg zinc finger nuclease (ZFN) was used, the cytotoxicity was the highest. * Indicates P>0.05 compared to donor (10 μg) EP, and # indicates P>0.01 compared to donor (10 μg) + zinc finger nuclease (ZFN) (10 μg). Data are mean ± SEM; n = 3. Therefore, the overall results described in (a)-(d) show that different doses of enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) and donor DNA were tested to determine the induction by quantification of cell viability and phosphorylated histone H2AX Conditions of minimal cytotoxicity. Overall results showed that co-electroporation of 5-10 μg enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) with 10 μg donor DNA induced minimal cytotoxicity.

圖 17(e) 所示為AAVS1 鋅指核酸酶(ZFN)對細胞增殖的劑量滴定。僅接受電穿孔(僅EP)的未經處理的活臍帶襯裡上皮細胞(CLEC WT)的MTS測定，在電穿孔後1天僅接受10 μg pZDonor供體DNA或pZDonor DNA以及增加劑量的鋅指核酸酶(ZFN)。MTS測定法是用於在增殖與細胞毒性中靈敏定量活細胞之方法。該方法基於活細胞還原MTS四唑鎓化合物以產生可溶於細胞培養基的有色甲臢產物。認為該轉化透過代謝活性細胞中的NAD(P)H依賴性脫氫酶進行。由活細胞產生的甲臢染料可透過測量490-500 nm處的吸光度來定量。因此，使用MRX II 96孔板讀數器(Dynex公司)定量490 nm處的吸光度。與圖10(a)-(d)中描述的實驗結果一致，在臍帶襯裡上皮細胞(CLECs)野生型、僅EP，以及僅供體處理的細胞中檢測到大多數活細胞，而以供體及鋅指核酸酶(ZFN)電穿孔的細胞顯示較少的增殖/活力。5-10 μg增強型Sharkey AAVS1 鋅指核酸酶(ZFN)與10 μg供體DNA的共電穿孔誘導最小的細胞毒性。*表示與供體(10 μg) EP相比P >0.01。數據為平均值±SEM；n = 4。 Figure 17(e) shows the dose titration of AAVS1 zinc finger nuclease (ZFN) for cell proliferation. MTS assay of untreated live umbilical cord lined epithelial cells (CLEC WT) that received electroporation only (EP only) and received only 10 μg pZDonor donor DNA or pZDonor DNA and increased doses of zinc finger nucleic acid 1 day after electroporation Enzyme (ZFN). The MTS assay is a method for sensitively quantifying living cells in proliferation and cytotoxicity. This method is based on the reduction of MTS tetrazolium compounds by living cells to produce colored formazan products that are soluble in cell culture media. It is thought that this conversion is performed by NAD(P)H-dependent dehydrogenase in metabolically active cells. The formazan dye produced by living cells can be quantified by measuring the absorbance at 490-500 nm. Therefore, an MRX II 96-well plate reader (Dynex Corporation) was used to quantify the absorbance at 490 nm. Consistent with the experimental results described in Figures 10(a)-(d), most of the live cells were detected in the umbilical cord lined epithelial cells (CLECs) wild-type, EP-only, and body-only treated cells. And zinc finger nuclease (ZFN) electroporated cells showed less proliferation/viability. Co-electroporation of 5-10 μg enhanced Sharkey AAVS1 zinc finger nuclease (ZFN) with 10 μg donor DNA induced minimal cytotoxicity. * Indicates P>0.01 compared to donor (10 μg) EP. Data are mean ± SEM; n = 4.

圖 18(a)-(b) 所示為臍帶襯裡上皮細胞(CLECs)中1.3-kb供體DNA的AAVS1位點特異性整合。 Figures 18(a)-(b) show the site-specific integration of AAVS1 of 1.3-kb donor DNA in umbilical cord lining epithelial cells (CLECs).

圖 18(a) 所示為同源重組調節的pAAVS-SA-2A-puro-pA供體(Addgene質體編號22075)整合到AAVS1基因座中的示意圖(未按比例繪製)。用於整合連接PCR的引子對為Puro LF；Puro LR(左側連接點)；以及Puro RF2；AAVS1 R (右側連接點)。在整合位點之外錨定長PCR的引子為Puro LF以及AAVS1 R。 Fig. 18(a) is a schematic diagram of homologous recombination-regulated pAAVS-SA-2A-puro-pA donor (Addgene plastid number 22075) integrated into the AAVS1 locus (not drawn to scale). The primer pairs used for integrated ligation PCR are Puro LF; Puro LR (left junction); and Puro RF2; AAVS1 R (right junction). The primers for anchoring long PCR outside the integration site are Puro LF and AAVS1 R.

圖 18(b) 所示為供體DNA的精確整合。在200 ng基因組DNA (相當於30440個細胞)上進行的左右整合連接PCR (左JPCR以及右JPCR)以及長PCR (跨越整合轉殖基因)穩定整合了與pAAVS-SA-2A-共電穿孔的嘌呤黴素抗性臍帶襯裡上皮細胞(CLECs) puro-pA供體(1-kb嘌呤黴素抗性基因)以及AAVS1 鋅指核酸酶(ZFN)。預測大小的擴增子(左JPCR擴增子，1.1kb；右JPCR擴增子，1.6kb)為AAVS1基因座供體DNA整合的證據。對照PCR擴增距離整合位點2kb的AAVS1基因座的900 bp區域。以錨定超出整合位點的基因組特異性引子進行的長PCR在沒有供體DNA整合的情況下產生2kb擴增子，並且在供體整合細胞中產生顯性3-kb擴增子。對未修飾的野生型臍帶襯裡上皮細胞(CLECs)的對照PCR反應僅顯示2-kb擴增子。透過定序確認整合連接PCR以及長PCR產物的身份。白色垂直線劃分合併的凝膠圖像區域。 Figure 18(b) shows the precise integration of donor DNA. The left-right integration ligation PCR (left JPCR and right JPCR) and long PCR (cross-integration transfer gene) performed on 200 ng genomic DNA (equivalent to 30440 cells) stably integrated the co-electroporation with pAAVS-SA-2A- Puromycin resistant umbilical cord lining epithelial cells (CLECs) puro-pA donor (1-kb puromycin resistance gene) and AAVS1 zinc finger nuclease (ZFN). The predicted size amplicon (left JPCR amplicon, 1.1 kb; right JPCR amplicon, 1.6 kb) is evidence of donor DNA integration of the AAVS1 locus. Control PCR amplified the 900 bp region of the AAVS1 locus 2 kb from the integration site. Long PCR with genome-specific primers anchored beyond the integration site produced a 2 kb amplicon without donor DNA integration, and a dominant 3-kb amplicon in donor integrated cells. Control PCR reactions on unmodified wild-type umbilical cord lining epithelial cells (CLECs) showed only 2-kb amplicons. Confirm the identity of integrated PCR and long PCR products by sequencing. White vertical lines divide the merged gel image area.

圖 19(a)-(c) 所示為臍帶襯裡上皮細胞(CLECs)中4.2-kb供體DNA的AAVS1位點特異性整合。 Figures 19(a)-(c) show the site-specific integration of AAVS1 of 4.2-kb donor DNA in umbilical cord lining epithelial cells (CLECs).

圖 19(a) 所示為同源重組調節的pAAV-CAGGS-EGFP (Addgene質體編號22212)整合到AAVS1基因座中的示意圖(未按比例繪製)。對於整合連接PCR，一種引子對載體特異性(供體DNA)，另一種引子對整合位點以外的基因組DNA特異(左側連接點：AAVS1F，Puro LR；右側連接點：GFP RF，Puro RR)。使用對整合位點以外的基因組DNA特異的外部引子，長PCR擴增兩個重疊區段以覆蓋供體DNA的整個長度(長PCR左：AAVS1F，CAGGS R；長PCR右：GFP F，Puro RR)。 FIG. 19(a) is a schematic diagram of homologous recombination-regulated pAAV-CAGGS-EGFP (Addgene plastid number 22212) integrated into the AAVS1 locus (not drawn to scale). For integration ligation PCR, one primer is specific to the vector (donor DNA), and the other primer is specific to genomic DNA outside the integration site (left junction: AAVS1F, Puro LR; right junction: GFP RF, Puro RR). Using external primers specific to genomic DNA outside the integration site, long PCR amplifies two overlapping segments to cover the entire length of the donor DNA (long PCR left: AAVS1F, CAGGS R; long PCR right: GFP F, Puro RR ).

圖 19(b) 所示為供體DNA的精確整合。整合連接PCR(左JPCR與右JPCR)以及重疊長PCR(跨越整合轉殖基因)在200 ng基因組DNA (相當於30440個細胞)上進行，嘌呤黴素抗性臍帶襯裡上皮細胞(CLECs)與pAAV-CAGGS-EGFP共同電穿孔(4.2 kb供體) 由無啟動子的嘌呤黴素抗性基因cDNA以及CAGGS啟動子-EGFP cDNA組成的DNA以及編碼左及右AAVS1 鋅指核酸酶(ZFN)的質體構築顯示在AAVS1基因座處的供體DNA整合。左及右整合連接PCR的產物(分別為1 kb與1.3 kb)具有正確的預測大小。對照PCR擴增距離整合位點2 kb的AAVS1基因座的900 bp區域。重疊長PCR包括預測大小的整合轉殖基因擴增產物(長PCR左；2.3 kb，長PCR右；2.5 kb)。在未修飾的野生型臍帶襯裡上皮細胞(CLECs)上進行的平行PCR中沒有產生擴增子。 Figure 19(b) shows the precise integration of donor DNA. Integrative ligation PCR (left JPCR and right JPCR) and overlapping long PCR (crossing the integrated transgene) were performed on 200 ng genomic DNA (equivalent to 30440 cells), puromycin-resistant umbilical cord lining epithelial cells (CLECs) and pAAV -CAGGS-EGFP co-electroporation (4.2 kb donor) DNA consisting of the promoter-free puromycin resistance gene cDNA and the CAGGS promoter-EGFP cDNA and the quality encoding the left and right AAVS1 zinc finger nuclease (ZFN) Somatic construction shows donor DNA integration at the AAVS1 locus. The left and right integrated PCR products (1 kb and 1.3 kb, respectively) have the correct predicted size. Control PCR was used to amplify the 900 bp region of the AAVS1 locus 2 kb from the integration site. Overlapping long PCR includes the predicted size of integrated transgene amplification products (long PCR left; 2.3 kb, long PCR right; 2.5 kb). Amplicons were not generated in parallel PCR performed on unmodified wild-type umbilical cord lined epithelial cells (CLECs).

圖 19(c) 所示為在AAVS1基因座中整合的持久轉殖基因表現。在以pAAV-CAGGS-EGFP電穿孔後第1天(嘌呤黴素篩選前)以及第9、21以及33天(嘌呤黴素篩選後)的臍帶襯裡上皮細胞(CLECs)的明場及螢光圖像顯示AAVS1基因座支持供體DNA的持久EGFP表現。比例尺=100 μm。 Figure 19(c) shows the performance of the persistent transgene integrated in the AAVS1 locus. Brightfield and fluorescence images of umbilical cord lined epithelial cells (CLECs) on day 1 (before puromycin screening) and on days 9, 21, and 33 (after puromycin screening) after electroporation with pAAV-CAGGS-EGFP The image shows that the AAVS1 locus supports persistent EGFP performance of donor DNA. Scale bar=100 μm.

圖 20 所示為透過目標深度定序檢測***缺失的靈敏度。將商業合成的DNA片段(GenScript公司，Piscataway，紐澤西州)與AAVS1基因座序列相似，除了鋅指核酸酶(ZFN)結合半位點之間的5-bp缺失外，以1:10、1:100、1:500以及1:1,000的莫爾比摻入野生型AAVS1基因座擴增子中，確定***或缺失(indel)檢測的靈敏度。表及圖顯示了突變擴增子與野生型擴增子的比例與百分比，實驗檢索的定位讀數的數量，在這些定位讀數中針對每個加標濃度檢測到的***缺失的數量及百分比。在加標對照中已知百分比的***缺失與透過深度定序實驗確定的***缺失百分比之間觀察到高度線性相關(R2 = 0.999)。這些數據確定了0.1%的檢測靈敏度。 Figure 20 shows the sensitivity of detecting insertion deletion through target depth sequencing. Sequences of commercially synthesized DNA fragments (GenScript, Piscataway, New Jersey) are similar to the AAVS1 locus, except for the 5-bp deletion between the zinc finger nuclease (ZFN) binding half-sites, at 1:10, The molar ratios of 1:100, 1:500, and 1:1,000 were incorporated into the wild-type AAVS1 locus amplicon to determine the sensitivity of indel detection. The table and graph show the ratio and percentage of mutant amplicons to wild-type amplicons, the number of positioning reads retrieved from the experiment, and the number and percentage of indels detected for each spiked concentration in these positioning reads. A high linear correlation was observed between the known percentage of indels in the spiked control and the percentage of indels determined by depth sequencing experiments (R2 = 0.999). These data determine the detection sensitivity of 0.1%.

圖 21 所示為puro-CLEC中90種失調的轉錄物的途徑分析。與野生型臍帶襯裡上皮細胞(CLECs)相比，在puro-CLEC中過度表現(n = 57)或表現不足(n = 33)由2倍的基因的DAVID分析僅映射到趨化因子-趨化因子受體相互作用。通路分析顯示10個失調的轉錄物透過DAVID分析(Benjamini校正的P = 0.011)定位於細胞因子-細胞因子受體相互作用，與PPP1R12C在發炎反應中的作用一致。雖然7個失調的基因為超過1,600個致癌基因的綜合目錄中潛在的原癌基因(http://www.bushmanlab.org/links/genelists)，但沒有一個定位於KEGG的任何經典癌症途徑。 Figure 21 shows the pathway analysis of 90 deregulated transcripts in puro-CLEC. Compared with wild-type umbilical cord lining epithelial cells (CLECs), over-expression (n = 57) or under-expression (n = 33) in puro-CLEC was mapped to chemokine-chemokine by 2-fold gene DAVID analysis Factor receptor interaction. Pathway analysis showed that 10 dysregulated transcripts were localized to cytokine-cytokine receptor interaction through DAVID analysis (Benjamini corrected P = 0.011), consistent with the role of PPP1R12C in the inflammatory response. Although the 7 dysregulated genes are potential proto-oncogenes in the comprehensive catalog of more than 1,600 oncogenes (http://www.bushmanlab.org/links/genelists), none of them is located in any classic cancer pathway of KEGG.

圖 22 所示為野生型-CLEC與puro-CLEC細胞增殖的比較。在野生型-CLEC後7天進行MTS測定，其檢測細胞的增殖，並且以每孔100個細胞的初始密度(96孔板)種植puro-CLEC。puro-CLEC的增殖略有但沒有顯著降低，可能反映了高DUSP6表現以及PPP1R12C單倍體缺乏的綜合影響，後者為完成有絲***及胞質***所必需的。數據為平均吸光度讀數±SD；每組n = 6。 P = 0.303。 Figure 22 shows a comparison of the proliferation of wild-type CLEC and puro-CLEC cells. An MTS assay was performed 7 days after wild-type CLEC, which detected the proliferation of cells, and puro-CLEC was planted at an initial density of 100 cells per well (96-well plate). The proliferation of puro-CLEC is slightly but not significantly reduced, which may reflect the combined effect of high DUSP6 performance and the lack of PPP1R12C haploid, which is necessary for the completion of mitosis and cytokinesis. The data is the mean absorbance reading ± SD; n = 6 per group. P = 0.303.

圖 23 所示為不同成體原代人類細胞中的位點特異性基因組修飾。(上 ) 成人原代人類細胞的轉染。以pmaxGFP (Lonza公司)在電穿孔後1天的原代人類細胞的明場及螢光圖像。明場圖片顯示細胞為存活的。下面的螢光圖片顯示GFP陽性細胞。因此，在所有測試的成年原代人類細胞中轉染是成功的。NF123及KF1為真皮纖維母細胞；BMSC1與BMSC2為骨髓來源的基質細胞；ADSC1及ADSC2為脂肪組織衍生的基質細胞。比例尺=100 μm。 (下 ) 成人原代人類細胞中的位點特異性AAVS1 鋅指核酸酶(ZFN)活性。跨越AAVS1整合位點的區域從以pZDonor電穿孔的BMSC1、BMSC2、ADSC1、ADSC2、NF123以及KF1萃取的基因組DNA中擴增。透過10%聚丙烯醯胺凝膠電泳解析CEL-1核酸酶消化的(+)或未消化的(-)PCR擴增子。CEL-1測定報告透過非同源末端連接進行DNA斷裂修復，從而間接檢測鋅指核酸酶(ZFN)活性。陽性整合對照是來自CompoZr®目標整合套組(Sigma-Aldrich公司)中提供的來自基因組DNA的PCR擴增子。顯示透過GFP陽性細胞的流式細胞儀分析確定的轉染效率以及透過切割及未切割的擴增子的光密度測定法確定的基因組修飾效率。還顯示標準化至100%轉染效率的基因組修飾百分比。在原代人類真皮纖維母細胞，人類骨髓以及脂肪組織衍生的基質細胞中容易誘導鋅指核酸酶(ZFN)調節的AAVS1基因座切割，總效率為20%或更高。數據為平均值±SD；每組n = 3。 Figure 23 shows site-specific genomic modifications in different adult primary human cells. ( Top ) Transfection of adult primary human cells. Brightfield and fluorescent images of primary human cells 1 day after electroporation with pmaxGFP (Lonza). Brightfield pictures show that the cells are alive. The fluorescent picture below shows GFP positive cells. Therefore, transfection in all tested adult primary human cells was successful. NF123 and KF1 are dermal fibroblasts; BMSC1 and BMSC2 are stromal cells derived from bone marrow; ADSC1 and ADSC2 are stromal cells derived from adipose tissue. Scale bar=100 μm. ( Bottom ) Site-specific AAVS1 zinc finger nuclease (ZFN) activity in adult primary human cells. The region spanning the AAVS1 integration site was amplified from genomic DNA extracted from BMSC1, BMSC2, ADSC1, ADSC2, NF123, and KF1 electroporated with pZDonor. The CEL-1 nuclease digested (+) or undigested (-) PCR amplicons were resolved by 10% polyacrylamide gel electrophoresis. The CEL-1 assay reports DNA break repair through non-homologous end ligation, thereby indirectly detecting zinc finger nuclease (ZFN) activity. The positive integration control is a PCR amplicon from genomic DNA provided in the CompoZr® target integration kit (Sigma-Aldrich). Shows transfection efficiency determined by flow cytometry analysis of GFP positive cells and genome modification efficiency determined by densitometry of cleaved and uncleaved amplicons. The percentage of genomic modifications normalized to 100% transfection efficiency is also shown. In primary human dermal fibroblasts, human bone marrow and adipose tissue-derived stromal cells, it is easy to induce cleavage of the AAVS1 locus regulated by zinc finger nuclease (ZFN), with a total efficiency of 20% or higher. Data are mean ± SD; n = 3 for each group.

圖 24 所示為不同原代人類細胞類型的FVIII轉殖基因分泌。 原代人類骨髓來源的基質細胞(BMSC1與BMSC2)，人類脂肪組織來源的基質細胞(ADSC2)以及臍帶襯裡上皮細胞(CLECs)與pmaxGFP (Lonza公司)以及質體共同電穿孔後分泌高含量的雜合FVIII 編碼由CMV啟動子表現的雜合FVIII cDNA的DNA。野生型表示未轉染的對照細胞。圖表顯示透過流式細胞儀測定的GFP陽性細胞(菱形；右軸)的百分比以及使用Coamatic®因子VIII套組測定(Chromogenix公司)測定的整夜條件培養基中的FVIII活性(條；左軸)。使用在臍帶襯裡上皮細胞(CLECs)中整合FVIII轉殖基因的相同條件，還可以誘導原代人類真皮纖維母細胞、骨髓以及脂肪組織衍生的基質細胞整合及分泌轉殖基因FVIII。數據為平均值±SEM；n = 3。 Figure 24 shows the FVIII transgene secretion of different primary human cell types. Primary human bone marrow-derived stromal cells (BMSC1 and BMSC2), human adipose tissue-derived stromal cells (ADSC2), umbilical cord lining epithelial cells (CLECs) and pmaxGFP (Lonza) and plastids secrete high levels of impurities after electroporation FVIII DNA encoding the hybrid FVIII cDNA expressed by the CMV promoter. Wild type means untransfected control cells. The graph shows the percentage of GFP-positive cells (diamonds; right axis) measured by flow cytometry and FVIII activity in the overnight conditioned medium (bar; left axis) measured using the Coamatic® Factor VIII kit assay (Chromogenix). Using the same conditions for integrating FVIII transgenes in umbilical cord lining epithelial cells (CLECs), primary human dermal fibroblasts, bone marrow, and adipose tissue-derived stromal cells can also be induced to integrate and secrete the transgene FVIII. Data are mean ± SEM; n = 3.

圖 25 所示為雜合的人-豬B結構域截短的FVIII cDNA以及組裝步驟。因子VIII蛋白由六個結構域組成：A1-A2-B-A3-C1-C2。A結構域與銅結合蛋白銅藍蛋白的A結構域同源。C結構域屬於磷脂結合的盤狀結構域家族，C2結構域調節膜結合。因子VIII對因子VIIIa的活化透過B結構域的切割及釋放來完成。上圖 ：B結構域截短的人-豬FVIII cDNA的示意圖，其由豬A1與A3結構域、人類訊息胜肽、A2、殘基B (包含前247個胺基酸以及6個糖基化位點)，C1及C2結構域所組成。底部 ：結構域組裝中使用的重疊PCR步驟。透過基於參考豬cDNA序列(NM_214167.1)的總豬肝RNA的RT-PCR獲得A1與A3結構域。在pSP64-VIII (美國典型培養物保藏中心)中從完整的人類FVIII cDNA擴增人類結構域。 Figure 25 shows the hybridized human-porcine B domain truncated FVIII cDNA and assembly steps. Factor VIII protein consists of six domains: A1-A2-B-A3-C1-C2. The A domain is homologous to the A domain of the copper-binding protein ceruloplasmin. The C domain belongs to the family of disc-shaped domains bound by phospholipids, and the C2 domain regulates membrane binding. Factor VIII activates factor VIIIa through cleavage and release of the B domain. Figure: B domain truncated human - a schematic view of porcine FVIII cDNA which (amino acids 247 comprises front and six glycosylation of porcine A1 and A3 domain, the human peptide message, A2, B residues Site), composed of C1 and C2 domains. Bottom : overlapping PCR steps used in domain assembly. The A1 and A3 domains were obtained by RT-PCR of total pig liver RNA based on the reference pig cDNA sequence (NM_214167.1). The human domain was amplified from the complete human FVIII cDNA in pSP64-VIII (American Type Culture Collection).

圖 26 所示為電腦預測的10個最可能的AAVS1鋅指核酸酶(ZFN)偏離目標位點的目標深度定序。透過來自野生型-CLEC與Puro-CLEC的基因組DNA的擴增子的目標深度定序來評估前10個預測的AAVS1 鋅指核酸酶(ZFN)(OT1-OT10)的偏離目標位點。對OT1-OT10的染色體位點、相應的染色體位置、總讀數圖譜、實驗檢測到的***或缺失(indel)類型，以及其相應的百分比進行了總結。僅在Puro-CLEC中出現的***或缺失(indel)以斜體顯示。所有基因組坐標均指hg19。預測的十個最可能的偏離目標位點(OT1-OT10)的目標深度定序顯示僅在OT1 (8q24.3基因座內)的低頻率(分別為1.36與1.37%)處的4 bp與1 bp缺失。沒有檢測到特異於puro-CLEC的OT2-OT10中的其他***缺失。 Figure 26 shows the computer-predicted target depth sequence for the 10 most likely AAVS1 zinc finger nucleases (ZFN) to deviate from the target site. The top 10 predicted AAVS1 zinc finger nucleases (ZFN) (OT1-OT10) deviated from the target site were evaluated by target depth sequencing of amplicons from genomic DNA of wild-type CLEC and Puro-CLEC. The OT1-OT10 chromosomal loci, the corresponding chromosomal location, the total reading map, the type of indels (indel) detected by the experiment, and their corresponding percentages are summarized. Indels that appear only in Puro-CLEC are shown in italics. All genomic coordinates refer to hg19. The predicted target depth sequencing of the ten most likely off-target sites (OT1-OT10) shows 4 bp and 1 at low frequencies (1.36 and 1.37%, respectively) only at OT1 (within the 8q24.3 locus) bp is missing. No other indels in OT2-OT10 specific for puro-CLEC were detected.

無no

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0015

Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0016

Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0017

Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0018

Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0019

Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0020

Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0021

Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0022

Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0023

Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0024

Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0025

Figure 12_A0101_SEQ_0026
Figure 12_A0101_SEQ_0026

Figure 12_A0101_SEQ_0027
Figure 12_A0101_SEQ_0027

Figure 12_A0101_SEQ_0028
Figure 12_A0101_SEQ_0028

Figure 12_A0101_SEQ_0029
Figure 12_A0101_SEQ_0029

Figure 12_A0101_SEQ_0030
Figure 12_A0101_SEQ_0030

Figure 12_A0101_SEQ_0031
Figure 12_A0101_SEQ_0031

Figure 12_A0101_SEQ_0032
Figure 12_A0101_SEQ_0032

Figure 12_A0101_SEQ_0033
Figure 12_A0101_SEQ_0033

Figure 12_A0101_SEQ_0034
Figure 12_A0101_SEQ_0034

Figure 12_A0101_SEQ_0035
Figure 12_A0101_SEQ_0035

Figure 12_A0101_SEQ_0036
Figure 12_A0101_SEQ_0036

Figure 12_A0101_SEQ_0037
Figure 12_A0101_SEQ_0037

Figure 12_A0101_SEQ_0038
Figure 12_A0101_SEQ_0038

Figure 12_A0101_SEQ_0039
Figure 12_A0101_SEQ_0039

Figure 12_A0101_SEQ_0040
Figure 12_A0101_SEQ_0040

Figure 12_A0101_SEQ_0041
Figure 12_A0101_SEQ_0041

Figure 12_A0101_SEQ_0042
Figure 12_A0101_SEQ_0042

Figure 12_A0101_SEQ_0043
Figure 12_A0101_SEQ_0043

Figure 12_A0101_SEQ_0044
Figure 12_A0101_SEQ_0044

Figure 12_A0101_SEQ_0045
Figure 12_A0101_SEQ_0045

Figure 12_A0101_SEQ_0046
Figure 12_A0101_SEQ_0046

Figure 12_A0101_SEQ_0047
Figure 12_A0101_SEQ_0047

Figure 12_A0101_SEQ_0048
Figure 12_A0101_SEQ_0048

Figure 12_A0101_SEQ_0049
Figure 12_A0101_SEQ_0049

Figure 12_A0101_SEQ_0050
Figure 12_A0101_SEQ_0050

Figure 12_A0101_SEQ_0051
Figure 12_A0101_SEQ_0051

Figure 12_A0101_SEQ_0052
Figure 12_A0101_SEQ_0052

Figure 12_A0101_SEQ_0053
Figure 12_A0101_SEQ_0053

Figure 12_A0101_SEQ_0054
Figure 12_A0101_SEQ_0054

Figure 12_A0101_SEQ_0055
Figure 12_A0101_SEQ_0055

Figure 12_A0101_SEQ_0056
Figure 12_A0101_SEQ_0056

Figure 12_A0101_SEQ_0057
Figure 12_A0101_SEQ_0057

Figure 12_A0101_SEQ_0058
Figure 12_A0101_SEQ_0058

Figure 12_A0101_SEQ_0059
Figure 12_A0101_SEQ_0059

Figure 12_A0101_SEQ_0060
Figure 12_A0101_SEQ_0060

Figure 12_A0101_SEQ_0061
Figure 12_A0101_SEQ_0061

Figure 12_A0101_SEQ_0062
Figure 12_A0101_SEQ_0062

Figure 12_A0101_SEQ_0063
Figure 12_A0101_SEQ_0063

Figure 12_A0101_SEQ_0064
Figure 12_A0101_SEQ_0064

Figure 12_A0101_SEQ_0065
Figure 12_A0101_SEQ_0065

Figure 12_A0101_SEQ_0066
Figure 12_A0101_SEQ_0066

Figure 12_A0101_SEQ_0067
Figure 12_A0101_SEQ_0067

Figure 12_A0101_SEQ_0068
Figure 12_A0101_SEQ_0068

Figure 12_A0101_SEQ_0069
Figure 12_A0101_SEQ_0069

Figure 12_A0101_SEQ_0070
Figure 12_A0101_SEQ_0070

Figure 12_A0101_SEQ_0071
Figure 12_A0101_SEQ_0071

Figure 12_A0101_SEQ_0072
Figure 12_A0101_SEQ_0072

Figure 12_A0101_SEQ_0073
Figure 12_A0101_SEQ_0073

Figure 12_A0101_SEQ_0074
Figure 12_A0101_SEQ_0074

Figure 12_A0101_SEQ_0075
Figure 12_A0101_SEQ_0075

Figure 12_A0101_SEQ_0076
Figure 12_A0101_SEQ_0076

Figure 12_A0101_SEQ_0077
Figure 12_A0101_SEQ_0077

Figure 12_A0101_SEQ_0078
Figure 12_A0101_SEQ_0078

Figure 12_A0101_SEQ_0079
Figure 12_A0101_SEQ_0079

Figure 12_A0101_SEQ_0080
Figure 12_A0101_SEQ_0080

Figure 12_A0101_SEQ_0081
Figure 12_A0101_SEQ_0081

Figure 12_A0101_SEQ_0082
Figure 12_A0101_SEQ_0082

Figure 12_A0101_SEQ_0083
Figure 12_A0101_SEQ_0083

Figure 12_A0101_SEQ_0084
Figure 12_A0101_SEQ_0084

Claims (36)

一種產生轉殖基因間質幹細胞群之方法，該方法包括透過酶調節的整合將轉殖基因***該間質幹細胞群的基因組中，其中該間質幹細胞群為臍帶羊膜的分離的間質幹細胞群，其中至少約90%或更多的間質幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105。A method of generating a population of transgenic mesenchymal stem cells, the method comprising inserting a transgenic gene into the genome of the mesenchymal stem cell population through enzyme-mediated integration, wherein the mesenchymal stem cell population is an isolated mesenchymal stem cell population of umbilical cord amniotic membrane , Wherein at least about 90% or more of the cells of the mesenchymal stem cell population exhibit each of the following markers: CD73, CD90, and CD105. 如請求項1之方法，其中該酶調節的整合將該轉殖基因***該間質幹細胞群的該基因組中是透過酵素而進行，該酵素係選自由重組酶、類轉錄活化劑效應子核酸酶以及核酸酶所組成之群組。The method of claim 1, wherein the enzyme-regulated integration inserts the transferred gene into the genome of the mesenchymal stem cell population through an enzyme selected from the group consisting of a recombinase, a transcription activator-like effector nuclease And a group of nucleases. 如請求項2之方法，其中該重組酶選自由酪胺酸重組酶(tyrosine recombinase，YR)以及絲胺酸重組酶(serine recombinase，SR)及其混合物所組成之群組。The method of claim 2, wherein the recombinase is selected from the group consisting of tyrosine recombinase (YR) and serine recombinase (SR) and mixtures thereof. 如請求項3之方法，其中該酪胺酸重組酶選自由Cre (Cre重組酶調節的整合)、Dre (Dre重組酶調節的整合)，以及Flp所組成之群組或其中該絲胺酸重組酶選自由重組酶phiC31 (phiC31整合酶調節的整合)、Bxb1，以及R4重組酶調節的整合所組成之群組。The method of claim 3, wherein the tyrosine recombinase is selected from the group consisting of Cre (Cre recombinase-regulated integration), Dre (Dre recombinase-regulated integration), and Flp or wherein the serine recombination The enzyme is selected from the group consisting of recombinase phiC31 (phiC31 integrase-mediated integration), Bxb1, and R4 recombinase-mediated integration. 如請求項2之方法，其中該核酸酶選自由鋅指核酸酶(zinc finger nuclease，ZFN)(鋅指核酸酶調節的整合)或Cas9 (CRISPR/Cas9調節的整合)所組成之群組。The method of claim 2, wherein the nuclease is selected from the group consisting of zinc finger nuclease (ZFN) (zinc finger nuclease-mediated integration) or Cas9 (CRISPR/Cas9-mediated integration). 如請求項1至5中任一項之方法，其中至少約90%或更多的分離的間質幹細胞群的細胞缺乏下列標記之表現：CD34、CD45以及HLA-DR。The method of any one of claims 1 to 5, wherein at least about 90% or more of the cells of the isolated mesenchymal stem cell population lack the performance of the following markers: CD34, CD45, and HLA-DR. 如請求項1至6中任一項之方法，其中至少約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多該分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，並且缺乏CD34、CD45以及HLA-DR各自的表現。The method of any one of claims 1 to 6, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, About 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population exhibit each of CD73, CD90, and CD105, and lack CD34 , CD45 and HLA-DR performance. 如請求項1至7中任一項之方法，其中該間質幹細胞群選自由人類幹細胞群、犬幹細胞群、貓幹細胞群、鼠幹細胞群、大鼠幹細胞群、豬幹細胞群、馬幹細胞群、猿幹細胞群，或獼猴幹細胞群所組成之群組。The method according to any one of claims 1 to 7, wherein the mesenchymal stem cell population is selected from the group consisting of human stem cell population, canine stem cell population, cat stem cell population, mouse stem cell population, rat stem cell population, porcine stem cell population, horse stem cell population, Ape stem cell population, or a group of macaque stem cell populations. 如請求項1至8中任一項之方法，其中該轉殖基因整合到該人類間質幹細胞群的AAVS1基因座(存在於人類染色體19 q13.3-qter上)或基因座8p22 (存在於人類染色體8上)的DLC1的內含子7中。The method of any one of claims 1 to 8, wherein the transgene is integrated into the AAVS1 locus of the human mesenchymal stem cell population (present on human chromosome 19 q13.3-qter) or locus 8p22 (present on Intron 7 of DLC1 on human chromosome 8). 如請求項5至9中任一項之方法，其中該方法包括透過突變的鋅指核酸酶或透過Cas9 (CRISPR/Cas9調節的整合)***該轉殖基因。The method of any one of claims 5 to 9, wherein the method comprises inserting the transgenic gene through a mutated zinc finger nuclease or through Cas9 (CRISPR/Cas9 regulated integration). 如前述請求項中任一項之方法，其中該轉殖基因選自由編碼凝血因子的核酸分子(基因)以及編碼內分泌腺分泌的蛋白激素的核酸分子(基因)所組成之群組。The method according to any one of the preceding claims, wherein the transgene is selected from the group consisting of nucleic acid molecules (genes) encoding coagulation factors and nucleic acid molecules (genes) encoding protein hormones secreted by endocrine glands. 如請求項11之方法，其中該凝血因子選自由因子VII、因子VIII，以及因子IX所組成之群組。The method of claim 11, wherein the coagulation factor is selected from the group consisting of factor VII, factor VIII, and factor IX. 如請求項12之方法，其中該由內分泌腺分泌的蛋白激素的表現或分泌之缺乏與內分泌缺乏有關。The method of claim 12, wherein the performance or lack of secretion of the protein hormone secreted by the endocrine glands is related to endocrine deficiency. 如請求項13之方法，其中該蛋白激素的缺乏與內分泌缺乏有關，該內分泌缺乏選自由胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常所組成之群組。The method of claim 13, wherein the protein hormone deficiency is associated with endocrine deficiency selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency , And a group of thyroid abnormalities. 如請求項12或13之方法，其中該轉殖基因為編碼嵌合因子VIII多胜肽的核酸分子。The method according to claim 12 or 13, wherein the transgene is a nucleic acid molecule encoding a chimeric factor VIII polypeptide. 如請求項15之方法，其中該轉殖基因編碼包含人類及豬結構域的嵌合因子VIII多胜肽。The method of claim 15, wherein the transgene encodes a chimeric factor VIII polypeptide comprising human and porcine domains. 如請求項16之方法，其中該轉殖基因編碼包含豬A1與A3結構域、人類訊息胜肽、人類A2結構域、殘留的人類B結構域，以及人類C1及C2結構域或由其組成的嵌合因子VIII多胜肽，及/或其中該轉殖基因包含如SEQ ID NO. 3所示的序列。The method according to claim 16, wherein the transgenic gene code comprises or consists of porcine A1 and A3 domains, human message peptides, human A2 domains, residual human B domains, and human C1 and C2 domains The chimeric factor VIII polypeptide, and/or wherein the transgene contains the sequence shown in SEQ ID NO.3. 一種轉殖基因間質幹細胞群，其攜帶透過如請求項1至17中任一項所述之方法而可獲得或獲得之轉殖基因。A population of transgenic mesenchymal stem cells carrying a transgenic gene obtainable or obtained by the method according to any one of claims 1 to 17. 如請求項18之轉殖基因間質幹細胞群，其中該間質幹細胞群選自由人類幹細胞群、犬幹細胞群、貓幹細胞群、馬幹細胞群、鼠幹細胞群、鼠幹細胞群、豬幹細胞群、猿幹細胞群，或獼猴幹細胞群所組成之群組。The transgenic gene mesenchymal stem cell population according to claim 18, wherein the mesenchymal stem cell population is selected from the group consisting of human stem cell population, canine stem cell population, cat stem cell population, horse stem cell population, murine stem cell population, murine stem cell population, porcine stem cell population, ape Stem cell populations, or groups of macaque stem cell populations. 如請求項18或19之轉殖基因間質幹細胞群，其中該轉殖基因被整合到該人類間質幹細胞群的AAVS1基因座(存在於人類染色體19 q13.3-qter上)或整合到基因座8p22 (存在於人類染色體8上)的DLC1的內含子7中。Transgenic gene mesenchymal stem cell population as claimed in claim 18 or 19, wherein the transgenic gene is integrated into the AAVS1 locus of the human mesenchymal stem cell population (present on human chromosome 19 q13.3-qter) or integrated into the gene Intron 7 of DLC1 at locus 8p22 (present on human chromosome 8). 如請求項18至20中任一項之轉殖基因間質幹細胞群，其中至少約81%或更多的該轉殖基因間質幹細胞群的細胞表現CD73、CD90以及CD105的每一種，而缺乏以下各種標記的表現：CD34、CD45以及HLA-DR。The transgenic mesenchymal stem cell population according to any one of claims 18 to 20, wherein at least about 81% or more of the cells of the transgenic mesenchymal stem cell population exhibit each of CD73, CD90, and CD105, and lack The performance of the following various markers: CD34, CD45 and HLA-DR. 如請求項21之轉殖基因間質幹細胞群，其中至少約81%或更多，約82%或更多，至少83%或更多，至少84%或更多，至少約85%，或約86%或更多，約87%或更多，約 88%或更多，約89%或更多，約90%或更多，約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多的分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，並且缺乏CD34、CD45以及HLA-DR各自的表現。The transgenic mesenchymal stem cell population of claim 21, wherein at least about 81% or more, about 82% or more, at least 83% or more, at least 84% or more, at least about 85%, or about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93 % Or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more The cells of the PMSCs express each of CD73, CD90, and CD105, and lack the respective performance of CD34, CD45, and HLA-DR. 如請求項18至22中任一項之轉殖基因間質幹細胞群，其中該轉殖基因為編碼嵌合因子VIII多胜肽的基因。The transgenic mesenchymal stem cell population according to any one of claims 18 to 22, wherein the transgenic gene is a gene encoding a chimeric factor VIII polypeptide. 如請求項18至23中任一項所定義之轉殖基因間質幹細胞群用於製備治療疾病的醫藥組合物之用途。Use of the transgenic mesenchymal stem cell population as defined in any one of claims 18 to 23 for the preparation of a pharmaceutical composition for treating diseases. 如請求項24之用途，其中該疾病為與基因缺陷或該基因表現缺陷相關的疾病，其中該基因選自由編碼凝血因子的基因以及編碼由內分泌腺分泌的蛋白激素的基因所組成之群組。The use according to claim 24, wherein the disease is a disease related to a gene defect or a defect in the gene expression, wherein the gene is selected from the group consisting of a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland. 如請求項25之用途，其中該凝血因子選自因子VII、因子VIII，以及因子IX。The use according to claim 25, wherein the coagulation factor is selected from factor VII, factor VIII, and factor IX. 如請求項24至26中任一項之用途，其中該疾病為血友病。The use according to any one of claims 24 to 26, wherein the disease is hemophilia. 如請求項27之用途，其中該血友病選自由A型血友病、B型血友病，以及C型血友病所組成之群組。The use according to claim 27, wherein the hemophilia is selected from the group consisting of hemophilia A, hemophilia B, and hemophilia C. 如請求項24之用途，其中該疾病與內分泌缺乏有關。The use according to claim 24, wherein the disease is related to endocrine deficiency. 如請求項29之用途，其中該疾病與由內分泌腺分泌的該蛋白激素之表現或分泌缺乏有關。The use according to claim 29, wherein the disease is related to a lack of expression or secretion of the protein hormone secreted by the endocrine glands. 如請求項30之用途，其中該蛋白激素的缺乏與內分泌缺乏有關，該內分泌缺乏選自由胰島素缺乏、與胰島素缺乏相關的糖尿病、***缺乏、貧血、低血糖、高血糖、胰腺缺乏、腎上腺缺乏，以及甲狀腺異常所組成之群組。The use according to claim 30, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from insulin deficiency, diabetes associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency , And a group of thyroid abnormalities. 如請求項24至31中任一項之用途，其中該轉殖基因間質幹細胞群透過植入或注射給藥。The use according to any one of claims 24 to 31, wherein the population of transgenic mesenchymal stem cells is administered by implantation or injection. 如請求項32之用途，其中該轉殖基因間質幹細胞群將以皮下植入。 第32項 一種含有如請求項18至23中任一項所定義之轉殖基因間質幹細胞群之醫藥組合物。 第33項 一種如請求項18至23中任一項所定義之轉殖基因間質幹細胞群用於製備用於基因療法治療的醫藥組合物之用途。The use according to claim 32, wherein the colony of mesenchymal stem cells of the transferred gene will be implanted subcutaneously. Item 32 A pharmaceutical composition containing a population of transgenic mesenchymal stem cells as defined in any one of claims 18 to 23. Item 33: Use of a transgenic mesenchymal stem cell population as defined in any one of claims 18 to 23 for the preparation of a pharmaceutical composition for gene therapy treatment. 臍帶羊膜的間質幹細胞群的活體外用途，其中至少約90%或更多的該間質幹細胞群的細胞表現以下標記中的每一種：CD73、CD90以及CD105，以用於產生如請求項18至23中任一項所定義之轉殖基因間質幹細胞群。In vitro use of the mesenchymal stem cell population of the umbilical cord amniotic membrane, wherein at least about 90% or more of the cells of the mesenchymal stem cell population exhibit each of the following markers: CD73, CD90, and CD105 for the production of claim 18 Transgenic mesenchymal stem cell population as defined in any of 23 to 23. 如請求項34之用途，其中至少約90%或更多的間質幹細胞群的細胞缺乏以下標記之表現：CD34、CD45以及HLA-DR。For the use of claim 34, at least about 90% or more of the cells of the mesenchymal stem cell population lack the performance of the following markers: CD34, CD45, and HLA-DR. 如請求項34及35中任一項之用途，其中至少約91%或更多，約92%或更多，約93%或更多，約94%或更多，約95%或更多，約96%或更多，約97%或更多，約98%或更多，約99%或更多該分離的間質幹細胞群的細胞表現CD73、CD90以及CD105中的每一種，並且缺乏CD34、CD45以及HLA-DR各自的表現。The use according to any one of claims 34 and 35, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, About 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population exhibit each of CD73, CD90, and CD105, and lack CD34 , CD45 and HLA-DR performance.
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