TWI758316B - Regulation of gene expression using engineered nucleases - Google Patents

Regulation of gene expression using engineered nucleases Download PDF

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TWI758316B
TWI758316B TW106129809A TW106129809A TWI758316B TW I758316 B TWI758316 B TW I758316B TW 106129809 A TW106129809 A TW 106129809A TW 106129809 A TW106129809 A TW 106129809A TW I758316 B TWI758316 B TW I758316B
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TW201825671A (en
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傑佛瑞 C. 米勒
愛德華 J. 里巴
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美商聖加莫治療股份有限公司
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The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.

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使用經工程改造之核酸酶調控基因表現Regulation of gene expression using engineered nucleases

本申請案主張2017年1月9日申請之美國臨時申請案第62/443,981號及2017年8月15日申請之美國臨時申請案第62/545,778號之權益，該等臨時申請案之揭示內容據此以全文引用之方式併入本文中。This application claims the benefit of U.S. Provisional Application No. 62/443,981, filed on January 9, 2017, and U.S. Provisional Application No. 62/545,778, filed on August 15, 2017, the disclosures of those provisional applications It is hereby incorporated by reference in its entirety.

本發明屬於基因組工程改造，尤其是對造血細胞之基因組進行靶向修飾之領域。The present invention belongs to the field of genome engineering, especially the targeted modification of the genome of hematopoietic cells.

當考慮到基因組定序工作已揭示人類基因組含有20,000至25,000個基因，但存在少於2000個轉錄調控基因時，顯而易見，許多因子必須相互作用以控制基因表現之所有不同的時間體現、發育體現及組織特異性體現。基因表現受與DNA元件相互作用之通用及特異性轉錄調節基因的非常複雜的混合物控制。此等DNA元件包括局部DNA元件，諸如核心啟動子及其相關轉錄因子結合位點，以及遠端元件，諸如增強子、緘默子、絕緣子及基因座控制區(LCR) (參見Matson等人,(2006)Ann Rev Genome Hum Genet 7: 29-50)。When one considers that genome sequencing efforts have revealed that the human genome contains 20,000 to 25,000 genes, but less than 2000 transcriptionally regulated genes, it becomes apparent that many factors must interact to control all the different temporal, developmental, and Tissue-specific manifestations. Gene expression is controlled by a very complex mixture of general and specific transcriptional regulatory genes interacting with DNA elements. Such DNA elements include local DNA elements, such as the core promoter and its associated transcription factor binding sites, and distal elements, such as enhancers, mutators, insulators, and locus control regions (LCRs) (see Matson et al., ( 2006) Ann Rev Genome Hum Genet 7: 29-50).

首先在SV40病毒基因組中鑑定出增強子元件，接著在人類免疫球蛋白重鏈基因座中發現增強子元件。現已知增強子在許多基因之表現中起調控作用，看似主要影響基因表現之時間及空間模式。亦已發現增強子可用於在距所靶向基因之核心啟動子較大距離處調控表現，而且不依賴關於該啟動子之任何特定序列取向。增強子可位於核心啟動子區域上游或下游數百鹼基處，在此情況下，其可位於內含子序列中，或甚至超出基因之3'端。Enhancer elements were first identified in the SV40 viral genome, followed by the discovery of enhancer elements in the human immunoglobulin heavy chain locus. Enhancers are now known to play regulatory roles in the expression of many genes and appear to primarily influence the temporal and spatial patterns of gene expression. Enhancers have also been found to be useful for regulating expression at large distances from the core promoter of the targeted gene, independent of any particular sequence orientation with respect to the promoter. The enhancer may be located several hundred bases upstream or downstream of the core promoter region, in which case it may be located in an intronic sequence, or even beyond the 3' end of the gene.

已描述用於使基因組DNA靶向裂解之多種方法及組合物。此種靶向裂解事件可用於例如誘導靶向突變誘發、誘導細胞DNA序列之靶向缺失及促進預定染色體基因座處之靶向重組。參見例如美國專利第9,255,250號、第9,200,266號、第9,045,763號、第9,005,973號、第9,150,847號、第8,956,828號、第8,945,868號、第8,703,489號、第8,586,526號、第6,534,261號、第6,599,692號、第6,503,717號、第6,689,558號、第7,067,317號、第7,262,054號、第7,888,121號、第7,972,854號、第7,914,796號、第7,951,925號、第8,110,379號、第8,409,861號、美國專利公開案第2003/0232410號、第2005/0208489號、第2005/0026157號、第2005/0064474號、第2006/0063231號、第2008/0159996號、第2010/00218264號、第2012/0017290號、第2011/0265198號、第2013/0137104號、第2013/0122591號、第2013/0177983號、第2013/0196373號、第2015/0056705號及第2015/0335708號，其揭示內容以全文引用之方式併入。Various methods and compositions have been described for targeted cleavage of genomic DNA. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, to induce targeted deletion of cellular DNA sequences, and to facilitate targeted recombination at predetermined chromosomal loci. See, eg, US Pat. Nos. 9,255,250, 9,200,266, 9,045,763, 9,005,973, 9,150,847, 8,956,828, 8,945,868, 8,703,489, 8,586,526, 6,595,276, 6,595,276, 6,595,276 No. 6,689,558, No. 7,067,317, No. 7,262,054, No. 7,888,121, No. 7,972,854, No. 7,914,796, No. 7,951,925, No. 8,110,379, No. 8,409,861, US Patent Publication No. 2003/02324 /0208489, 2005/0026157, 2005/0064474, 2006/0063231, 2008/0159996, 2010/00218264, 2012/0017290, 2011/0265198, 2013/0137104 2013/0122591, 2013/0177983, 2013/0196373, 2015/0056705 and 2015/0335708, the disclosures of which are incorporated by reference in their entirety.

此等方法通常涉及使用經工程改造之裂解系統在靶DNA序列中誘導雙鏈斷裂(DSB)或鏈裂，使得藉由諸如非同源末端接合(NHEJ)、非同源性引導末端俘獲供體之誤差產生法修復該斷裂或使用修復模板進行修復(同源性引導修復或HDR)可能導致基因敲除或相關序列***(靶向整合)。參見例如美國專利第9,045,763號、第9,200,266號、第9,005,973號及第8,703,489號。此等技術亦可用於藉由使用供體寡核苷酸在基因組序列中引入位點特異性變化，包括引入基因組區域之特異性缺失或特定點突變或定位變化(亦稱為基因矯正)。裂解可藉由使用諸如經工程改造之鋅指核酸酶(ZFN)、轉錄活化因子樣效應因子核酸酶(TALEN)之特定核酸酶或使用具有經工程改造之crRNA/tracr RNA (『單嚮導RNA』)之CRISPR/Cas系統來指導特異性裂解而發生。此外，正在基於Argonaute系統(例如，得自於嗜熱棲熱菌(T. thermophilus )，稱為『TtAgo』，參見Swarts等人,(2014)Nature 507(7491): 258-261)開發靶向核酸酶，其亦可能具有用於基因組編輯及基因療法之潛力。These methods typically involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or strand breaks in the target DNA sequence such that the donor is captured by non-homologous leading ends such as non-homologous end joining (NHEJ), Repair of the break by error-generating methods or repair using a repair template (homology-guided repair or HDR) may result in gene knockout or insertion of related sequences (targeted integration). See, eg, US Patent Nos. 9,045,763, 9,200,266, 9,005,973, and 8,703,489. These techniques can also be used to introduce site-specific changes in genomic sequences through the use of donor oligonucleotides, including the introduction of specific deletions or specific point mutations in genomic regions or changes in location (also known as gene correction). Cleavage can be achieved by the use of specific nucleases such as engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or by using engineered crRNA/tracr RNA ("single guide RNA"). ) of the CRISPR/Cas system to direct specific cleavage. In addition, targeting is being developed based on the Argonaute system (eg, from T. thermophilus , known as "TtAgo", see Swarts et al., (2014) Nature 507(7491): 258-261 ) Nucleases, which may also have potential for genome editing and gene therapy.

在人類中，紅血細胞(RBC)或紅血球為血液之主要細胞組件並且佔細胞之四分之一。成熟RBC缺乏細胞核及許多其他細胞器並且充滿血紅素，亦即，用於將氧自肺攜帶至組織以及將二氧化碳自該組織中攜帶出來並且攜帶回至肺以便排出的金屬蛋白質。此蛋白質組成RBC乾重之大約97%，並且其使血液之氧攜帶能力增加約七十倍。血紅素為包含兩個α (alpha)樣球蛋白鏈及兩個β (beta)樣球蛋白鏈及4個原血紅素基團之異型四聚體。在成人中，α2β2四聚體被稱為血紅素A (HbA)或成年血紅素。典型地，以大約1:1比率合成α及β球蛋白鏈，且就血紅素及RBC穩定而言，此比率似乎非常重要。在正在發育之胎兒中，產生一種不同的血紅素形式，亦即胎兒血紅素(HbF)，其對氧之結合親和力高於血紅素A，從而可經由媽媽之血流將氧遞送至寶寶之系統。有兩種基因編碼胎兒球蛋白，其在序列上非常相似，並且基於其在β球蛋白基因座中之排列順序而稱為HBG1 (亦稱為Gγ)及HBG2 (Aγ)。如同成年血紅素，胎兒血紅素蛋白含有兩個α球蛋白鏈，但其具有兩個胎兒γ (gamma)球蛋白鏈而不是成年β球蛋白鏈(亦即，胎兒血紅素為α2γ2)。在妊娠大約30週時，胎兒中之γ球蛋白合成開始下降，同時β球蛋白之產生增加。截至大約10月齡時，新生兒之血紅素幾乎均為α2β2，但一些HbF持續進入成熟期(佔總血紅素之大約1-3%)。自產生γ球蛋白轉至產生β球蛋白之調控相當複雜，且主要涉及下調γ球蛋白轉錄，同時上調β球蛋白轉錄。In humans, red blood cells (RBCs) or erythrocytes are the major cellular component of blood and make up one quarter of the cells. Mature RBCs lack a nucleus and many other organelles and are filled with heme, the metalloproteins used to carry oxygen from the lung to tissue and carbon dioxide out of the tissue and back to the lung for expulsion. This protein constitutes approximately 97% of the dry weight of RBCs, and it increases the oxygen carrying capacity of the blood approximately seventy-fold. Heme is a heterotetramer comprising two alpha (alpha)-like globulin chains and two beta (beta)-like globulin chains and four pro-heme groups. In adults, the α2β2 tetramer is called heme A (HbA) or adult heme. Typically, alpha and beta globulin chains are synthesized in an approximately 1:1 ratio, and this ratio appears to be very important for heme and RBC stabilization. In the developing fetus, a different form of heme is produced, fetal heme (HbF), which has a higher binding affinity for oxygen than heme A, allowing oxygen to be delivered to the baby's system via the mother's bloodstream . There are two genes encoding fetal globulins that are very similar in sequence and are called HBG1 (also known as Gγ) and HBG2 (Aγ) based on their order in the beta globulin locus. Like adult heme, fetal heme protein contains two alpha globin chains, but it has two fetal gamma (gamma) globin chains instead of adult beta globin chains (ie, fetal heme is α2γ2). At approximately 30 weeks of gestation, gamma globulin synthesis in the fetus begins to decline, while beta globulin production increases. By about 10 months of age, neonatal hemoglobin is nearly all α2β2, but some HbF continues to mature (approximately 1-3% of total hemoglobin). The regulation of switching from gamma globulin production to beta globulin production is rather complex and primarily involves downregulation of gamma globulin transcription while upregulation of beta globulin transcription.

編碼血紅素鏈之序列中的基因缺陷可能負責一組稱為血紅素病之疾病，包括鐮形血球貧血症以及α型及β型地中海型貧血。在大多數血紅素病患者中，由於如以上所描述在分娩前後發生正常基因壓抑之故，編碼γ球蛋白之基因仍然存在，但表現相對較低。Defects in the gene encoding the heme chain may be responsible for a group of diseases called heme diseases, including sickle cell anemia and alpha and beta thalassemias. In most patients with heme disease, the gene encoding gamma globulin is still present but relatively underrepresented due to normal gene repression that occurs around delivery as described above.

據估計，在美國，每5000人中便有1人患有鐮形血球貧血症(SCD)，主要在具有撒哈拉以南非洲血統之人士中(Roseff (2009)Immunohematology 25(2):67)。鐮形血球突變之異型接合載體由於可防治瘧疾而似乎有一定益處，故可能隨時間而積極地選擇此特質，使得據估計，在撒哈拉以南非洲有高達28%之人口具有鐮形血球特質(Elguero等人,(2015)PNAS USA 112 (22): 7051)。鐮形血球貧血症係由β球蛋白基因中由於第6個胺基酸處纈胺酸取代麩胺酸(DNA層面上之GAG至GTG突變)引起之突變所致，其中所得血紅素被稱為「血紅素S」或「HbS」。在低氧條件下，HbS之去氧形式中之構形變化使該蛋白質上之疏水性區塊暴露在E螺旋與F螺旋之間。血紅素中β鏈6位上之纈胺酸之疏水性殘基能夠與該疏水性區塊締合，從而引起HbS分子聚集並且形成纖維性沈澱物。此等聚集物又造成RBC之異常或『鐮形化』，從而喪失細胞可撓性。在鐮形血球患者中，鐮形化RBC不再能夠擠入微血管床中，而且可能導致血管阻塞危機。另外，鐮形化RBC比正常RBC更脆弱並且傾向於溶血，最終導致患者貧血。It is estimated that 1 in 5000 people in the United States suffer from sickle cell anemia (SCD), mainly in those of sub-Saharan African ancestry (Roseff (2009) Immunohematology 25(2):67). Heteroconjugation vectors for the sickle cell mutation appear to have some benefit due to their ability to prevent malaria, so this trait may have been actively selected over time, leading to estimates that up to 28% of the population in sub-Saharan Africa has the sickle cell trait ( Elguero et al., (2015) PNAS USA 112(22): 7051). Sickle cell anemia is caused by a mutation in the beta globulin gene due to the substitution of valine for glutamic acid at the sixth amino acid (a GAG to GTG mutation at the DNA level), where the resulting heme is called "Heme S" or "HbS". Under hypoxic conditions, conformational changes in the deoxygenated form of HbS expose a hydrophobic block on the protein between the E and F helices. The hydrophobic residue of valine at position 6 of the beta chain in heme is able to associate with this hydrophobic block, causing HbS molecules to aggregate and form fibrous precipitates. These aggregates in turn cause abnormal or "sickle" of RBCs, thereby losing cell flexibility. In sickle cell patients, sickle RBCs are no longer able to crowd into the microvascular bed and may lead to a vaso-occlusive crisis. Additionally, sickle-shaped RBCs are more fragile than normal RBCs and are prone to hemolysis, ultimately leading to anemia in the patient.

鐮形血球患者之治療及控制為一個終生命題，涉及抗生素治療、疼痛控制及在急性發作期間進行輸血。一種方法為使用羥基脲，其部分藉由增加γ球蛋白產生來發揮其作用。然而，長期羥基脲療法之長期副作用仍為未知的，且治療產生不需要之副作用從而導致低患者順應性，而且所具有之效力因患者而各異(Brandow及Panepinto, (2011)Am J Hematol 86(9):804-806)。儘管鐮形血球治療之效力有所增加，但患者之壽命預期仍然僅在50歲中晚期，而且該疾病之相關發病對患者之生活品質具有深遠影響。Treatment and management of sickle cell patients is a life-long challenge involving antibiotic therapy, pain management, and blood transfusions during exacerbations. One approach is to use hydroxyurea, which exerts its effect in part by increasing gamma globulin production. However, the long-term side effects of long-term hydroxyurea therapy are still unknown, and the treatment produces unwanted side effects resulting in low patient compliance, and its efficacy varies from patient to patient (Brandow and Panepinto, (2011) Am J Hematol 86 (9):804-806). Despite the increased efficacy of sickle cell therapy, the life expectancy of patients is still only in the mid to late 50s, and the associated morbidity of the disease has a profound impact on patients' quality of life.

地中海型貧血亦為與血紅素有關之疾病且典型地涉及球蛋白鏈表現減少。此可能因基因調控區中之突變或因球蛋白編碼序列中導致功能球蛋白表現減少或水準降低之突變而發生。由α球蛋白基因座中之突變導致之α型地中海型貧血主要與具有西非及南亞血統之人士相關，並且可能賦予瘧疾抗性。由β球蛋白基因座中之突變導致之β型地中海型貧血主要與具有地中海血統，典型地來自希臘以及土耳其及意大利沿海地區之人士相關。在輕度地中海型貧血中，β球蛋白對偶基因中僅有一個攜帶突變。個體將罹患小紅血球性貧血，且偵測通常涉及低於正常平均紅血球容積(＜80 fL)。患有輕度地中海型貧血之個體的對偶基因為β+/β或β0/β (其中『β+』係指允許一定量之β鏈形成發生的對偶基因，『β』係指野生型β球蛋白對偶基因，且『β0』係指與完全不存在β球蛋白表現相關之β球蛋白突變)。中度地中海型貧血個體通常可維持正常生活，但可能需要偶爾輸血，尤其在疾病或懷孕時，視其貧血嚴重程度而定。此等患者之對偶基因可為β+/β+或βo/β+。當兩個對偶基因皆具有地中海型貧血突變時，發生重度地中海型貧血。此為嚴重小紅血球性低血色性貧血。在不加治療之情況下，其導致貧血、脾腫大及嚴重骨畸形，並且在20歲前便進展至死亡。治療由週期性輸血、脾腫大時進行脾切除術及螯合輸血致鐵過載組成。若可鑑定適當供體，則骨髓移植亦可用於治療患有嚴重地中海型貧血之人士，但此程序可能存在巨大風險。Thalassemia is also a heme-related disorder and typically involves a decrease in the expression of globulin chains. This may occur due to mutations in the regulatory regions of the gene or due to mutations in the globulin coding sequence resulting in reduced expression or level of functional globulin. Thalassemia alpha, caused by mutations in the alpha globulin locus, is primarily associated with people of West African and South Asian ancestry and may confer malaria resistance. Beta thalassemia, caused by mutations in the beta globin locus, is primarily associated with individuals of Mediterranean ancestry, typically from Greece and coastal regions of Turkey and Italy. In thalassemia minor, only one of the β-globin pair genes carries a mutation. Individuals will suffer from microcytic anemia, and detection usually involves subnormal mean corpuscular volume (<80 fL). Individuals with thalassemia minor have dual genes that are either β+/β or β0/β (where "β+" refers to the dual gene that allows a certain amount of β-strand formation to occur, and "β" refers to wild-type beta globules. protein pair gene, and "β0" refers to a β-globin mutation associated with the complete absence of β-globin expression). Individuals with thalassemia moderate can usually lead normal lives, but may require occasional blood transfusions, especially during illness or pregnancy, depending on the severity of their anemia. The paired genes in these patients can be β+/β+ or βo/β+. Thalassemia major occurs when both paired genes have a thalassemia mutation. This is severe microcytic hypochromic anemia. Left untreated, it causes anemia, splenomegaly, and severe bone deformities, and progresses to death before age 20. Treatment consists of periodic blood transfusions, splenectomy in the case of splenomegaly, and iron overload from chelation transfusions. Bone marrow transplants can also be used to treat people with thalassemia severe if a suitable donor can be identified, but this procedure can carry significant risks.

一種已提議用於治療SCD及β型地中海型貧血之方法為增加γ球蛋白之表現，目標在於讓HbF在功能上替換異常成年血紅素。如以上所提及，用羥基脲治療SCD患者之所以被認為是成功的，部分係由於其對增加γ球蛋白表現之效應。影響γ球蛋白表現之第一組已發現化合物為細胞毒性藥物。首先在實驗動物中使用5-氮雜胞苷顯示藉由藥理學操縱引起從頭合成γ球蛋白之能力(DeSimone (1982)Proc Nat'l Acad Sci USA 79(14):4428-31)。後續研究證實5-氮雜胞苷增加患有β型地中海型貧血及鐮形血球貧血症之患者的HbF的能力(Ley等人, (1982)N. Engl. J. Medicine , 307: 1469-1475；及Ley等人, (1983)Blood 62: 370-380)。另外，短鏈脂肪酸(例如丁酸及衍生物)已在實驗系統中顯示增加HbF (Constantoulakis等人, (1988)Blood 72(6):1961-1967)。亦存在患有稱為『遺傳性胎兒血紅素持續存在』(HPFH)之病狀的人類群體的區段，其中升高量之HbF在成年期持續存在(10-40%呈HPFH異型接合子形式(參見Thein等人,(2009)Hum. Mol. Genet 18 (R2): R216-R223)。此為罕見病狀，但不存在任何相關β球蛋白異常，即使當個體之血紅素100%為HbF時亦不與任何顯著臨床體現相關。當患有β型地中海型貧血之個體亦患有共發性HPFH時，HbF之表現可減輕該疾病之嚴重程度(Potoka及Gladwin (2015) Am J Physiol Lung Cell Mol Physiol. 308(4): L314-L324)。此外，鐮形血球貧血症之自然病程之嚴重程度可能因患者而明顯各異，且此變異性部分可上溯至一些患有輕度疾病之個體表現較高水準之HbF。One approach that has been proposed for the treatment of SCD and beta thalassemia is to increase the expression of gamma globulin with the goal of allowing HbF to functionally replace abnormal adult heme. As mentioned above, the treatment of SCD patients with hydroxyurea is considered successful in part due to its effect on increased gamma globulin expression. The first group of discovered compounds that affect gamma globulin expression are cytotoxic drugs. The ability to induce de novo synthesis of gamma globulin by pharmacological manipulation was first shown in experimental animals using 5-azacytidine (DeSimone (1982) Proc Nat'l Acad Sci USA 79(14):4428-31). Subsequent studies demonstrated the ability of 5-azacytidine to increase HbF in patients with beta thalassemia and sickle cell anemia (Ley et al., (1982) N. Engl. J. Medicine , 307: 1469-1475 and Ley et al., (1983) Blood 62: 370-380). Additionally, short chain fatty acids such as butyric acid and derivatives have been shown to increase HbF in experimental systems (Constantoulakis et al., (1988) Blood 72(6):1961-1967). There is also a segment of the human population with a condition known as "hereditary fetal hemoglobin persistence" (HPFH), in which elevated amounts of HbF persist into adulthood (10-40% in the form of HPFH heterozygotes). (See Thein et al., (2009) Hum. Mol. Genet 18(R2): R216-R223.) This is a rare condition without any associated beta globulin abnormalities, even when the individual's heme is 100% HbF was not associated with any significant clinical manifestations. When individuals with β-thalassemia also have co-occurring HPFH, manifestations of HbF can reduce the severity of the disease (Potoka and Gladwin (2015) Am J Physiol Lung Cell Mol Physiol. 308(4): L314-L324). In addition, the severity of the natural history of sickle cell anemia may vary markedly from patient to patient, and some of this variability can be traced back to some individuals with mild disease The individuals showed higher levels of HbF.

一種增加HbF表現之方法涉及鑑定產物在調控γ球蛋白表現方面起作用之基因。一個此種基因為BCL11A，由於其在淋巴細胞發育中之作用而首先被鑑定。BCL11A編碼被認為參與γ球蛋白表現之發育階段特異性調控的鋅指蛋白。BCL11A表現於成年紅血球前驅細胞中，且下調其表現導致γ球蛋白表現增加。另外，BCL11A mRNA之剪接受發育調控。在胚細胞中，主要表現稱為BCL11A-S及BCL11A-XS之較短BCL11A mRNA變異體，而在成年細胞中，主要表現較長BCL11A-L及BCL11A-XL mRNA變異體。參見Sankaran等人,(2008)Science 322 p. 1839。BCL11A蛋白看似與β球蛋白基因座相互作用，從而在不同的發育階段改變其構形且因而改變其表現。已提議使用靶向BCL11A基因之抑制性RNA (參見例如美國專利公開案第2011/0182867號)，但此技術存在若干潛在缺點，即不能達成完全敲低，此種RNA之遞送可能存在問題及該等RNA必須持續存在，從而在一生中需要進行多次治療。One approach to increasing HbF expression involves identifying genes whose products play a role in regulating gamma globulin expression. One such gene, BCL11A, was first identified for its role in lymphocyte development. BCL11A encodes a zinc finger protein thought to be involved in the developmental stage-specific regulation of gamma globulin expression. BCL11A is expressed in adult erythrocyte precursor cells, and down-regulation of its expression results in increased gamma globulin expression. In addition, the splicing of BCL11A mRNA is under developmental regulation. In blast cells, the shorter BCL11A mRNA variants termed BCL11A-S and BCL11A-XS were predominantly expressed, while in adult cells the longer BCL11A-L and BCL11A-XL mRNA variants were predominantly expressed. See Sankaran et al., (2008) Science 322 p. 1839. The BCL11A protein appears to interact with the beta globin locus, altering its conformation and thus its expression at different developmental stages. The use of inhibitory RNAs targeting the BCL11A gene has been proposed (see, eg, U.S. Patent Publication No. 2011/0182867), but this technique has several potential drawbacks, namely that complete knockdown cannot be achieved, the delivery of such RNAs may be problematic and the Other RNAs must persist, requiring multiple treatments over a lifetime.

靶向BCL11A增強子序列提供一種增加HbF之機制。參見例如美國專利公開案第2015/0132269號及PCT公開案第WO 2016/183298號。全基因組關聯研究已鑑定BCL11A基因座處與HbF水準增加相關之一組基因變異。此等變異為在充當階段特異性譜系限制增強子區之BCL11A非編碼區中發現之小核苷酸多型現象(SNP)之集合。進一步研究揭示紅血球細胞中需要此BCL11A增強子以進行BCL11A表現，但B細胞中不需要其表現(參見Bauer等人, (2013)Science 343:253-257)。在BCL11A基因之內含子2內發現增強子區，且鑑定內含子2中之三個DNAseI過敏區域(通常指示與調控潛力相關之染色質狀態)。根據距BCL11A之轉錄起始位點之距離(以千鹼基計)將此三個區域鑑定為「+62」、「+58」及「+55」。此等增強子區之長度大致為350 (+55)、550 (+58)及350 (+62)個核苷酸(Bauer 2013,如上 )。Targeting the BCL11A enhancer sequence provides a mechanism to increase HbF. See, eg, US Patent Publication No. 2015/0132269 and PCT Publication No. WO 2016/183298. Genome-wide association studies have identified a group of genetic variants at the BCL11A locus that are associated with increased levels of HbF. These variants are a collection of small nucleotide polymorphisms (SNPs) found in the noncoding region of BCL11A that serve as stage-specific lineage-restricted enhancer regions. Further studies revealed that this BCL11A enhancer is required for BCL11A expression in red blood cells, but not in B cells (see Bauer et al., (2013) Science 343:253-257). An enhancer region was found within intron 2 of the BCL11A gene, and three DNAseI hypersensitive regions (generally indicative of chromatin states associated with regulatory potential) were identified in intron 2. The three regions were identified as "+62", "+58" and "+55" based on their distance (in kilobases) from the transcription start site of BCL11A. These enhancer regions are approximately 350 (+55), 550 (+58) and 350 (+62) nucleotides in length (Bauer 2013, supra ).

當開發核酸酶以用於對人類進行治療性治療時，核酸酶有必要具有最大限度之安全特徵。特定言之，核酸酶必須具有極低水準之脫靶裂解。除使用者規定之標靶以外的位置上的大量雙鏈切口可引起對脫靶基因及在罕見情況下發生之染色體易位的壓抑(參見Hoban及Bauer, (2016)Blood , 127(21):2525-2535；及Tsang等人, (2017)Nature Methods , 出版中)。可藉由消除經工程改造之核酸酶與基因組DNA之間的非特異性相互作用來達成特異性之改良(參見美國臨時專利申請案第62/378,978號及第62/443,981號)。When developing nucleases for therapeutic treatment in humans, it is necessary for the nucleases to have the greatest possible safety profile. In particular, nucleases must have very low levels of off-target cleavage. Numerous double-stranded nicks at positions other than user-specified targets can cause repression of off-target genes and, in rare cases, chromosomal translocations (see Hoban and Bauer, (2016) Blood , 127(21):2525 -2535; and Tsang et al., (2017) Nature Methods , in press). Improvement in specificity can be achieved by eliminating nonspecific interactions between engineered nucleases and genomic DNA (see US Provisional Patent Application Nos. 62/378,978 and 62/443,981).

因而，仍需要用於改變BCL11A基因表現之其他高特異性方法及組合物，例如用以治療諸如鐮形血球貧血症及β型地中海型貧血之血紅素病。Thus, there remains a need for other highly specific methods and compositions for altering the expression of the BCL11A gene, eg, for the treatment of heme diseases such as sickle cell anemia and beta thalassemia.

本發明描述用於基因療法及基因組工程改造之高特異性組合物及方法。特定言之，所描述之方法及組合物係關於使BCL11A基因，例如，充當一或多個其他基因之調節基因的基因不活化(例如例如，藉由完全或部分廢除其表現)。特定言之，本發明描述用於在特定細胞譜系(例如，紅血球)中干擾BCL11A基因中之增強子功能以降低或敲除其活性的方法及組合物。另外，本發明提供用於干擾BCL11A增強子功能之方法及組合物，其中增強子序列不位於BCL11A基因之編碼序列內，且其中所提供之試劑展現高特異性活性。此等情形下之所得BCL11A基因下調引起γ球蛋白表現增加及脫靶裂解事件數目減少。The present invention describes highly specific compositions and methods for gene therapy and genome engineering. In particular, the described methods and compositions relate to inactivating the BCL11A gene, eg, a gene that acts as a regulatory gene for one or more other genes (eg, by completely or partially abrogating its expression). In particular, the present invention describes methods and compositions for interfering with enhancer function in the BCL11A gene to reduce or knock out its activity in specific cell lineages (eg, red blood cells). Additionally, the present invention provides methods and compositions for interfering with BCL11A enhancer function, wherein the enhancer sequence is not located within the coding sequence of the BCL11A gene, and wherein the provided agents exhibit highly specific activity. The resulting BCL11A gene downregulation under these conditions resulted in increased gamma globulin expression and decreased number of off-target cleavage events.

在一些態樣中，本發明包含一種非天然存在之鋅指蛋白，其包括包含4、5或6個指之鋅指蛋白(ZFP)，各指包含識別DNA標靶亞位點之識別螺旋區，其中該識別螺旋區按表1之單列中所示之順序包含諸多序列。在各鋅指內，將鋅指主鏈(具有大約30個殘基，包括鋅配位殘基)內具有7個胺基酸之識別螺旋區編號為-1至+6。在某些實施例中，本文中所描述之鋅指蛋白之組件鋅指中有1、2、3個以上進一步包含識別螺旋區外之一或多個殘基之突變，包括但不限於在-5位、-14位或在-5位與-14位(編號自用於識別螺旋區之-1至+6編號繼續)兩者上之胺基酸突變之突變。參見例如美國臨時專利申請案62/378,978及62/443,981中所描述之Qm4及Qm14突變。鋅指蛋白之組件鋅指可由任何連接子連接，例如，如美國專利第8,772,453號中所描述。在某些實施例中，ZFP包含如表1中針對如下命名之蛋白質所示之識別螺旋：63014 (其結合SEQ ID NO:1中所示之標靶位點)及65722 (其結合SEQ ID NO:2中所示之標靶位點)。In some aspects, the invention comprises a non-naturally occurring zinc finger protein, comprising a zinc finger protein (ZFP) comprising 4, 5 or 6 fingers, each finger comprising a recognition helical region that recognizes a DNA target subsite , wherein the recognition helical region comprises sequences in the order shown in the individual columns of Table 1. Within each zinc finger, the recognition helix region with 7 amino acids within the zinc finger backbone (having approximately 30 residues, including the zinc coordinating residues) is numbered from -1 to +6. In certain embodiments, 1, 2, 3 or more of the component zinc fingers of the zinc finger proteins described herein further comprise mutations that recognize one or more residues outside the helical region, including but not limited to- Mutations of amino acid mutations at positions 5, -14, or both at positions -5 and -14 (numbering continues from -1 to +6 numbering used to identify the helical region). See, eg, the Qm4 and Qm14 mutations described in US Provisional Patent Applications 62/378,978 and 62/443,981. Components of Zinc Finger Proteins Zinc fingers can be linked by any linker, eg, as described in US Pat. No. 8,772,453. In certain embodiments, the ZFP comprises the recognition helices shown in Table 1 for the proteins named: 63014 (which binds the target site shown in SEQ ID NO: 1) and 65722 (which binds SEQ ID NO: 1) : target site shown in 2).

在某些實施例中，如本文中所描述之鋅指蛋白與功能結構域(例如，轉錄活化結構域、轉錄壓抑結構域、裂解結構域(以形成鋅指核酸酶)等)融合。任何連接子均可用於可操作地連接裂解結構域與鋅指蛋白，包括但不限於如美國專利第9,394,531號及第9,567,609號中所描述之連接子。此外，當使用FokI裂解結構域時，可能存在催化結構域中、二聚結構域中、磷酸酯接觸殘基(不在二聚或催化結構域中)之其他突變以及催化結構域、二聚結構域及磷酸酯接觸殘基中任一者中之突變之組合，包括但不限於二聚結構域之ELD或KKR突變、FokI結構域之殘基525之突變(K至S)及二聚結構域之ELD或KKR突變與FokI結構域之殘基525之突變(K至S)的組合，相對於野生型進行編號。參見美國專利第7,888,121號、第7,914,796號、第8,034,598號、第8,623,618號及美國專利公開案第2011/0201055號以及美國臨時專利申請案第62/378,978號及第62/443,981號。In certain embodiments, zinc finger proteins as described herein are fused to functional domains (eg, transcriptional activation domains, transcriptional repression domains, cleavage domains (to form zinc finger nucleases), etc.). Any linker can be used to operably link the cleavage domain to the zinc finger protein, including, but not limited to, linkers as described in US Pat. Nos. 9,394,531 and 9,567,609. In addition, when using the FokI cleavage domain, there may be other mutations in the catalytic domain, in the dimerization domain, phosphate contact residues (not in the dimerization or catalytic domains) as well as in the catalytic domain, dimerization domain and combinations of mutations in any of the phosphate contact residues, including but not limited to ELD or KKR mutations in the dimerization domain, mutations at residue 525 (K to S) in the FokI domain, and in the dimerization domain Combinations of ELD or KKR mutations and mutations at residue 525 of the FokI domain (K to S) are numbered relative to wild type. See US Patent Nos. 7,888,121, 7,914,796, 8,034,598, 8,623,618 and US Patent Publication No. 2011/0201055 and US Provisional Patent Application Nos. 62/378,978 and 62/443,981.

在某些實施例中，鋅指核酸酶(ZFN)可用於使配對二聚，以便在配對之ZFN之標靶位點之一或兩者處或附近裂解，例如，表1之「左搭配物」(例如，63014)可與表1之「右搭配物」(例如，65722)形成二聚體以使BCL11A增強子序列裂解。在某些實施例中，ZFN配對包含以下胺基酸序列： 63014：MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFARNFSLTMHTKIHTGSQKPFQCRICMQNFSSTGNLTNHIRTHTGEKPFACDICGRKFATSGSLTRHTKIHTHPRAPIPKPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFAAQCCLFHHTKIH-連接子- ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQTRDKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS (SEQ ID NO:3)；及 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMQKFARNDHRTTHTKIHTGEKPFQCRICMQNFSQKAHLIRHIRTHTGEKPFACDICGRKFAQKGTLGEHTKIHTGSQKPFQCRICMQNFSRGRDLSRHIRTHTGEKPFACDICGRKFARRDNLHSHTKIH-連接子- ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFSGNYKAQLTRLNRKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO:4)，其中該連接子序列可為此項技術中已知的任何連接子序列，例如，如美國專利第9,394,531號及第9,567,609號中所描述。在某些實施例中，用於63014之連接子包含L7c5連接子(LRGSISRARPLNPHP (SEQ ID NO:5))或由其組成，且用於65722之連接子包含L0連接子(LRGSQLVKS (SEQ ID NO:6)，參見美國專利第9,567,609號)或由其組成。以上所示之序列之連接子之C末端FokI裂解結構域序列亦可包含與鋅指蛋白可操作地連接之替代FokI結構域。在某些實施例中，FokI裂解結構域可包括催化結構域、二聚結構域、磷酸酯接觸殘基之替代或額外突變以及催化結構域、二聚結構域及磷酸酯接觸殘基中任一者之突變之組合。In certain embodiments, zinc finger nucleases (ZFNs) can be used to dimerize pairs for cleavage at or near one or both of the target sites of the paired ZFNs, e.g. " (eg, 63014) can dimerize with the "right partner" of Table 1 (eg, 65722) to cleave the BCL11A enhancer sequence. In certain embodiments, ZFN pair containing the following amino acid sequence: 63014: MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFARNFSLTMHTKIHTGSQKPFQCRICMQNFSSTGNLTNHIRTHTGEKPFACDICGRKFATSGSLTRHTKIHTHPRAPIPKPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFAAQCCLFHHTKIH- linker - ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQTRDKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS (SEQ ID NO: 3); and MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMQKFARNDHRTTHTKIHTGEKPFQCRICMQNFSQKAHLIRHIRTHTGEKPFACDICGRKFAQKGTLGEHTKIHTGSQKPFQCRICMQNFSRGRDLSRHIRTHTGEKPFACDICGRKFARRDNLHSHTKIH- linker - ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFSGNYKAQLTRLNRKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 4), wherein the connecting The subsequence can be any linker sequence known in the art, eg, as described in US Pat. Nos. 9,394,531 and 9,567,609. In certain embodiments, the linker for 63014 comprises or consists of the L7c5 linker (LRGSISRARPLNPHP (SEQ ID NO: 5)) and the linker for 65722 comprises the L0 linker (LRGSQLVKS (SEQ ID NO: 5) 6), see US Pat. No. 9,567,609) or consist thereof. The C-terminal FokI cleavage domain sequence of the linker of the sequences shown above may also comprise an alternative FokI domain operably linked to a zinc finger protein. In certain embodiments, a FokI cleavage domain can include a catalytic domain, a dimerization domain, substitutions or additional mutations of phosphate contact residues, and any of the catalytic domain, dimerization domain, and phosphate contact residues A combination of mutations.

在另一態樣中，本發明包括出於基因組工程改造之目的遞送至少一種核酸酶(例如，結合BCL11A增強子序列之核酸酶)至人類幹細胞或前驅細胞(HSC/PC)。在某些實施例中，該核酸酶包含有包含4、5或6個指之鋅指蛋白(ZFP)，各指包含識別標靶亞位點之識別螺旋區，其中該識別螺旋區按表1之單列中所示之順序包含諸多序列。在其他實施例中，該ZFN核酸酶包含命名為63014/65722之核酸酶配對。如本文中所描述之核酸酶可進一步包含連接子(例如，在DNA結合結構域與裂解結構域之間)，例如，如9,567,609中所示之連接子，包括但不限於LRGSISRARPLNPHP (SEQ ID NO:5)或(LRGSQLVKS (SEQ ID NO:6))。In another aspect, the invention includes delivery of at least one nuclease (eg, a nuclease that binds a BCL11A enhancer sequence) to human stem or precursor cells (HSC/PC) for the purpose of genome engineering. In certain embodiments, the nuclease comprises a zinc finger protein (ZFP) comprising 4, 5 or 6 fingers, each finger comprising a recognition helical region that recognizes a target subsite, wherein the recognition helical region is according to Table 1 The sequence shown in a single column of , contains a number of sequences. In other embodiments, the ZFN nuclease comprises the nuclease pair designated 63014/65722. Nucleases as described herein can further comprise a linker (e.g., between the DNA binding domain and the cleavage domain), e.g., as shown in 9,567,609, including but not limited to LRGSISRARPLNPHP (SEQ ID NO: 5) or (LRGSQLVKS (SEQ ID NO: 6)).

在一些實施例中，該核酸酶作為肽進行遞送，而在其他實施例中，其作為編碼至少一種核酸酶之核酸進行遞送。在一些實施例中，使用超過一種核酸酶。在一些較佳實施例中，編碼核酸酶之核酸為mRNA，且在一些情況下，對該mRNA加以保護。在一些態樣中，可對該mRNA進行化學修飾(參見例如Kormann等人, (2011)Nature Biotechnology 29(2):154-157)。在其他態樣中，該mRNA可包含ARCA帽(參見美國專利第7,074,596號及第8,153,773號)。在其他實施例中，該mRNA可包含未經修飾與經修飾之核苷酸的混合物(參見美國專利公開案第2012/0195936號)。在一較佳實施例中，經由電穿孔將該編碼核酸酶之核酸遞送至HSC/PC。在一些實施例中，該核酸酶在轉錄因子結合位點處或附近裂解。在一些態樣中，該轉錄因子為GATA-1。In some embodiments, the nuclease is delivered as a peptide, while in other embodiments, it is delivered as a nucleic acid encoding at least one nuclease. In some embodiments, more than one nuclease is used. In some preferred embodiments, the nucleic acid encoding the nuclease is mRNA, and in some cases, the mRNA is protected. In some aspects, the mRNA can be chemically modified (see, eg, Kormann et al., (2011) Nature Biotechnology 29(2):154-157). In other aspects, the mRNA may comprise an ARCA cap (see US Pat. Nos. 7,074,596 and 8,153,773). In other embodiments, the mRNA may comprise a mixture of unmodified and modified nucleotides (see US Patent Publication No. 2012/0195936). In a preferred embodiment, the nucleic acid encoding the nuclease is delivered to the HSC/PC via electroporation. In some embodiments, the nuclease cleaves at or near the transcription factor binding site. In some aspects, the transcription factor is GATA-1.

在其他態樣中，本發明包含一種細胞或細胞株，其中內源BCL11A增強子序列係藉由如本文中所描述(例如，表1中所示)之核酸酶加以基因修飾，例如與該細胞之野生型序列相比。對BCL11A增強子之基因修飾導致對球蛋白(β及γ)基因表現之修飾。如本文中所描述之經核酸酶修飾之細胞或細胞株在結構、功能及結構與功能兩者之組合方面不同於野生型。對於修飾而言，經基因修飾之細胞或細胞株可為異型接合或同型接合的。修飾可包含***(例如，轉殖基因***)、缺失及***與缺失之組合；此種***、缺失及***與缺失之組合共同稱為「***缺失」。在一些較佳實施例中，***缺失導致對轉錄因子結合位點之破壞。在某些實施例中，修飾處於核酸酶結合位點、裂解位點及結合位點與裂解位點之組合處或附近，例如在裂解位點上游或下游1至300 (或介於其之間的任何值)個鹼基對內，更佳在表1中所示之結合位點、裂解位點及結合位點與裂解位點之組合的任一側的1至100個鹼基對(或介於其之間的任何值)內，甚至更佳在結合位點、裂解位點及結合位點與裂解位點之組合的任一側上1至50個鹼基對(或介於其之間的任何值)內。在某些實施例中，BCL11A增強子序列之基因修飾在表1中所示之序列(靶位點)內及/或之間。該修飾亦可包括對裂解位點中之一或多個核苷酸的修飾。該修飾亦可包括對結合位點中之一或多個核苷酸的修飾。該修飾可進一步包括對裂解位點中及一或多個結合位點中之一或多個核苷酸的修飾。在某些實施例中，一或多個核酸酶靶位點未經修飾。在其他實施例中，至少一個核酸酶靶位點受到修飾。在某些實施例中，該修飾處於BCL11A增強子「+58」區域處或附近，例如，處於SEQ ID NO:1及SEQ ID NO:2中之任一者中所示之核酸酶結合位點處或附近。任何細胞或細胞株均可藉由如本文中所描述之核酸酶加以修飾，例如幹細胞(造血幹細胞，諸如CD34+造血幹細胞)或紅血細胞(RBC)前驅細胞。In other aspects, the invention comprises a cell or cell line wherein the endogenous BCL11A enhancer sequence is genetically modified by a nuclease as described herein (eg, as shown in Table 1), eg, with the cell compared to the wild-type sequence. Genetic modification of the BCL11A enhancer results in modification of the expression of the globulin (beta and gamma) genes. Nuclease-modified cells or cell lines as described herein differ from wild-type in structure, function, and a combination of both. For modification, the genetically modified cell or cell line may be heterozygous or homozygous. Modifications can include insertions (eg, transgene insertions), deletions, and combinations of insertions and deletions; such insertions, deletions, and combinations of insertions and deletions are collectively referred to as "indels." In some preferred embodiments, the indel results in disruption of the transcription factor binding site. In certain embodiments, the modification is at or near a nuclease binding site, a cleavage site, and a combination of binding and cleavage sites, such as 1 to 300 (or in between) upstream or downstream of the cleavage site any value) base pairs, more preferably 1 to 100 base pairs on either side of the binding site, cleavage site, and combination of binding and cleavage sites shown in Table 1 (or within any value in between), even more preferably 1 to 50 base pairs (or in between) on either side of the binding site, the cleavage site, and the combination of the binding site and the cleavage site any value in between). In certain embodiments, the genetic modification of the BCL11A enhancer sequence is within and/or between the sequences shown in Table 1 (target sites). The modification may also include modification of one or more nucleotides in the cleavage site. The modification may also include modification of one or more nucleotides in the binding site. The modification may further include modification of one or more nucleotides in the cleavage site and in the one or more binding sites. In certain embodiments, one or more nuclease target sites are unmodified. In other embodiments, at least one nuclease target site is modified. In certain embodiments, the modification is at or near the BCL11A enhancer "+58" region, eg, at the nuclease binding site set forth in any of SEQ ID NO:1 and SEQ ID NO:2 at or near. Any cell or cell line can be modified by nucleases as described herein, eg stem cells (hematopoietic stem cells, such as CD34+ hematopoietic stem cells) or red blood cell (RBC) precursor cells.

亦描述藉由如本文中所描述之核酸酶進行修飾後獲得之細胞或細胞株，例如自如本文中所描述之經核酸酶修飾之細胞或細胞株傳代而來之細胞或細胞株。亦提供自如本文中所描述之經修飾之幹細胞傳代而來的部分或完全分化之細胞(例如，RBC或RBC前驅細胞)。自經核酸酶修飾之細胞傳代而來之細胞可在試管內進行繁殖、分化及繁殖與分化兩者之組合(培養)，或可在活個體內分化，例如在擬體內投與經核酸酶修飾之幹細胞後。本文中所揭示之任何經基因修飾之細胞或細胞株均可顯示增加之γ球蛋白表現。亦提供包含如本文中所描述之經基因修飾之細胞的組合物，諸如醫藥組合物。Also described are cells or cell lines obtained after modification by nucleases as described herein, eg, cells or cell lines passaged from nuclease-modified cells or cell lines as described herein. Partially or fully differentiated cells (eg, RBCs or RBC precursor cells) derived from passage of modified stem cells as described herein are also provided. Cells passaged from nuclease-modified cells can be propagated, differentiated, and a combination of propagation and differentiation (culture) in vitro, or can be differentiated in vivo, such as in vivo by administration of nuclease-modified cells after modified stem cells. Any of the genetically modified cells or cell lines disclosed herein can exhibit increased gamma globulin expression. Compositions, such as pharmaceutical compositions, comprising genetically modified cells as described herein are also provided.

在其他態樣中，本發明包含遞送供體核酸至靶細胞以獲得經基因修飾之細胞，其中該供體被整合至該細胞中。該供體可在表1之編碼核酸酶之核酸之前、之後或隨其一起遞送。供體核酸可包含欲整合至細胞之基因組，例如內源基因座中的外源序列(轉殖基因)。在一些實施例中，該供體可包含側接有與靶向裂解位點具有同源性之區域的全長基因或其片段。在一些實施例中，該供體缺乏同源區域且藉由非同源性依賴性機制(亦即，NHEJ)整合至靶基因座中。該供體可包含任何核酸序列，例如當用作對核酸酶誘導之雙鏈斷裂進行同源性引導修復之受質時導致將在內源染色體基因座(例如，BCL11A增強子區域)處產生之供體特異性缺失或替代地(或另外)將產生之內源基因座之新穎對偶基因形式(例如，摘除轉錄因子結合位點之點突變)的核酸。在一些態樣中，該供體核酸為寡核苷酸，其中整合導致基因矯正事件或靶向缺失。In other aspects, the invention comprises delivering a donor nucleic acid to a target cell to obtain a genetically modified cell, wherein the donor is integrated into the cell. The donor can be delivered before, after, or with the nucleic acid encoding the nuclease of Table 1. The donor nucleic acid may comprise an exogenous sequence (transgenic gene) to be integrated into the genome of the cell, eg, in an endogenous locus. In some embodiments, the donor may comprise a full-length gene or a fragment thereof flanked by regions of homology to the targeted cleavage site. In some embodiments, the donor lacks a homology region and integrates into the target locus by a homology-independent mechanism (ie, NHEJ). The donor may comprise any nucleic acid sequence that results in a donor that will be produced at an endogenous chromosomal locus (eg, the BCL11A enhancer region) when used as a substrate for homology-guided repair of nuclease-induced double-strand breaks, for example A body-specific deletion or alternatively (or additionally) a nucleic acid that will generate a novel counterpart of an endogenous locus (eg, a point mutation that removes a transcription factor binding site). In some aspects, the donor nucleic acid is an oligonucleotide, wherein integration results in a gene corrective event or targeted deletion.

在其他態樣中，藉由病毒基因轉移法、非病毒基因轉移法及病毒基因轉移法與非病毒基因轉移法之組合來遞送核酸酶、供體及核酸酶與供體兩者之組合。在較佳實施例中，經由腺病毒相關病毒(AAV)將該供體遞送至細胞。在一些情況下，該AAV包含與殼體血清型相比具有異種血清型之LTR。In other aspects, nucleases, donors, and combinations of both nucleases and donors are delivered by viral gene transfer methods, non-viral gene transfer methods, and combinations of viral and non-viral gene transfer methods. In a preferred embodiment, the donor is delivered to the cell via an adeno-associated virus (AAV). In some cases, the AAV comprises an LTR with a heterologous serotype compared to the capsid serotype.

在一些態樣中，使用如表1中所示之一或多種核酸酶製造增強子之DNAseI過敏區域(例如，BCL11A增強子之+58區域)內包含缺失之區域。此等缺失可包含約1個核苷酸至約551個核苷酸。因而，缺失可包含1、5、10、15、20、25、30、40、50、100、150、200、250、300、350、400、450、500、550個核苷酸或介於其之間的任何值。在一些實施例中，缺失包含一或多個轉錄因子之結合區域。在一些較佳實施例中，缺失包含GATA-1結合位點或GATA-1結合位點與其他因子之組合。In some aspects, one or more nucleases as shown in Table 1 are used to create the DNAseI hypersensitive region of the enhancer (eg, the +58 region of the BCL11A enhancer) comprising the deleted region. Such deletions can comprise from about 1 nucleotide to about 551 nucleotides. Thus, deletions may comprise 1, 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 nucleotides or in between any value in between. In some embodiments, the deletion comprises the binding region of one or more transcription factors. In some preferred embodiments, the deletion comprises a GATA-1 binding site or a combination of a GATA-1 binding site and other factors.

在一些實施例中，表1之DNA結合結構域與功能結構域融合。一些態樣包括使DNA結合結構域與能夠調控基因表現之結構域融合。在一些實施例中，該等融合蛋白質包含表1之DNA結合結構域與調節因子壓抑基因表現之基因表現調節結構域融合。In some embodiments, the DNA binding domains of Table 1 are fused to functional domains. Some aspects include fusing a DNA binding domain to a domain capable of regulating gene expression. In some embodiments, the fusion proteins comprise the DNA binding domains of Table 1 fused to a gene expression regulatory domain that represses gene expression by a regulator.

在一些實施例中，使HSC/PC細胞與本發明(亦即，如表1中所示之ZFP)之核酸酶、DNA結合蛋白質及核酸酶與DNA結合蛋白質之組合接觸。在一些實施例中，將核酸酶、DNA結合蛋白質及核酸酶與DNA結合蛋白質之組合作為核酸遞送，且在其他實施例中，將其作為蛋白質遞送。在一些實施例中，該等核酸為編碼該等核酸酶、該等DNA結合蛋白質及該等核酸酶與該等DNA結合蛋白質之組合的mRNA，且在其他實施例中，該等mRNA可能受到保護。在一些實施例中，該mRNA可經化學修飾，可包含ARCA帽、未經修飾與經修飾核苷酸之混合物及ARCA帽跟未經修飾與經修飾核苷酸之混合物的組合。亦提供自此等細胞傳代而來之細胞或細胞株，包括部分或完全分化之細胞。In some embodiments, HSC/PC cells are contacted with nucleases, DNA binding proteins, and combinations of nucleases and DNA binding proteins of the invention (ie, ZFPs as shown in Table 1). In some embodiments, nucleases, DNA-binding proteins, and combinations of nucleases and DNA-binding proteins are delivered as nucleic acids, and in other embodiments, are delivered as proteins. In some embodiments, the nucleic acids are mRNAs encoding the nucleases, the DNA-binding proteins, and combinations of the nucleases and the DNA-binding proteins, and in other embodiments, the mRNAs may be protected . In some embodiments, the mRNA can be chemically modified and can include an ARCA cap, a mixture of unmodified and modified nucleotides, and a combination of an ARCA cap and a mixture of unmodified and modified nucleotides. Cells or cell lines passaged from these cells are also provided, including partially or fully differentiated cells.

在一些態樣中，在自個體分離HSC/PC或自所收集之骨髓純化後，使HSC/PC與本發明之核酸酶、DNA結合蛋白質及核酸酶與DNA結合蛋白質之組合擬體內接觸。在一些實施例中，本文中所描述之核酸酶引起BCL11A增強子區內之修飾，例如，產生在結構上、在功能上及在結構上與在功能上之組合與野生型細胞、其他經修飾(例如，經核酸酶修飾)之細胞及野生型與其他經修飾之細胞之組合不同的經基因修飾之細胞。在其他實施例中，將含有BCL11A增強子區修飾之HSC/PC引入回個體體內。在一些情況下，在引入之前對含有BCL11A增強子區修飾之HSC/PC進行擴增。在其他態樣中，在骨髓移植中將經基因修飾之HSC/PC給與個體，其中該HSC/PC在活體內植入、分化並且成熟。在一些實施例中，在G-CSF誘導之動員、普樂沙福(plerixafor)誘導之動員及G-CSF誘導之動員與普樂沙福誘導之動員之組合後自個體分離HSC/PC，且在其他實施例中，自人類骨髓或人類臍帶分離該等細胞。在一些態樣中，在引入包含經修飾之HSC/PC的移植物之前對個體提供輕度骨髓淨除程序，而在其他態樣中，用強力骨髓淨除處理方案治療個體。在一些實施例中，本發明之方法及組合物用於治療或預防血紅素病。在一些態樣中，該血紅素病為地中海型貧血。在一些態樣中，該血紅素病為β型地中海型貧血，而在其他態樣中，該血紅素病為鐮形血球貧血症。In some aspects, HSCs/PCs are contacted ex vivo with nucleases, DNA binding proteins, and combinations of nucleases and DNA binding proteins of the invention following isolation of HSCs/PCs from individuals or purification from collected bone marrow. In some embodiments, the nucleases described herein cause modifications within the enhancer region of BCL11A, eg, resulting in structural, functional, and a combination of structural and functional and wild-type cells, other modified (eg, nuclease-modified) cells and genetically modified cells in which the combination of wild-type and other modified cells differs. In other embodiments, HSCs/PCs containing modifications to the BCL11A enhancer region are introduced back into the subject. In some cases, HSCs/PCs containing modifications to the BCL11A enhancer region were amplified prior to introduction. In other aspects, the genetically modified HSC/PC is administered to the individual in a bone marrow transplant, wherein the HSC/PC engrafts, differentiates and matures in vivo. In some embodiments, the HSCs/PCs are isolated from the individual following G-CSF-induced mobilization, plerixafor-induced mobilization, and a combination of G-CSF-induced mobilization and plerixafor-induced mobilization, and In other embodiments, the cells are isolated from human bone marrow or human umbilical cord. In some aspects, the subject is provided with a mild myeloablative procedure prior to introduction of the graft comprising the modified HSC/PC, while in other aspects, the subject is treated with a vigorous myeloablative treatment regimen. In some embodiments, the methods and compositions of the present invention are used to treat or prevent heme disorders. In some aspects, the heme disease is thalassemia. In some aspects, the heme disease is beta thalassemia, and in other aspects, the heme disease is sickle cell anemia.

在一些實施例中，使該等HSC/PC進一步與供體分子接觸。在一些實施例中，該供體分子由病毒載體遞送。該供體分子可包含一或多個編碼功能多肽且有或無啟動子之序列(例如，cDNA或其片段)。當供體分子用於不活化時，可包括其他序列(編碼或非編碼序列)，包括但不限於編碼2A肽、SA位點、IRES等之序列。In some embodiments, the HSCs/PCs are further contacted with donor molecules. In some embodiments, the donor molecule is delivered by a viral vector. The donor molecule may comprise one or more sequences (eg, cDNA or fragments thereof) encoding a functional polypeptide, with or without a promoter. When the donor molecule is used for inactivation, other sequences (coding or non-coding sequences) may be included, including but not limited to sequences encoding 2A peptides, SA sites, IRES, and the like.

在一個態樣中，本發明之方法及組合物包含用於活體內接觸HSC/PC之方法。藉由此項技術中已知的方法將核酸酶、DNA結合蛋白或核酸酶與DNA結合蛋白之組合原位遞送至HSC/PC。在一些實施例中，本發明之核酸酶及/或DNA結合蛋白包含在有需要時投與個體之病毒粒子，而在其他實施例中，核酸酶、DNA結合蛋白或核酸酶與DNA結合蛋白之組合包含奈米粒子(例如脂質體)。在一些實施例中，將病毒粒子、奈米粒子或病毒粒子與奈米粒子之組合遞送至HSC/PC駐留之器官(例如骨髓)。In one aspect, the methods and compositions of the present invention comprise methods for contacting HSC/PC in vivo. Nucleases, DNA-binding proteins, or a combination of nucleases and DNA-binding proteins are delivered to HSCs/PCs in situ by methods known in the art. In some embodiments, the nucleases and/or DNA-binding proteins of the invention comprise viral particles that are administered to an individual in need, while in other embodiments, the nucleases, DNA-binding proteins, or the combination of nucleases and DNA-binding proteins The combination includes nanoparticles (eg, liposomes). In some embodiments, virions, nanoparticles, or a combination of virions and nanoparticles are delivered to the organ in which the HSC/PC resides (eg, bone marrow).

在另一態樣中，本文中描述經由非同源性依賴性機制將供體核酸整合至細胞基因組中之方法。該等方法包括在細胞之基因組中產生雙鏈斷裂(DSB)及使用如本文中所描述之核酸酶使供體分子裂解，以便將供體核酸整合在DSB位點。在某些實施例中，經由非同源性依賴性方法(例如NHEJ)來整合供體核酸。如以上所指出，在活體內裂解後，供體序列可在DSB位置上以靶向方式整合至細胞之基因組中。供體序列可包括用於產生DSB之核酸酶中之一或多者的相同靶位點中之一或多個。因而，可藉由用於使需要整合至其中之內源基因裂解的相同核酸酶中之一或多者使供體序列裂解。在某些實施例中，供體序列包括與用於誘導DSB之核酸酶不同的核酸酶靶位點。靶細胞基因組中之DSB可藉由任何機制來產生。在某些實施例中，藉由一或多個鋅指核酸酶(ZFN)、包含經工程改造以結合相關區域內之序列的鋅指結合結構域及裂解結構域或裂解半結構域之融合蛋白質來產生DSB。In another aspect, described herein are methods of integrating a donor nucleic acid into a cellular genome via a homology-independent mechanism. These methods include generating a double-strand break (DSB) in the genome of the cell and cleaving the donor molecule using a nuclease as described herein to integrate the donor nucleic acid at the DSB site. In certain embodiments, the donor nucleic acid is integrated via a non-homology-dependent method (eg, NHEJ). As noted above, following in vivo cleavage, the donor sequence can be integrated into the genome of the cell in a targeted manner at the DSB position. The donor sequence may include one or more of the same target sites of one or more of the nucleases used to generate the DSB. Thus, the donor sequence can be cleaved by one or more of the same nucleases used to cleave the endogenous gene into which it is desired to be integrated. In certain embodiments, the donor sequence includes a different nuclease target site than the nuclease used to induce the DSB. DSBs in the target cell genome can be produced by any mechanism. In certain embodiments, fusion proteins comprising one or more zinc finger nucleases (ZFNs), zinc finger binding domains and cleavage domains or cleavage half domains that are engineered to bind to sequences within the relevant regions to generate DSBs.

在一個態樣中，該供體可編碼能結合相關基因、調節其表現或結合相關基因並且調節其表現之相關調控蛋白質(例如，ZFP TF、TALE TF或CRISPR/Cas TF)。在一個實施例中，該等調控蛋白質結合DNA序列且防止結合其他調控因子。在另一實施例中，調控蛋白質之結合可調節(亦即誘導或壓抑)靶DNA之表現。In one aspect, the donor can encode a related regulatory protein (eg, a ZFP TF, TALE TF, or CRISPR/Cas TF) capable of binding the related gene, modulating its expression, or binding the related gene and modulating its expression. In one embodiment, the regulatory proteins bind DNA sequences and prevent binding of other regulatory factors. In another embodiment, binding of the regulatory protein can modulate (ie induce or repress) the expression of the target DNA.

在一些實施例中，轉殖基因HSC/PC細胞、轉殖基因動物或轉殖基因HSC/PC細胞與動物之組合包括編碼人類基因之轉殖基因。在一些情況下，轉殖基因動物包含內源基因座處之敲除及內源基因經其人類對應物置換，從而允許開發可孤立地研究人類蛋白質之活體內系統。此種轉殖基因模型可用於篩檢目的以鑑定可與相關人類蛋白質相互作用或修飾相關人類蛋白質之小分子或大生物分子或其他實體。在一些態樣中，將轉殖基因整合至所選基因座(例如安全港)中進入幹細胞(例如，胚胎幹細胞、誘導型多能幹細胞、造血幹細胞等)或藉由本文中所描述之任何方法獲得之動物胚胎中，接著移植該胚胎，使得活動物得以出生。接著將該動物餵養至性成熟且允許產生後代，其中至少一些後代包含經編輯之內源基因序列或所整合之轉殖基因。In some embodiments, the transgenic HSC/PC cells, transgenic animals, or combinations of transgenic HSC/PC cells and animals include a transgenic gene encoding a human gene. In some cases, transgenic animals contain knockouts at endogenous loci and replacement of endogenous genes with their human counterparts, allowing the development of in vivo systems that can study human proteins in isolation. Such transgenic gene models can be used for screening purposes to identify small or large biomolecules or other entities that can interact with or modify relevant human proteins. In some aspects, the transgenic gene is integrated into a selected locus (eg, safe harbor) into stem cells (eg, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, etc.) or by any of the methods described herein Of the animal embryos obtained, the embryos are then transferred, resulting in the birth of live animals. The animal is then fed to sexual maturity and allowed to produce offspring, at least some of which comprise the edited endogenous gene sequence or the integrated transgenic gene.

在另一態樣中，本文中提供一種改變細胞中之基因表現(例如，BCL11A、球蛋白基因及BCL11A與球蛋白基因之組合)之方法，該方法包括：在使得一或多種蛋白質得以表現且基因表現得以改變之條件下將一或多種如本文中所描述之核酸酶(如表1中所示)引入細胞中。在某些實施例中，球蛋白基因(例如γ球蛋白或β球蛋白)之表現得以改變(例如增加)。本文中所描述之任何方法均可進一步包括將供體序列(例如，在外源或內源啟動子控制下之轉殖基因或其片段)整合至細胞之基因組中，例如將供體整合在BCL11A基因中之核酸酶裂解位點處或附近。使用病毒載體、作為寡核苷酸、在質體上以及選自使用病毒載體、作為寡核苷酸或在質體上之一或多種方法之組合將供體序列引入細胞中。基因表現得以改變之細胞可為例如紅血細胞(RBC)前驅細胞、造血幹細胞(例如，CD34+細胞)及RBC前驅細胞與造血幹細胞之組合。In another aspect, provided herein is a method of altering gene expression (eg, BCL11A, a globulin gene, and a combination of BCL11A and a globulin gene) in a cell, the method comprising: after causing expression of one or more proteins and One or more nucleases as described herein (as shown in Table 1) are introduced into cells under conditions in which gene expression is altered. In certain embodiments, the expression of globulin genes (eg, gamma globulin or beta globulin) is altered (eg, increased). Any of the methods described herein can further comprise integrating a donor sequence (eg, a transgenic gene or fragment thereof under the control of an exogenous or endogenous promoter) into the genome of the cell, eg, integrating the donor into the BCL11A gene at or near the nuclease cleavage site. The donor sequence is introduced into the cell using a viral vector, as an oligonucleotide, on a plastid, and a combination of one or more methods selected from using a viral vector, as an oligonucleotide, or on a plastid. Cells with altered gene expression can be, for example, red blood cell (RBC) precursor cells, hematopoietic stem cells (eg, CD34+ cells), and combinations of RBC precursor cells and hematopoietic stem cells.

在其他實施例中，本文中提供一種產生在內源BCL11A增強子序列內包含基因組修飾(對BCL11A增強子序列之核苷酸序列之修飾)之經基因修飾之細胞的方法，該方法包括以下步驟：a)使細胞與編碼包含4、5或6個鋅指結構域之鋅指核酸酶的聚核苷酸(例如DNA或mRNA)接觸，其中各鋅指結構域按表1之單列中所示之順序包含識別螺旋區；b)使該細胞處於有助於自該聚核苷酸表現該鋅指蛋白質之條件下；及c)利用足以產生該經基因修飾之細胞的已表現之鋅指蛋白質來修飾該內源BCL11A增強子序列。在某些實施例中，用至少一種細胞因子刺激該等細胞(例如，在步驟(a)之前)。可使用任何適合之方法使該聚核苷酸與該細胞接觸，包括但不限於經由轉染、使用非病毒載體、使用病毒載體、藉由化學手段或藉由暴露於電場(例如電穿孔)。In other embodiments, provided herein is a method of producing a genetically modified cell comprising a genomic modification (modification of the nucleotide sequence of the BCL11A enhancer sequence) within an endogenous BCL11A enhancer sequence, the method comprising the steps of : a) contacting the cell with a polynucleotide (eg DNA or mRNA) encoding a zinc finger nuclease comprising 4, 5 or 6 zinc finger domains, wherein each zinc finger domain is as indicated in a separate column of Table 1 comprising a recognition helical region; b) subjecting the cell to conditions conducive to expression of the zinc finger protein from the polynucleotide; and c) utilizing the expressed zinc finger protein sufficient to produce the genetically modified cell to modify the endogenous BCL11A enhancer sequence. In certain embodiments, the cells are stimulated with at least one cytokine (eg, prior to step (a)). The polynucleotide can be contacted with the cell using any suitable method, including, but not limited to, via transfection, using a non-viral vector, using a viral vector, by chemical means, or by exposure to an electric field (eg, electroporation).

亦提供包含本文中所描述之基因組修飾之一或組合的細胞，包括自藉由本文中所描述之方法產生之細胞傳代而來之細胞。Cells comprising one or a combination of the genomic modifications described herein are also provided, including cells passaged from cells produced by the methods described herein.

亦提供一種治療需要增加球蛋白基因表現之患者的方法，該方法包括以足以增加該患者之球蛋白基因表現之量對該患者投與如本文中所描述之醫藥製劑，其中該醫藥製劑包含經基因修飾之細胞、蛋白質、聚核苷酸以及選自經基因修飾之細胞、蛋白質及聚核苷酸之一或多者的組合。在某些實施例中，已知該患者患有、疑似患有或處於發展地中海型貧血或鐮形血球貧血症之風險下。Also provided is a method of treating a patient in need of increased globulin gene expression, the method comprising administering to the patient a pharmaceutical formulation as described herein in an amount sufficient to increase globulin gene expression in the patient, wherein the pharmaceutical formulation comprises a Genetically modified cells, proteins, polynucleotides, and combinations of one or more selected from the group consisting of genetically modified cells, proteins, and polynucleotides. In certain embodiments, the patient is known to have, is suspected of having, or is at risk of developing thalassemia or sickle cell anemia.

亦提供一種套組，其包含本發明之核酸、蛋白質、經基因修飾之細胞及選自該等核酸、蛋白質及經基因修飾之細胞之一或多者的組合。該套組可包含編碼核酸酶之核酸(例如，RNA分子或包含於適合表現載體中之ZFN、TALEN或CRISPR/Cas系統編碼基因)、核酸酶蛋白質之等分試樣、供體分子、幹細胞自我更新(「幹性」)之適合調節因子、細胞、緩衝液、說明書(例如，用於進行本發明之方法)及其類似物，包括此等套組組件之多種組合。本發明包括但不限於包含至少一個由核酸酶製造之基因組修飾(例如，如表1之單列中所示)的經基因修飾之細胞(例如幹細胞，諸如造血(CD34+)幹細胞或RBC前驅細胞)，其中該基因組修飾處於內源BCL11A增強子序列內，且此外其中該基因組修飾係選自由***、缺失及其組合組成之群，且在SEQ ID NO:1及SEQ ID NO:2中之任一者處、附近或之間包含修飾。在某些實施例中，該細胞為自如本文中所描述之幹細胞傳代而來之經基因修飾之已分化細胞(例如，自造血幹細胞或RBC前驅細胞傳代而來之RBC)。Also provided is a kit comprising the nucleic acids, proteins, genetically modified cells of the present invention, and combinations of one or more selected from the group consisting of nucleic acids, proteins, and genetically modified cells. The kit may comprise nucleic acids encoding nucleases (eg, RNA molecules or genes encoding ZFNs, TALENs or CRISPR/Cas systems contained in suitable expression vectors), aliquots of nuclease proteins, donor molecules, stem cell self Renewed ("dry") suitable regulators, cells, buffers, instructions (eg, for carrying out the methods of the invention) and the like, including various combinations of these kit components. The invention includes, but is not limited to, genetically modified cells (e.g., stem cells, such as hematopoietic (CD34+) stem cells or RBC precursor cells) comprising at least one genome modification made by a nuclease (e.g., as shown in a single column of Table 1), wherein the genomic modification is within the endogenous BCL11A enhancer sequence, and further wherein the genomic modification is selected from the group consisting of insertions, deletions, and combinations thereof, and is in any of SEQ ID NO: 1 and SEQ ID NO: 2 contains modifiers at, near, or between. In certain embodiments, the cells are genetically modified differentiated cells derived from passage of stem cells as described herein (eg, RBCs derived from passage of hematopoietic stem cells or RBC precursor cells).

核酸酶可包含至少一個鋅指核酸酶(ZFN) (例如，如表1中所示)、至少一個TALEN及至少一個ZFN與至少一個TALEN之組合。可將核酸酶以蛋白質形式、作為編碼核酸酶之聚核苷酸或作為蛋白質形式與編碼核酸酶之聚核苷酸的組合引入細胞中。在某些實施例中，該基因組修飾包含***，該***包括整合編碼轉殖基因之供體聚核苷酸。亦提供包含一或多種如本文中所描述之經基因修飾之細胞的醫藥組合物。Nucleases can include at least one zinc finger nuclease (ZFN) (eg, as shown in Table 1), at least one TALEN, and a combination of at least one ZFN and at least one TALEN. Nucleases can be introduced into cells in protein form, as nuclease-encoding polynucleotides, or as a combination of protein form and nuclease-encoding polynucleotides. In certain embodiments, the genomic modification comprises an insertion comprising the integration of a donor polynucleotide encoding the transgenic gene. Also provided are pharmaceutical compositions comprising one or more genetically modified cells as described herein.

亦提供一種包含鋅指蛋白之DNA結合蛋白質，該鋅指蛋白包含4、5或6個包含識別螺旋區之鋅指結構域，其中該等鋅指蛋白按表1之單列中所示之順序包含識別螺旋區。亦提供一種TALE蛋白，其包含複數個重複序列，該等重複序列可結合包含表1中所示之靶位點之部分(例如至少4、5、6個以上)鹼基對的序列。亦提供一種包含如本文中所描述之鋅指蛋白或TALE蛋白及野生型或經工程改造之裂解結構域或裂解半結構域的融合蛋白質，連同編碼如本文中所描述之蛋白質(ZFP、TALE、ZFN、TALEN)的聚核苷酸。亦提供包含一或多種如本文中所描述之聚核苷酸、蛋白質及聚核苷酸與蛋白質之組合的細胞(例如，經分離之幹細胞，諸如造血(CD34+)幹細胞)。亦提供包含一或多種如本文中所描述之蛋白質、聚核苷酸、細胞或其組合之套組。Also provided is a DNA binding protein comprising a zinc finger protein comprising 4, 5 or 6 zinc finger domains comprising a recognition helical region, wherein the zinc finger proteins comprise in the order shown in the individual columns of Table 1 Identify helical regions. Also provided is a TALE protein comprising a plurality of repeats that can bind to sequences comprising a portion (eg, at least 4, 5, 6 or more) base pairs of the target sites shown in Table 1. Also provided is a fusion protein comprising a zinc finger protein or TALE protein as described herein and a wild-type or engineered cleavage domain or cleavage half-domain, together with encoding a protein as described herein (ZFP, TALE, ZFN, TALEN) polynucleotides. Also provided are cells (eg, isolated stem cells, such as hematopoietic (CD34+) stem cells) comprising one or more polynucleotides, proteins, and combinations of polynucleotides and proteins as described herein. Kits comprising one or more proteins, polynucleotides, cells, or combinations thereof as described herein are also provided.

亦描述一種改變細胞(例如RBC前驅細胞、造血幹細胞及RBC前驅細胞與造血幹細胞之組合)中之球蛋白基因表現之方法，該方法包括：在使得一或多種蛋白質得以表現且球蛋白基因(例如，γ球蛋白、β球蛋白及γ球蛋白與β球蛋白之組合)之表現得以改變(例如增加)的條件下向該細胞中引入如本文中所描述之編碼一或多種核酸酶之一或多種聚核苷酸。在某些實施例中，該等方法進一步包括例如使用病毒載體、作為寡核苷酸或在質體上將供體序列整合至細胞之基因組中。該供體序列可包含受內源或外源啟動子控制之轉殖基因。Also described is a method of altering the expression of globulin genes in cells such as RBC precursor cells, hematopoietic stem cells, and combinations of RBC precursor cells and hematopoietic stem cells, the method comprising: after allowing one or more proteins to be expressed and a globulin gene (e.g. introducing into the cell one of the encoding one or more nucleases as described herein or various polynucleotides. In certain embodiments, the methods further comprise integrating the donor sequence into the genome of the cell, eg, using a viral vector, as an oligonucleotide, or on a plastid. The donor sequence may comprise a transgenic gene under the control of an endogenous or exogenous promoter.

亦提供一種產生在內源BCL11A增強子序列(例如，如表1中所示之靶位點)內包含基因組修飾之經基因修飾之細胞的方法，該方法包括以下步驟：(a)使細胞與編碼融合蛋白質之聚核苷酸接觸，該融合蛋白質包含有包含4、5或6個鋅指結構域之鋅指核酸酶，其中各鋅指結構域按表1之單列中所示之順序包含識別螺旋區；(b)使該細胞處於有助於自該聚核苷酸表現該融合蛋白質之條件下；及(c)利用足以產生該經基因修飾之細胞的已表現之融合蛋白質來修飾該內源BCL11A增強子序列。在某些實施例中，該方法進一步包括用至少一種細胞因子刺激該等細胞。可例如使用非病毒遞送系統、病毒遞送系統、遞送媒劑及選自非病毒遞送系統、病毒遞送系統及遞送媒劑之組合在細胞內遞送聚核苷酸，且可包括使該等細胞處於電場下或採用細胞膜破環作為遞送機制(所謂的『壓擠技術』，參見例如Sharei等人, (2015)PLOS ONE doi: 10.1371/journal/pone.0118803)。Also provided is a method of generating a genetically modified cell comprising a genomic modification within an endogenous BCL11A enhancer sequence (eg, a target site as shown in Table 1), the method comprising the steps of: (a) causing the cell to bind to Contacts of polynucleotides encoding fusion proteins comprising zinc finger nucleases comprising 4, 5 or 6 zinc finger domains, wherein each zinc finger domain comprises recognition in the order shown in the individual columns of Table 1 helical region; (b) subjecting the cell to conditions conducive to expression of the fusion protein from the polynucleotide; and (c) modifying the internal Source BCL11A enhancer sequence. In certain embodiments, the method further comprises stimulating the cells with at least one cytokine. The polynucleotides can be delivered intracellularly, for example, using non-viral delivery systems, viral delivery systems, delivery vehicles, and combinations selected from the group consisting of non-viral delivery systems, viral delivery systems, and delivery vehicles, and can include subjecting the cells to an electric field or employ membrane disruption as a delivery mechanism (the so-called "squeeze technique", see eg Sharei et al., (2015) PLOS ONE doi: 10.1371/journal/pone.0118803).

亦提供治療需要增加球蛋白基因表現之患者(例如，已知患有、疑似患有或處於發展諸如地中海型貧血(例如，β型地中海型貧血)或鐮形血球貧血症之血紅素病之風險下的患者)的方法，該方法包括以足以增加該患者中之球蛋白基因表現的量對該患者投與如本文中所描述之醫藥組合物(例如蛋白質、聚核苷酸、細胞或選自蛋白質、聚核苷酸及細胞之組合)。Treatment is also provided for patients in need of increased expression of globulin genes (eg, known to have, suspected of having, or at risk of developing heme diseases such as thalassemia (eg, beta thalassemia) or sickle cell anemia patient), the method comprising administering to the patient a pharmaceutical composition as described herein (e.g., a protein, polynucleotide, cell or selected from the group consisting of an amount sufficient to increase expression of globulin genes in the patient combination of proteins, polynucleotides and cells).

根據整個揭示內容，此等及其他態樣對熟練技術人員將顯而易見。These and other aspects will be apparent to the skilled artisan in view of the entire disclosure.

本文中揭示用於基因組工程改造之組合物及方法，其用於調節BCL11A表現、γ球蛋白表現及BCL11A表現與γ球蛋白表現之組合且用於治療、預防或治療及預防血紅素病。特定言之，經由利用包含具有如表1之單列中所示之識別螺旋區之ZFP的核酸酶進行靶向，在HSC/PC中有效地達成破環BCL11A增強子且引起後續紅血球生成期間相關γ球蛋白表現之變化。此BCL11A及γ球蛋白表現調節尤其適用於治療血紅素病(例如β型地中海型貧血、鐮形血球貧血症)，其中存在不足β球蛋白表現或β球蛋白突變形式表現。使用本發明之方法及組合物，可藉由改變紅血球前驅細胞中之γ球蛋白表現來克服由異常β球蛋白導致之併發症及疾病相關後遺症。 概述Disclosed herein are compositions and methods for genome engineering for modulating BCL11A expression, gamma globulin expression, and combinations of BCL11A expression and gamma globulin expression, and for treating, preventing, or treating and preventing heme disorders. Specifically, disrupting the BCL11A enhancer and causing subsequent erythropoiesis during subsequent erythropoiesis was efficiently achieved in HSC/PC via targeting with a nuclease comprising a ZFP with a recognition helical region as shown in the single column of Table 1. Changes in globulin expression. This modulation of BCL11A and gamma globulin expression is particularly useful in the treatment of heme diseases (eg beta thalassemia, sickle cell anemia) in which there is insufficient beta globulin expression or expression of mutant forms of beta globulin. Using the methods and compositions of the present invention, complications and disease-related sequelae resulting from abnormal beta globulins can be overcome by altering gamma globulin expression in red blood cell precursor cells. Overview

除非另外指示，否則本文中所揭示之方法之實施以及組合物之製備及使用將採用此項技術範圍內之分子生物學、生物化學、染色質結構及分析、計算化學、細胞培養、重組DNA及相關領域內之習知技術。此等技術充分說明於文獻中。參見例如Sambrook等人, MOLECULAR CLONING: A LABORATORY MANUAL, 第二版, Cold Spring Harbor Laboratory Press, 1989及第三版, 2001；Ausubel等人, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987及週期性更新；叢書METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, 第三版, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, 第304卷, 「Chromatin」 (P.M. Wassarman及A. P. Wolffe編), Academic Press, San Diego, 1999；及METHODS IN MOLECULAR BIOLOGY, 第119卷, 「Chromatin Protocols」 (P.B. Becker編) Humana Press, Totowa, 1999。 定義Unless otherwise indicated, the performance of the methods disclosed herein and the preparation and use of the compositions will employ molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and Known technology in the relevant field. Such techniques are well described in the literature. See, e.g., Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 1989 and Third Edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, 3rd edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Cromatin" (P.M. Wassarman and A. P. Wolffe, ed.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (ed. P.B. Becker) Humana Press, Totowa, 1999. definition

術語「核酸」、「聚核苷酸」及「寡核苷酸」可互換使用，並且係指呈線性或環狀構形且呈單鏈或雙鏈形式之去氧核糖核苷酸或核糖核苷酸聚合物。出於本發明之目的，此等術語不應被視為對聚合物之長度具限制性。該等術語可涵蓋天然核苷酸之已知類似物以及在鹼基、糖、磷酸酯部分(例如硫代磷酸酯主鏈主鏈)及選自鹼基、糖及磷酸酯部分之組合中經修飾之核苷酸。一般而言，特定核苷酸之類似物具有相同的鹼基配對特異性；亦即，A之類似物將與T進行鹼基配對。The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleic acid in a linear or circular configuration and in single- or double-stranded form Glycolic acid polymer. For the purposes of the present invention, these terms should not be construed as limiting the length of the polymer. These terms can encompass known analogs of natural nucleotides as well as those modified in bases, sugars, phosphate moieties (eg, phosphorothioate backbone backbones), and combinations selected from the group consisting of bases, sugars, and phosphate moieties. Modified Nucleotides. In general, analogs of a particular nucleotide have the same base pairing specificity; that is, analogs of A will base pair with T.

術語「多肽」、「肽」及「蛋白質」在本文中可互換用於指胺基酸殘基之聚合物。該術語亦適用於一或多個胺基酸為相應天然存在胺基酸之化學類似物或經修飾衍生物的胺基酸聚合物。The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

「結合」係指大分子之間(例如，蛋白質與核酸之間)的序列特異性非共價相互作用。結合相互作用之所有組件並非均需要具有序列特異性(例如，與DNA主鏈主鏈中之磷酸酯殘基接觸)，只要該相互作用總體上具有序列特異性即可。此種相互作用一般以10-6 M-1 以下之解離常數(Kd )為特徵。「親和力」係指結合強度：結合親和力增加與Kd 較低相關。"Binding" refers to sequence-specific non-covalent interactions between macromolecules (eg, between a protein and a nucleic acid). Not all components of a binding interaction need to be sequence-specific (eg, contacts with phosphate residues in the DNA backbone), so long as the interaction is sequence-specific in general. Such interactions are generally characterized by dissociation constants (K d ) below 10 -6 M -1 . "Affinity" refers to binding strength: increased binding affinity correlates with lower Kd .

「結合蛋白」為能夠結合另一分子之蛋白質。結合蛋白可結合例如DNA分子(DNA結合蛋白)、RNA分子(RNA結合蛋白)、蛋白質分子(蛋白質結合蛋白)，或可結合選自DNA分子、RNA分子或蛋白質之分子的組合。在蛋白質結合蛋白之情況下，其可結合自身(以形成同源二聚體、同源三聚體等)，其可結合一或多個不同的蛋白質分子，或其可結合自身與一或多個不同的蛋白質分子。結合蛋白可具有超過一種類型之結合活性。舉例而言，鋅指蛋白具有DNA結合活性、RNA結合活性及蛋白質結合活性。A "binding protein" is a protein capable of binding another molecule. A binding protein can bind, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein), a protein molecule (protein binding protein), or a combination of molecules selected from DNA molecules, RNA molecules, or proteins. In the case of protein binding proteins, it can bind itself (to form homodimers, homotrimers, etc.), it can bind one or more different protein molecules, or it can bind itself with one or more different protein molecules. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding activity, RNA-binding activity, and protein-binding activity.

「鋅指DNA結合蛋白」(或結合結構域)為經由一或多個鋅指以序列特異性方式結合DNA之蛋白質或較大蛋白質內結構域，該一或多個鋅指為藉由鋅離子配位來穩定結構之結合結構域內的胺基酸序列區域。術語鋅指DNA結合蛋白通常縮寫為鋅指蛋白或ZFP。A "zinc finger DNA-binding protein" (or binding domain) is a protein or a larger intraprotein domain that binds DNA in a sequence-specific manner via one or more zinc fingers that bind to DNA via zinc ions Coordinate to stabilize regions of amino acid sequences within the binding domain of the structure. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

「TALE DNA結合結構域」或「TALE」為包含一或多個TALE重複結構域/單位之多肽。重複結構域參與TALE與其同源靶DNA序列之結合。單一「重複單元」(亦稱為「重複序列」)典型地為33至35個胺基酸長且展現與天然存在之TALE蛋白內之其他TALE重複序列具有至少一定程度之序列同源性。A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repeat domain is involved in the binding of TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33 to 35 amino acids long and exhibits at least some degree of sequence homology with other TALE repeats within a naturally occurring TALE protein.

鋅指及TALE結合結構域可經工程改造以結合預定核苷酸序列，例如經由工程改造天然存在之鋅指或TALE蛋白之識別螺旋區(改變其一或多個胺基酸)。因此，經工程改造之DNA結合蛋白(鋅指或TALE)為非天然存在之蛋白質。設計並選擇為用於工程改造DNA結合蛋白之方法的非限制性實例。所設計之DNA結合蛋白為自然界中不存在之蛋白質，其設計/組成主要源自於合理準則。設計之合理準則包括應用替代規則及電腦算法來處理儲存現有ZFP及/或TALE設計及結合資料之資訊的資料庫中的資訊。參見例如美國專利第6,140,081號、第6,453,242號、第6,534,261號及第8,585,526號；亦參見PCT公開案第WO 98/53058號、第WO 98/53059號、第WO 98/53060號、第WO 02/016536號及第WO 03/016496號。Zinc finger and TALE binding domains can be engineered to bind to predetermined nucleotide sequences, eg, by engineering a naturally occurring zinc finger or the recognition helical region of a TALE protein (by altering one or more amino acids thereof). Thus, engineered DNA binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins. Design and selection are non-limiting examples of methods for engineering DNA binding proteins. The designed DNA-binding proteins are proteins that do not exist in nature, and their design/composition is mainly derived from rational criteria. Reasonable design criteria include the application of substitution rules and computer algorithms to process information in databases storing information on existing ZFP and/or TALE design and binding data. See, eg, US Patent Nos. 6,140,081, 6,453,242, 6,534,261, and 8,585,526; see also PCT Publication Nos. WO 98/53058, WO 98/53059, WO 98/53060, WO 02/ 016536 and WO 03/016496.

「所選」鋅指蛋白或TALE為自然界中未發現之蛋白質，其產生主要源自於諸如噬菌體呈現、相互作用陷阱或雜交體選擇之經驗性方法。參見例如美國專利第5,789,538號、第5,925,523號、第6,007,988號、第6,013,453號、第6,200,759號、第8,586,526號、PCT公開案第WO 95/19431號、第WO 96/06166號、第WO 98/53057號、第WO 98/54311號、第WO 00/27878號、第WO 01/60970號、第WO 01/88197號、第WO 02/099084號。"Selected" zinc finger proteins, or TALEs, are proteins not found in nature, the production of which is largely derived from empirical methods such as phage display, interaction traps, or hybrid selection. See, eg, US Patent Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, 8,586,526, PCT Publication Nos. WO 95/19431, WO 96/06166, WO 98/53057 WO 98/54311, WO 00/27878, WO 01/60970, WO 01/88197, WO 02/099084.

「TtAgo」為認為參與基因緘默之原核Argonaute蛋白。TtAgo來源於細菌嗜熱棲熱菌。參見例如Swarts等人,如上 ；G. Sheng等人, (2013)Proc. Natl. Acad. Sci. U.S.A. 111, 652)。「TtAgo系統」為所需要之所有組件，包括例如被TtAgo酶裂解之嚮導DNA。"TtAgo" is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo is derived from the bacterium Thermus thermophilus. See, eg, Swarts et al, supra ; G. Sheng et al, (2013) Proc. Natl. Acad. Sci. USA 111, 652). The "TtAgo system" is all components required, including, for example, guide DNA that is cleaved by the TtAgo enzyme.

「重組」係指兩個聚核苷酸之間交換基因資訊之過程，包括但不限於藉由非同源末端接合(NHEJ)進行供體捕獲及同源重組。出於本發明之目的，「同源重組(HR)」係指例如在經由同源性引導修復機制修復雙鏈斷裂期間發生的此種交換之特化形式。此過程需要核苷酸序列同源性，使用「供體」分子來進行「標靶」分子(亦即，已經歷雙鏈斷裂之分子)之模板修復，且被冠以不同的名稱如「無交叉基因轉化(non-crossover gene conversion)」或「短道基因轉化(short tract gene conversion)」，因為其引起基因資訊自供體轉移至標靶。不希望受任何特定理論束縛，此種轉移可能涉及已斷裂標靶與供體之間形成的異源雙鏈體DNA之錯配矯正、使用供體來再合成將變成標靶之一部分的遺傳資訊的「合成依賴性鏈黏接(synthesis-dependent strand annealing)」「」或其組合。此種特化HR通常引起靶分子之序列改變，從而供體聚核苷酸序列部分或全部併入靶聚核苷酸中。"Recombination" refers to the process of exchanging genetic information between two polynucleotides, including but not limited to donor capture and homologous recombination by non-homologous end joining (NHEJ). For the purposes of the present invention, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a "donor" molecule for template repair of a "target" molecule (that is, a molecule that has undergone a double-strand break), and is given various names such as "no "non-crossover gene conversion" or "short tract gene conversion" because it results in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of the heteroduplex DNA formed between the fragmented target and the donor, use of the donor to resynthesize the genetic information that will become part of the target "synthesis-dependent strand annealing" "" or a combination thereof. Such specialized HRs typically result in sequence changes of the target molecule, whereby the donor polynucleotide sequence is partially or fully incorporated into the target polynucleotide.

在本發明之方法中，一或多種如本文中所描述之靶向核酸酶在靶序列(例如，細胞染色質)中在預定位點產生雙鏈斷裂(DSB)。DSB可藉由同源性引導修復或藉由非同源性引導修復機制而引起***缺失。缺失可包括許多鹼基對。類似地，***可包括許多鹼基對，包括例如整合視情況與斷裂區域中之核苷酸序列具有同源性的「供體」聚核苷酸。可物理整合供體序列，或替代地，使用供體聚核苷酸作為模板以便經由同源重組來修復斷裂，從而將供體中之核苷酸序列全部或部分依原樣引入細胞染色質中。因而，可改變細胞染色質中之第一序列，且在某些實施例中可轉化成供體聚核苷酸中所存在之序列。因而，術語「置換」之使用可理解為表示以一個核苷酸序列置換另一核苷酸序列(亦即，在資訊意義上置換序列)，而未必需要以一個聚核苷酸物理或化學置換另一聚核苷酸。In the methods of the invention, one or more targeted nucleases as described herein create double-strand breaks (DSBs) at predetermined sites in a target sequence (eg, cellular chromatin). DSBs can cause indels by homologous guided repair or by non-homologous guided repair mechanisms. Deletions can include many base pairs. Similarly, insertions can include many base pairs, including, for example, the integration of a "donor" polynucleotide that optionally has homology to the nucleotide sequence in the fragmented region. The donor sequence can be physically integrated, or alternatively, the nucleotide sequence in the donor can be introduced as such, in whole or in part, into the cellular chromatin using the donor polynucleotide as a template to repair the break via homologous recombination. Thus, the first sequence in the cellular chromatin can be altered, and in certain embodiments, can be converted to the sequence present in the donor polynucleotide. Thus, the use of the term "replacement" can be understood to mean the replacement of one nucleotide sequence with another nucleotide sequence (ie, the replacement of the sequence in an informative sense), without necessarily requiring physical or chemical replacement with a polynucleotide another polynucleotide.

在本文中所描述之任何方法中，鋅指蛋白或TALEN之額外配對可用於對細胞內之額外靶位點進行額外雙鏈裂解。In any of the methods described herein, additional pairings of zinc finger proteins or TALENs can be used to perform additional double-stranded cleavage at additional target sites within the cell.

本文中所描述之任何方法均可用於***任何尺寸之供體，或者藉由靶向整合能破壞相關基因之表現的供體序列而使細胞中之一或多個靶序列部分或完全不活化。亦提供具有部分或完全不活化之基因的細胞株。Any of the methods described herein can be used to insert a donor of any size, or to partially or completely inactivate one or more target sequences in a cell by targeted integration of a donor sequence capable of disrupting the expression of the gene of interest. Cell lines with partially or completely inactivated genes are also provided.

在本文中所描述之任何方法中，外源核苷酸序列(「供體序列」或「轉殖基因」)可含有與相關區域中之基因組序列同源但不一致之序列，從而刺激同源重組，以便在相關區域中***不一致序列。因而，在某些實施例中，供體序列中與相關區域中之序列同源之部分展現與所置換之基因組序列具有約80%至99% (或介於其之間的任何整數)序列一致性。在其他實施例中，供體與及基因組序列之間的同源性高於99%，例如在具有超過100個連續鹼基對之供體與基因組序列之間僅有1個核苷酸不同時。在某些情況下，供體序列之非同源部分可含有相關區域中不存在之序列，從而將新的序列引入相關區域中。在此等情況下，非同源序列一般側接具有50至1,000個鹼基對(或介於其之間的任何整數值)或大於1,000之任何數目之鹼基對的序列，該等序列與相關區域中之序列同源或一致。在其他實施例中，供體序列與第一序列不同源，且藉由非同源重組機制***基因組中。In any of the methods described herein, the exogenous nucleotide sequence ("donor sequence" or "transgenic gene") may contain sequences that are homologous but not identical to the genomic sequence in the region of interest, thereby stimulating homologous recombination , in order to insert discordant sequences in the relevant regions. Thus, in certain embodiments, the portion of the donor sequence that is homologous to the sequence in the relevant region exhibits about 80% to 99% (or any integer in between) sequence identity to the replaced genomic sequence sex. In other embodiments, the homology between the donor and the genomic sequence is greater than 99%, such as when only 1 nucleotide differs between the donor and the genomic sequence with more than 100 contiguous base pairs . In some cases, non-homologous portions of the donor sequence may contain sequences that are not present in the region of interest, thereby introducing new sequences into the region of interest. In these cases, the non-homologous sequences are typically flanked by sequences of 50 to 1,000 base pairs (or any integer value in between), or any number of base pairs greater than 1,000, which are associated with The sequences in the related regions are homologous or identical. In other embodiments, the donor sequence is not homologous to the first sequence and inserted into the genome by a non-homologous recombination mechanism.

「裂解」係指DNA分子之共價主鏈斷裂。可藉由多種方法引發裂解，包括但不限於磷酸二酯鍵之酶促或化學水解。單鏈裂解及雙鏈裂解皆有可能，且可能因為兩個不同的單鏈裂解事件而發生雙鏈裂解。DNA裂解可產生平頭末端或交錯狀末端。在某些實施例中，將融合多肽用於靶向雙鏈DNA裂解。"Cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two distinct single-strand cleavage events. DNA cleavage can produce blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used to target double-stranded DNA cleavage.

「裂解半結構域」為連同第二多肽(一致的或不同的)一起形成具有裂解活性(較佳雙鏈裂解活性)之複合物的多肽序列。術語「第一及第二裂解半結構域」、「+及-裂解半結構域」及「右及左裂解半結構域」可互換用於指能二聚之裂解半結構域配對。A "cleavage half-domain" is a polypeptide sequence that, together with a second polypeptide (identical or different), forms a complex with cleavage activity, preferably double-strand cleavage activity. The terms "first and second cleavage half-domains," "+ and - cleavage half-domains," and "right and left cleavage half-domains" are used interchangeably to refer to pairs of cleavage half-domains capable of dimerization.

「經工程改造之裂解半結構域」為已經修飾以便與另一裂解半結構域(例如，另一經工程改造之裂解半結構域)形成絕對異源二聚體的裂解半結構域。亦參見美國專利第7,888,121號、第7,914,796號、第8,034,598號、第8,623,618號及美國專利公開案第2011/0201055號，各案均以全文引用之方式併入本文中。An "engineered cleavage half-domain" is a cleavage half-domain that has been modified to form an absolute heterodimer with another cleavage half-domain (eg, another engineered cleavage half-domain). See also US Patent Nos. 7,888,121, 7,914,796, 8,034,598, 8,623,618, and US Patent Publication No. 2011/0201055, each of which is incorporated herein by reference in its entirety.

術語「序列」係指任何長度之核苷酸序列，其可為DNA或RNA；可為線性、環狀或分枝的，且可為單鏈或雙鏈的。術語「供體序列」係指被***基因組中之核苷酸序列。供體序列可具有任何長度，例如長度在2個與100,000,000個核苷酸之間(或者介於其之間或在其之上的任何整數值)，較佳長度在約100個與100,000個核苷酸之間(或介於其之間的任何整數)，更佳長度在約2000個與20,000個核苷酸之間(或介於其之間的任何值)，且甚至更佳在約5與15 kb之間(或介於其之間的任何值)。The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA; may be linear, circular or branched, and may be single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence inserted into the genome. The donor sequence can be of any length, for example between 2 and 100,000,000 nucleotides in length (or any integer value between or above), preferably between about 100 and 100,000 nuclei in length nucleotides (or any integer therebetween), preferably between about 2000 and 20,000 nucleotides in length (or any value therebetween), and even more preferably about 5 and 15 kb (or anything in between).

「染色質」為包含細胞基因組之核蛋白結構。細胞染色質包含核酸(主要是DNA)及蛋白質，包括組蛋白及非組蛋白染色體蛋白質。大多數真核生物細胞染色質以核小體形式存在，其中核小體核心包含大約150個鹼基對之DNA，該DNA與包含兩份各具有組蛋白H2A、H2B、H3及H4之八聚體締合；且連接子DNA (具有可變長度，視生物體而定)在核小體核心之間延伸。組蛋白H1分子一般與連接子DNA締合。出於本發明之目的，術語「染色質」意在涵蓋細胞核蛋白之所有類型，原核生物的與真核生物的兩者。細胞染色質包括染色體型染色質及附加體型染色質兩者。"Chromatin" is the nuclear protein structure comprising the genome of a cell. Cellular chromatin contains nucleic acids (primarily DNA) and proteins, including histones and non-histone chromosomal proteins. Most eukaryotic cellular chromatin exists in the form of nucleosomes, where the core of the nucleosome contains approximately 150 base pairs of DNA with an octamer containing two copies of histones H2A, H2B, H3, and H4 each and linker DNA (of variable length, depending on the organism) extends between the nucleosome cores. Histone H1 molecules are generally associated with linker DNA. For the purposes of the present invention, the term "chromatin" is intended to encompass all types of nuclear proteins, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin.

「染色質」為包含全部或部分細胞基因組之染色質複合物。細胞基因組通常以其核型為特徵，其為構成細胞基因組之所有染色體之集合。細胞基因組可包含一或多個染色體。"Chromatin" is the chromatin complex that contains all or part of a cell's genome. The genome of a cell is usually characterized by its karyotype, which is the collection of all the chromosomes that make up the genome of a cell. The genome of a cell can contain one or more chromosomes.

「附加體」為複製型核酸、核蛋白複合物或包含並非細胞染色體核型之一部分的核酸的其他結構。附加體之實例包括質體及某些病毒基因組。An "episome" is a replicating nucleic acid, nucleoprotein complex, or other structure comprising nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plastids and certain viral genomes.

「可及區」為細胞染色質中之位點，其中核酸中所存在之靶位點可由能識別該靶位點之外源分子結合。不希望受任何特定理論束縛，據信可及區為未包封在核小體結構中之區域。不同的可及區結構通常可藉由其對化學探針及酶探針(例如，核酸酶)之敏感性而加以偵測。An "accessible region" is a site in cellular chromatin where a target site present in a nucleic acid can be bound by a foreign molecule capable of recognizing the target site. Without wishing to be bound by any particular theory, it is believed that the accessible regions are regions that are not encapsulated in the nucleosome structure. Different accessible region structures can generally be detected by their sensitivity to chemical and enzymatic probes (eg, nucleases).

「靶位點」或「靶序列」為限定結合分子在存在足以發生結合之條件時將結合之核酸之一部分的核酸序列。A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind in the presence of conditions sufficient for binding to occur.

「外源」分子為正常情況下不存在於細胞中但可藉由一或多種遺傳學、生物化學或其他方法引入細胞中之分子。「正常情況下存在於細胞中」係相對於該細胞之特定發育階段及環境條件而確定。因而，舉例而言，僅在肌肉之胚胎發育期間存在之分子相對於成年肌肉細胞為外源分子。類似地，由熱休克誘導之分子相對於非熱休克細胞為外源分子。外源分子可包含例如功能錯誤內源分子之功能型式或功能正常內源分子之功能錯誤型式。A "foreign" molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods. "Normally present in a cell" is determined relative to the particular developmental stage and environmental conditions of the cell. Thus, for example, molecules that are present only during embryonic development of muscle are foreign to adult muscle cells. Similarly, molecules induced by heat shock are exogenous molecules relative to non-heat shock cells. An exogenous molecule may comprise, for example, a functional version of a malfunctioning endogenous molecule or a malfunctioning version of a properly functioning endogenous molecule.

外源分子尤其可為小分子，諸如藉由組合化學方法產生者；或大分子，諸如蛋白質、核酸、碳水化合物、脂質、糖蛋白、脂蛋白、聚醣、以上分子之任何經修飾衍生物或包含一或多種以上分子之任何複合物。核酸包括DNA及RNA，可為單鏈或雙鏈的；可為線性、分枝或環狀的；且可具有任何長度。核酸包括能夠形成雙鏈體之核酸以及形成三鏈體之核酸。參見例如美國專利第5,176,996號及第5,422,251號。蛋白質包括但不限於DNA結合蛋白、轉錄因子、染色質重塑因子、甲基化DNA結合蛋白、聚合酶、甲基化酶、去甲基酶、乙醯化酶、去乙醯基酶、激酶、磷酸酶、整合酶、重組酶、連接酶、拓撲異構酶、迴旋酶及解旋酶。Exogenous molecules can be, inter alia, small molecules, such as those produced by combinatorial chemical methods; or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, glycans, modified derivatives of any of the above, or Any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include nucleic acids capable of forming duplexes as well as nucleic acids that form triplexes. See, eg, US Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases , phosphatase, integrase, recombinase, ligase, topoisomerase, gyrase and helicase.

外源分子可為與內源分子相同類型之分子，例如外源蛋白質或核酸。舉例而言，外源核酸可包含感染性病毒基因組、引入細胞中之質體或附加體或正常情況下不存在於細胞中之染色體。用於將外源分子引入細胞中之方法對於熟習此項技術者為已知的，並且包括但不限於脂質介導之轉移(亦即，脂質體，包括中性脂質及陽離子脂質)、電穿孔、直接注入、細胞融合、粒子轟擊、磷酸鈣共沈澱、DEAE-葡聚糖介導之轉移及病毒載體介導之轉移。外源分子亦可為與內源分子相同類型之分子，但來源於與細胞所來源之物種不同的物種。舉例而言，可將人類核酸序列引入最初來源於小鼠或倉鼠之細胞株中。An exogenous molecule can be the same type of molecule as an endogenous molecule, such as an exogenous protein or nucleic acid. For example, the exogenous nucleic acid may comprise an infectious viral genome, a plastid or episome introduced into a cell, or a chromosome that is not normally present in a cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (ie, liposomes, including neutral lipids and cationic lipids), electroporation , direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule, but derived from a different species than the one from which the cell is derived. For example, human nucleic acid sequences can be introduced into cell lines originally derived from mice or hamsters.

相形之下，「內源」分子為正常在特定環境條件下、特定發育階段存在於特定細胞中之分子。舉例而言，內源核酸可包含染色體、粒線體、葉綠體或其他細胞器之基因組或天然存在之附加體型核酸。其他內源分子可包括蛋白質，例如轉錄因子及酶。In contrast, "endogenous" molecules are molecules that are normally present in specific cells under specific environmental conditions and at specific stages of development. For example, an endogenous nucleic acid may comprise the genome of a chromosome, mitochondria, chloroplast, or other organelle, or a naturally occurring episomal nucleic acid. Other endogenous molecules can include proteins such as transcription factors and enzymes.

如本文中所使用，術語「外源核酸之產物」包括聚核苷酸及多肽產物兩者，例如，轉錄產物(聚核苷酸，諸如RNA)及轉譯產物(多肽)。As used herein, the term "product of exogenous nucleic acid" includes both polynucleotide and polypeptide products, eg, transcription products (polynucleotides, such as RNA) and translation products (polypeptides).

「融合」分子為兩個以上次單元連接(較佳共價地)之分子。次單元分子可為相同化學類型之分子，或可為不同化學類型之分子。第一種類型之融合分子之實例包括但不限於融合蛋白質(例如，ZFP或TALE DNA結合結構域與一或多個活化結構域之間的融合物)及融合核酸(例如，編碼如上所描述之融合蛋白質的核酸)。第二種類型之融合分子之實例包括但不限於形成三鏈體之核酸與多肽之間的融合物及小溝結合子與核酸之間的融合物。A "fusion" molecule is a molecule in which two or more subunits are linked, preferably covalently. Subunit molecules can be molecules of the same chemical type, or can be molecules of different chemical types. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (eg, fusions between a ZFP or TALE DNA binding domain and one or more activation domains) and fusion nucleic acids (eg, encoding as described above) fusion protein nucleic acid). Examples of the second type of fusion molecule include, but are not limited to, fusions between triplex-forming nucleic acids and polypeptides and fusions between minor groove binders and nucleic acids.

在細胞中表現融合蛋白質可由將該融合蛋白質遞送至該細胞或藉由將編碼該融合蛋白質之聚核苷酸遞送至細胞，在其中轉錄該聚核苷酸並且轉譯轉錄物以產生該融合蛋白質而發生。在細胞中表現蛋白質亦可涉及反式剪接、多肽裂解及多肽連接。本發明在其他部分提供聚核苷酸及多肽遞送至細胞之方法。Expression of a fusion protein in a cell can be achieved by delivering the fusion protein to the cell or by delivering a polynucleotide encoding the fusion protein to the cell, where the polynucleotide is transcribed and the transcript is translated to produce the fusion protein occur. Expression of proteins in cells can also involve trans-splicing, polypeptide cleavage, and polypeptide ligation. The invention provides, in other parts, methods of delivering polynucleotides and polypeptides to cells.

出於本發明之目的，「基因」包括編碼基因產物之DNA區域(參見下文)以及調控基因產物產生之所有DNA區域，無論此種調控序列與編碼序列、轉錄序列及編碼序列與轉錄序列之組合相鄰與否。相應地，基因包括但未必限於啟動子序列、終止子、轉譯調控序列(諸如核糖體結合位點及內部核糖體進入位點)、增強子、緘默子、絕緣子、邊界元件、複製起點、基質附接位點及基因座控制區。For the purposes of the present invention, a "gene" includes a region of DNA encoding a gene product (see below) and all regions of DNA that regulate the production of a gene product, regardless of the combination of such regulatory and coding sequences, transcribed sequences, and coding and transcribed sequences adjacent or not. Accordingly, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences (such as ribosome binding sites and internal ribosomal entry sites), enhancers, silencers, insulators, border elements, origins of replication, matrix attachments Junction sites and locus control regions.

「基因表現」係指基因中所含之資訊轉化至基因產物中。基因產物可為基因之直接轉錄產物(例如mRNA、tRNA、rRNA、反義RNA、mRNA、核糖酶、結構RNA或任何其他類型之RNA)或由轉譯mRNA而產生之蛋白質。基因產物亦包括藉由諸如加帽、聚腺苷酸化、甲基化及編輯之方法加以修飾之RNA以及藉由例如甲基化、乙醯化、磷酸化、泛素化、ADP核糖基化、肉豆蔻基化及糖基化加以修飾之蛋白質。"Gene expression" refers to the transformation of the information contained in a gene into a gene product. A gene product can be a direct transcription product of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, mRNA, ribozyme, structural RNA, or any other type of RNA) or a protein resulting from translation of mRNA. Gene products also include RNAs modified by methods such as capping, polyadenylation, methylation, and editing and by methods such as methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, Proteins modified by myristylation and glycosylation.

「調節」基因表現係指改變基因之活性。調節表現可包括但不限於基因活化及基因壓抑。基因組編輯(例如，裂解、改變、不活化、隨機突變)可用於調節表現。基因不活化係指與不包括如本文中所描述之ZFP、TALE或CRISPR/Cas系統之細胞相比在基因表現方面之任何減少。因而，基因不活化可部分的或完全的。"Regulating" gene expression refers to altering the activity of a gene. Regulated expression can include, but is not limited to, gene activation and gene repression. Genome editing (eg, cleavage, alteration, inactivation, random mutagenesis) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression compared to cells that do not include ZFP, TALE or CRISPR/Cas systems as described herein. Thus, gene inactivation can be partial or complete.

「受保護」之mRNA為已用某種方式改變mRNA以增加mRNA之穩定性或轉譯的mRNA。保護之實例包括使用以2-硫代尿苷(s2U)及5-甲基胞苷(m5C)置換至多25%胞苷及尿苷殘基。所得mRNA與其未經修飾之對應物相比展現較低免疫原性及較大穩定性。(參見Karikó等人, ((2012),Molecular Therapy , 第16卷, 第11號, 第1833-1844頁)。其他變化包括添加所謂的ARCA帽，其增加試管內產生之mRNA之轉譯能力(參見美國專利第7,074,596號)。A "protected" mRNA is one that has been altered in some way to increase the stability or translation of the mRNA. Examples of protection include the use of substitutions of up to 25% of cytidine and uridine residues with 2-thiouridine (s2U) and 5-methylcytidine (m5C). The resulting mRNA exhibits lower immunogenicity and greater stability compared to its unmodified counterpart. (See Karikó et al., ((2012), Molecular Therapy , Vol. 16, No. 11, pp. 1833-1844). Other changes include the addition of so-called ARCA caps, which increase the translational capacity of mRNA produced in vitro (see US Patent No. 7,074,596).

「相關區域」為期望與外源分子結合之任何細胞染色質區域，舉例而言，諸如基因或者在基因內或與基因相鄰之非編碼序列。結合可出於靶向DNA裂解、靶向重組及靶向DNA裂解與靶向重組之組合的目的。舉例而言，相關區域可存在於染色體、附加體、細胞器基因組(例如，粒線體、葉綠體)或感染性病毒基因組中。相關區域可在基因之編碼區內、在所轉錄之非編碼區(舉例而言，諸如前導序列、尾部序列或內含子)內或在編碼區上游或下游之非轉錄區內。相關區域之長度可小至單一核苷酸配對或高達2,000個核苷酸配對，或任何整數值之核苷酸配對。A "region of interest" is any region of cellular chromatin that is desired to bind to a foreign molecule, such as, for example, a gene or a noncoding sequence within or adjacent to a gene. Binding can be for the purpose of targeted DNA cleavage, targeted recombination, and a combination of targeted DNA cleavage and targeted recombination. For example, a region of interest can be present in a chromosome, episome, organelle genome (eg, mitochondria, chloroplast), or infectious virus genome. A region of interest can be within a coding region of a gene, within a transcribed non-coding region (such as, for example, a leader sequence, a trailer sequence, or an intron), or within a non-transcribed region upstream or downstream of a coding region. The length of a region of interest can be as small as a single nucleotide pair or as high as 2,000 nucleotide pairs, or any integer value of nucleotide pairs.

「真核生物」細胞包括但不限於真菌細胞(諸如酵母)、植物細胞、動物細胞、哺乳動物細胞及人類細胞(例如，T細胞)。"Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (eg, T cells).

術語「可操作連接」及「可操作地連接」在關於兩個以上組件(諸如序列元件)並列時可互換使用，其中排列該等組件，以使得兩個組件均正常發揮功能且允許該等組件中之至少一個可介導施加於其他組件中之至少一個的功能的可能性。舉例而言，若轉錄調控序列響應於存在或不存在一或多種轉錄調控因子而控制編碼序列之轉錄水準，則該轉錄調控序列(諸如啟動子)可操作地連接至該編碼序列。轉錄調控序列一般順式地與編碼序列可操作地連接，但不必與其直接相鄰。舉例而言，增強子為可操作地連接至編碼序列之轉錄調控序列，即使其並非連續的。The terms "operably linked" and "operably linked" are used interchangeably with respect to the juxtaposition of two or more components, such as sequential elements, wherein the components are arranged so that both components function properly and allow the components The likelihood that at least one of the components may mediate a function imposed on at least one of the other components. For example, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Transcriptional regulatory sequences are typically operably linked to the coding sequence in cis, but need not be directly adjacent thereto. For example, an enhancer is a transcriptional regulatory sequence operably linked to a coding sequence, even if it is not contiguous.

關於融合多肽，術語「可操作地連接」可能係指各組件在與另一組件連接時執行與其未如此連接時相同的功能。舉例而言，關於ZFP、TALE或Cas DNA結合結構域與活化結構域融合之融合多肽，若在該融合多肽中，ZFP、TALE或Cas DNA結合結構域部分能夠結合其靶位點、其結合位點及其靶位點與結合位點之組合，而活化結構域能夠上調基因表現，則ZFP、TALE或Cas DNA結合結構域及活化結構域呈可操作連接。當ZFP、TALE或Cas DNA結合結構域與裂解結構域融合之融合多肽時，若在該融合多肽中，ZFP、TALE或Cas DNA結合結構域部分能夠結合其靶位點、其結合位點及其靶位點與其結合位點之組合，而裂解結構域能夠裂解靶位點附近之DNA，則ZFP、TALE或Cas DNA結合結構域及裂解結構域呈可操作連接。With respect to fusion polypeptides, the term "operably linked" may mean that each component when linked to another component performs the same function as when it is not so linked. For example, with respect to a fusion polypeptide in which a ZFP, TALE or Cas DNA binding domain is fused to an activation domain, if in the fusion polypeptide, the portion of the ZFP, TALE or Cas DNA binding domain is capable of binding its target site, its binding site ZFP, TALE or Cas DNA binding domain and activation domain are operably linked. When a ZFP, TALE or Cas DNA binding domain is fused to a fusion polypeptide with a cleavage domain, if in the fusion polypeptide, the portion of the ZFP, TALE or Cas DNA binding domain is capable of binding its target site, its binding site and its The combination of the target site and its binding site, and the cleavage domain is capable of cleaving DNA near the target site, the ZFP, TALE or Cas DNA binding domain and the cleavage domain are operably linked.

蛋白質、多肽或核酸之「功能片段」為序列與全長蛋白質、多肽或核酸不一致卻保留與全長蛋白質、多肽或核酸相同之功能的蛋白質、多肽或核酸。功能片段可具有與相應天然分子相比更多、更少或相同之殘基數目，可含有一或多個胺基酸或核苷酸取代，且可為具有與相應天然分子相比更多、更少或相同之殘基數目與含有一或多個胺基酸或核苷酸取代的組合。用於測定核酸功能(例如，編碼功能、與另一核酸雜交之能力)的方法在此項技術中為眾所周知的。類似地，用於測定蛋白質功能之方法為眾所周知的。舉例而言，可例如藉由過濾器結合、電泳遷移率變化或免疫沈澱分析法來測定多肽之DNA結合功能。可藉由凝膠電泳來分析DNA裂解。參見Ausubel等人,同上 。蛋白質與另一蛋白質相互作用之能力可例如藉由共免疫沈澱、雙雜交分析或互補(遺傳學及生物化學)來測定。A "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid that is not identical in sequence to the full-length protein, polypeptide or nucleic acid but retains the same function as the full-length protein, polypeptide or nucleic acid. Functional fragments may have more, less, or the same number of residues compared to the corresponding native molecule, may contain one or more amino acid or nucleotide substitutions, and may have more, less, or the same number of residues compared to the corresponding native molecule. Fewer or the same number of residues and combinations containing one or more amino acid or nucleotide substitutions. Methods for determining nucleic acid function (eg, encoding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining protein function are well known. For example, the DNA binding function of a polypeptide can be determined, eg, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be analyzed by gel electrophoresis. See Ausubel et al, supra . The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid analysis, or complementation (genetics and biochemistry).

「載體」能夠將基因序列轉移至靶細胞。典型地，「載體構建體」、「表現載體」及「基因轉移載體」意謂能夠指導相關基因之表現且可將基因序列轉移至靶細胞的任何核酸構建體。因而，該術語包括選殖及表現媒劑以及整合載體。"Vectors" are capable of transferring gene sequences to target cells. Typically, "vector construct", "expression vector" and "gene transfer vector" mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer a gene sequence to a target cell. Thus, the term includes cloning and expression vehicles as well as integrating vectors.

術語「個體」與「患者」可互換使用，並且係指哺乳動物，諸如人類患者及非人類靈長類動物，以及實驗動物，諸如豬、牛、兔、犬、貓、大鼠、小鼠及其他動物。相應地，術語「個體」或「患者」在用於本文中時意謂可投與本發明之幹細胞的任何哺乳動物患者或個體。本發明之個體包括已暴露於一或多種化學毒素，包括例如神經毒素之彼等個體。The terms "individual" and "patient" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as experimental animals, such as pigs, cows, rabbits, dogs, cats, rats, mice, and other animals. Accordingly, the terms "individual" or "patient" as used herein mean any mammalian patient or individual to which the stem cells of the invention may be administered. Individuals of the present invention include those that have been exposed to one or more chemical toxins, including, for example, neurotoxins.

「幹性」係指任何細胞以類似幹細胞之方式起作用的相對能力，亦即，全潛能性、多潛能性(pluripotency)、多重潛能性(multipotency)或寡潛能性程度，及任何特定幹細胞可具有之擴增或無限自我更新。 核酸酶"Stemness" refers to the relative ability of any cell to function in a stem cell-like manner, that is, the degree of pluripotency, pluripotency, multipotency, or oligopotency, and any particular stem cell may Possesses the augmentation or infinite self-renewal. Nuclease

本文中描述適用於活體內裂解攜帶轉殖基因之供體分子的組合物，特定言之核酸酶，及用於裂解細胞基因組從而以靶向方式將轉殖基因整合至基因組中的核酸酶。在某些實施例中，該等核酸酶中之一或多種為天然存在的。在其他實施例中，該等核酸酶中之一或多種為非天然存在的，亦即，在DNA結合結構域、裂解結構域及DNA結合結構域與裂解結構域之組合中經工程改造。舉例而言，可改變天然存在之核酸酶的DNA結合結構域以結合所選靶位點(例如，已經工程改造以結合與同源結合位點不同的位點的兆鹼基核酸酶)。在其他實施例中，該核酸酶包含異源DNA結合結構域及裂解結構域(例如，鋅指核酸酶；TAL效應因子結構域DNA結合蛋白；具有異源裂解結構域之兆鹼基核酸酶DNA結合結構域)。 A. DNA結合結構域Described herein are compositions suitable for in vivo cleavage of donor molecules carrying transgenic genes, in particular nucleases, and nucleases for lysing cellular genomes to integrate the transgenic genes into the genome in a targeted manner. In certain embodiments, one or more of the nucleases are naturally occurring. In other embodiments, one or more of the nucleases are non-naturally occurring, ie, engineered in DNA binding domains, cleavage domains, and combinations of DNA binding and cleavage domains. For example, the DNA binding domain of a naturally occurring nuclease can be altered to bind to a target site of choice (eg, a meganuclease that has been engineered to bind to a site different from the cognate binding site). In other embodiments, the nuclease comprises a heterologous DNA binding domain and a cleavage domain (eg, a zinc finger nuclease; a TAL effector domain DNA binding protein; a megabase nuclease DNA with a heterologous cleavage domain) binding domain). A. DNA binding domain

在某些實施例中，用於活體內裂解、細胞基因組靶向裂解及活體內裂解與細胞基因組靶向裂解之組合的一或多種核酸酶之DNA結合結構域包含鋅指蛋白。單一鋅指蛋白由多個鋅指結構域(例如，3、4、5、6個以上鋅指結構域)組成。各鋅指結構域之長度為約30個胺基酸，其含有保持呈允許該蛋白質結合靶序列之特定構形的β轉折(含有兩個鋅配位殘基)及α螺旋(含有兩個不變鋅配位殘基)。可使用β轉折中有兩個半胱胺酸(Cys)鋅配位殘基且α螺旋中有兩個組胺酸(His)鋅配位殘基之典型(C2H2)鋅指結構域或非典型(CH3)。參見例如美國專利第9,234,187號。具有7個胺基酸之識別螺旋包含在β轉折之鋅配位殘基與α螺旋之鋅配位殘基之間。將識別螺旋區編號為鋅指結構域內-1至+6，且此識別區外之胺基酸(且不包括鋅配位殘基)被稱為主鏈殘基。In certain embodiments, the DNA binding domain of one or more nucleases for in vivo lysis, cellular genome targeted cleavage, and a combination of in vivo cleavage and cellular genome targeted cleavage comprises a zinc finger protein. A single zinc finger protein consists of multiple zinc finger domains (eg, 3, 4, 5, 6 or more zinc finger domains). Each zinc finger domain is about 30 amino acids in length and contains a beta turn (containing two zinc-coordinating residues) and an alpha helix (containing two different Zinc coordinating residues). A canonical (C2H2) zinc finger domain or atypical with two cysteine (Cys) zinc coordinating residues in the beta turn and two histidine (His) zinc coordinating residues in the alpha helix can be used (CH3). See, eg, US Patent No. 9,234,187. A recognition helix with 7 amino acids is contained between the zinc coordinating residues of the beta turn and the zinc coordinating residues of the alpha helix. The recognition helix region is numbered -1 to +6 within the zinc finger domain, and amino acids outside this recognition region (and excluding zinc coordinating residues) are referred to as backbone residues.

鋅指蛋白較佳為非天然的，其中識別螺旋經工程改造以結合所選靶位點。參見例如Beerli等人, (2002) Nature Biotechnol. 20 :135-141；Pabo等人, (2001) Ann. Rev. Biochem.70 :313-340；Isalan等人, (2001) Nature Biotechnol.19 :656-660；Segal等人, (2001) Curr. Opin. Biotechnol.12 :632-637；Choo等人, (2000) Curr. Opin. Struct. Biol.10:411-416；美國專利第6,453,242號、第6,534,261號、第6,599,692號、第6,503,717號、第6,689,558號、第7,030,215號、第6,794,136號、第7,067,317號、第7,262,054號、第7,070,934號、第7,361,635號、第7,253,273號及美國專利公開案第2005/0064474號、第2007/0218528號、第2005/0267061號，均以全文引用之方式併入本文中。Zinc finger proteins are preferably non-native, wherein the recognition helix is engineered to bind to the target site of choice. See, eg, Beerli et al, (2002) Nature Biotechnol. 20:135-141; Pabo et al, (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al, (2001) Nature Biotechnol. 19:656 -660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; No. 0064474, No. 2007/0218528, No. 2005/0267061, all incorporated herein by reference in their entirety.

與天然存在之鋅指蛋白相比，經工程改造之鋅指結合結構域可具有新穎結合特異性。工程改造方法包括但不限於合理設計及不同的選擇類型。合理設計包括例如使用包含三鏈體(或四鏈體)核苷酸序列及個別鋅指胺基酸序列之資料庫，其中各三鏈體或四鏈體核苷酸序列與可結合特定三鏈體或四鏈體序列之一或多個鋅指胺基酸序列締合。參見例如共有美國專利第6,453,242號及第6,534,261號，該等專利以全文引用之方式併入本文中。Engineered zinc finger binding domains can have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include but are not limited to rational design and different selection types. Rational design includes, for example, the use of databases comprising triplex (or quadruplex) nucleotide sequences and individual zinc finger amino acid sequences, where each triplex or quadruplex nucleotide sequence is associated with a specific triplex One or more zinc finger amino acid sequences are associated with one or more of the sequences of the body or quadruplex. See, eg, co-owned US Pat. Nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entirety.

此外，在某些實施例中，ZFP DNA結合結構域進一步包含對一或多個組件鋅指結構域之主鏈的一或多個修飾。ZFP對靶DNA序列之特異性視鋅指結構域與特定DNA鹼基之間，特定言之，識別螺旋區與靶位點之間的序列特異性接觸而定(典型地，各識別螺旋結合具有3個核苷酸之標靶亞位點。另外，鋅指結構域亦包含參與同DNA主鏈之磷酸酯之非特異性相互作用的胺基酸殘基。Elrod-Erickson等人((1996)Structure 4:1171)藉由使鋅指蛋白及其同源DNA標靶共結晶而證明存在能夠藉由形成氫鍵而與DNA主鏈上之磷酸酯相互作用的特定胺基酸。採用眾所周知的Zif268主鏈之鋅指蛋白典型地具有精胺酸作為其β片第二股鏈之胺基末端殘基，其亦為第二不變半胱胺酸之羧基末端之第二位置。此位置因為其為α螺旋起點前之第5個殘基而可稱為各鋅指結構域內之(-5)(及相對於編號-1至+6之識別螺旋之位置-5)。此位置上之精胺酸可經由與其側鏈胍基形成帶電氫鍵而與DNA主鏈上之磷酸酯相互作用。Zif268主鏈中之鋅指蛋白亦通常在與第一不變半胱胺酸之胺基末端相距4個殘基之位置上具有離胺酸。此位置因為其對於在鋅配位半胱胺酸殘基之間有兩個殘基之鋅指為α螺旋起點之前的第14個殘基而可稱為各指內之(-14) (及相對於編號-1至+6之識別螺旋區之位置-14)。該離胺酸可經由與其側鏈胺基形成水介導之帶電氫鍵而與DNA主鏈上之磷酸酯相互作用。因為發現磷酸酯基均沿著DNA主鏈，故鋅指與DNA分子之間的此種類型之相互作用一般被視為非序列特異性的(J. Miller, Massachusetts Institute of Technology Ph.D. Thesis, 2002)。Furthermore, in certain embodiments, the ZFP DNA binding domain further comprises one or more modifications to the backbone of one or more component zinc finger domains. The specificity of ZFPs for target DNA sequences depends on the sequence-specific contacts between the zinc finger domains and specific DNA bases, in particular, between the recognition helix region and the target site (typically, each recognition helix binds with Targeting subsite of 3 nucleotides. In addition, the zinc finger domain also contains amino acid residues that participate in nonspecific interactions with phosphates of the DNA backbone. Elrod-Erickson et al. ((1996) Structure 4:1171) demonstrated the existence of specific amino acids capable of interacting with phosphates on the DNA backbone by forming hydrogen bonds by co-crystallizing zinc finger proteins and their cognate DNA targets. Using the well-known Zif268 Zinc finger proteins of the backbone typically have arginine as the amino-terminal residue of the second strand of their beta sheet, which is also the second position of the carboxy-terminus of the second invariant cysteine. is the 5th residue before the start of the alpha helix and may be referred to as (-5) within each zinc finger domain (and position -5 relative to the recognition helix numbered -1 to +6). Amino acids can interact with phosphates on the DNA backbone via the formation of charged hydrogen bonds with their side chain guanidine groups. The zinc finger proteins in the Zif268 backbone are also typically located at a distance from the amino terminus of the first invariant cysteine lysine at a 4-residue position. This position is possible because it is the 14th residue before the start of the alpha helix for the zinc finger with two residues between the zinc-coordinating cysteine residues. Referred to as (-14) within each finger (and position -14 relative to the recognition helical regions numbered -1 to +6). This lysine can be formed by water-mediated charged hydrogen bonding with its side chain amine group. Interacts with phosphates on the DNA backbone. Since the phosphate groups are found along the DNA backbone, this type of interaction between zinc fingers and DNA molecules is generally regarded as non-sequence specific (J. Miller, Massachusetts Institute of Technology Ph.D. Thesis, 2002).

最近之研究已假定一些核酸酶中之非特異性磷酸酯接觸側鏈亦可佔彼等核酸酶之非特異性之一定量(Kleinstiver等人, (2016)Nature 529(7587):490-5；Guilinger等人,(2014)Nat Meth : 429-435)。研究人員已提出此等核酸酶可能具有『過量DNA結合能』，意謂該等核酸酶對其DNA標靶之親和力可能超過實質上結合並裂解標靶位點所需之親和力。因而，試圖減少TALE DNA結合結構域(Guilinger,如上 )或Cas9 DNA結合結構域(Kleinstiver,如上 )中之陽離子電荷以降低此等核酸酶之DNA結合能，從而增加試管內裂解特異性。然而，其他研究(Sternberg等人,(2015)Nature 527(7576):110-113)亦表明Cas9 DNA結合結構域之在Kleinstiver研究中突變之一些陽離子胺基酸在Cas9核酸酶結構域之適當摺疊及活化中的作用。因而，此等胺基酸在Cas9活性中之確切作用為未知的。Recent studies have postulated that nonspecific phosphate contacting side chains in some nucleases can also account for a quantification of their nonspecificity (Kleinstiver et al., (2016) Nature 529(7587):490-5; Guilinger et al., (2014) Nat Meth : 429-435). Researchers have suggested that these nucleases may have "excess DNA binding energy," meaning that the affinity of the nucleases for their DNA targets may exceed that required to substantially bind and cleave the target site. Thus, attempts were made to reduce the cationic charge in the TALE DNA binding domain ( Guilinger , supra) or the Cas9 DNA binding domain ( Kleinstiver , supra) to reduce the DNA binding energy of these nucleases, thereby increasing in vitro cleavage specificity. However, other studies (Sternberg et al., (2015) Nature 527(7576):110-113) have also demonstrated proper folding of some cationic amino acids of the Cas9 DNA binding domain mutated in the Kleintiver study in the Cas9 nuclease domain and activation. Thus, the exact role of these amino acids in Cas9 activity is unknown.

本發明之方法及組合物因而包括ZFP DNA結合結構域(『ZFP主鏈』)內可與DNA主鏈上之磷酸酯發生非特異性相互作用之胺基酸的突變，但其在DNA識別螺旋中不包含變化。因而，本發明包括ZFP主鏈中不為核苷酸標靶特異性所需之陽離子胺基酸殘基的突變。在一些實施例中，ZFP主鏈中之此等突變包含使陽離子胺基酸殘基突變至中性或陰離子胺基酸殘基。在一些實施例中，ZFP主鏈中之此等突變包含使極性胺基酸殘基突變至中性或非極性胺基酸殘基。在較佳實施例中，相對於DNA結合螺旋，進行位置(-5)、位置(-9)、位置(-14)上之突變以及選自位置(-5)、位置(-9)及位置(-14)上之突變的突變的組合。在一些實施例中，鋅指可包含位置(-5)、位置(-9)、位置(-14)上之一或多個突變以及選自位置(-5)、位置(-9)及位置(-14)上之突變的突變的組合。在其他實施例中，多指型鋅指蛋白中之一或多個鋅指可在(-5)、(-9)、(-14)以及選自(-5)、(-9)及(-14)之組合處包含突變。在一些實施例中，使(-5)、(-9)、(-14)以及選自(-5)、(-9)及(-14)之組合處的胺基酸(例如精胺酸(R)或離胺酸(K))突變至丙胺酸(A)、白胺酸(L)、Ser (S)、Asp (D)、Glu (E)、Tyr (Y)及/或麩醯胺酸(Q)。The methods and compositions of the invention thus include mutations within the ZFP DNA-binding domain ("ZFP backbone") of amino acids that can nonspecifically interact with phosphates on the DNA backbone, but which are in the DNA recognition helix does not contain changes. Thus, the present invention includes mutation of cationic amino acid residues in the ZFP backbone that are not required for nucleotide target specificity. In some embodiments, such mutations in the ZFP backbone comprise mutating cationic amino acid residues to neutral or anionic amino acid residues. In some embodiments, such mutations in the ZFP backbone comprise mutating polar amino acid residues to neutral or non-polar amino acid residues. In a preferred embodiment, the mutations at position (-5), position (-9), position (-14) and are selected from the group consisting of position (-5), position (-9) and position relative to the DNA binding helix A combination of mutations above (-14). In some embodiments, the zinc finger may comprise one or more mutations at position (-5), position (-9), position (-14) and selected from the group consisting of position (-5), position (-9), and position A combination of mutations above (-14). In other embodiments, one or more zinc fingers in the polydactyly zinc finger protein can be in (-5), (-9), (-14) and selected from (-5), (-9) and ( The combination of -14) contains mutations. In some embodiments, amino acids (eg, arginine) at (-5), (-9), (-14), and combinations selected from (-5), (-9), and (-14) are made (R) or lysine (K)) to Alanine (A), Leucine (L), Ser (S), Asp (D), Glu (E), Tyr (Y) and/or Gluten amino acid (Q).

在本文中所描述之此等融合多肽中之任一者中，ZFP搭配物可進一步包含處於鋅指DNA結合結構域中之(-5)、(-9)、(-14)位置上之突變以及選自(-5)、(-9)及(-14)上之突變的突變的組合。在一些實施例中，位置-5處之Arg (R)變成Tyr (Y)、Asp (D)、Glu (E)、Leu (L)、Gln (Q)或Ala (A)。在其他實施例中，用Ser (S)、Asp (D)或Glu (E)置換位置(-9)處之Arg (R)。在其他實施例中，用Ser (S)或Gln (Q)置換位置(-14)處之Arg (R)。在其他實施例中，融合多肽可包含處於鋅指DNA結合結構域中之突變，其中(-5)、(-9)、(-14)位置以及選自(-5)、(-9)及(-14)處之突變的突變的組合變成以上任何組合中列出之胺基酸中之任一者。In any of these fusion polypeptides described herein, the ZFP partner may further comprise mutations at (-5), (-9), (-14) positions in the zinc finger DNA binding domain and combinations of mutations selected from mutations on (-5), (-9) and (-14). In some embodiments, Arg (R) at position -5 becomes Tyr (Y), Asp (D), Glu (E), Leu (L), Gln (Q), or Ala (A). In other embodiments, Arg (R) at position (-9) is replaced with Ser (S), Asp (D) or Glu (E). In other embodiments, Arg (R) at position (-14) is replaced with Ser (S) or Gln (Q). In other embodiments, the fusion polypeptide may comprise a mutation in a zinc finger DNA binding domain, wherein (-5), (-9), (-14) positions and are selected from the group consisting of (-5), (-9) and The combination of mutations at (-14) becomes any of the amino acids listed in any combination above.

例示性選擇方法，包括噬菌體呈現及雙雜交體系統，揭示於美國專利5,789,538、5,925,523、6,007,988、6,013,453、6,410,248、6,140,466、6,200,759及6,242,568以及WO 98/37186、WO 98/53057、WO 00/27878、WO 01/88197及GB 2,338,237中。另外，增強對鋅指結合結構域之結合特異性已描述於例如共有WO 02/077227中。Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in US Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 01/88197 and GB 2,338,237. Additionally, enhancing binding specificity to zinc finger binding domains has been described, for example, in consensus WO 02/077227.

標靶位點之選擇、用於設計及構建融合蛋白質(及編碼其之聚核苷酸)之ZFP及方法對熟習此項技術者為已知的且詳細描述於美國專利第6,140,081號、第5,789,538號、第6,453,242號、第6,534,261號、第5,925,523號、第6,007,988號、第6,013,453號、第6,200,759號、PCT公開案第WO 95/19431號、第WO 96/06166號、第WO 98/53057號、第WO 98/54311號、第WO 00/27878號、第WO 01/60970號、第WO 01/88197號、第WO 02/099084號、第WO 98/53058號、第WO 98/53059號、第WO 98/53060號、第WO 02/016536號及第WO 03/016496中。Selection of target sites, ZFPs and methods for designing and constructing fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are described in detail in US Pat. Nos. 6,140,081, 5,789,538 No., No. 6,453,242, No. 6,534,261, No. 5,925,523, No. 6,007,988, No. 6,013,453, No. 6,200,759, PCT Publication No. WO 95/19431, No. WO 96/06166, No. WO 98/53057, WO 98/54311, WO 00/27878, WO 01/60970, WO 01/88197, WO 02/099084, WO 98/53058, WO 98/53059, In WO 98/53060, WO 02/016536 and WO 03/016496.

幾乎任何連接子(間隔基)均可用在DNA結合結構域之一或多個組件(例如，鋅指)之間、一或多個DNA結合結構域之間、DNA結合結構域與功能結構域(例如核酸酶)之間以及一或多個DNA結合結構域之間及DNA結合結構域與功能結構域之間。適合之連接子序列的非限制性實例包括美國專利第8,772,453號、第7,888,121號、第6,479,626號、第6,903,185號及第7,153,949號以及美國專利公開案第2009/0305419號、第2015/0064789號及第2015/0132269號。因而，本文中所描述之蛋白質可包括介於本文中所描述之組合物之個別DNA結合組件之間、DNA結合結構域與功能結構域之間或一或多個DNA結合結構域之間及DNA結合結構域與功能結構域之間的適合連接子之任何組合。 B. 裂解結構域Almost any linker (spacer) can be used between one or more components of a DNA-binding domain (eg, zinc fingers), between one or more DNA-binding domains, between a DNA-binding domain and a functional domain ( For example, between nucleases) and between one or more DNA binding domains and between DNA binding domains and functional domains. Non-limiting examples of suitable linker sequences include US Patent Nos. 8,772,453; 7,888,121; 6,479,626; 6,903,185; No. 2015/0132269. Thus, proteins described herein can include DNA between individual DNA-binding components, between DNA-binding domains and functional domains, or between one or more DNA-binding domains of the compositions described herein, and DNA Any combination of suitable linkers between binding and functional domains. B. Cleavage Domain

任何適合之裂解結構域均可能可操作地連接至如本文中所描述之DNA結合結構域以形成核酸酶。裂解結構域與DNA結合結構域可為異源的，例如鋅指DNA結合結構域與來自於核酸酶之裂解結構域。異源裂解結構域可獲自任何內切核酸酶或外切核酸酶。可得到裂解結構域之例示性內切核酸酶包括但不限於限制內切核酸酶及歸巢內切核酸酶。參見例如2002-2003 Catalogue, New England Biolabs, Beverly, MA；及Belfort等人, (1997)Nucleic Acids Res . 25:3379-3388。可使DNA裂解之其他酶為已知的(例如，S1核酸酶；綠豆核酸酶；胰臟DNA酶I；微球菌核酸酶；酵母HO內切核酸酶；亦參見Linn等人(編),Nucleases , Cold Spring Harbor Laboratory Press, 1993)。此等酶(或其功能片段)中之一或多者可用作裂解結構域及裂解半結構域之來源。Any suitable cleavage domain may be operably linked to a DNA binding domain as described herein to form a nuclease. The cleavage domain and the DNA binding domain can be heterologous, eg, a zinc finger DNA binding domain and a cleavage domain from a nuclease. A heterologous cleavage domain can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which cleavage domains can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, eg, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al., (1997) Nucleic Acids Res . 25:3379-3388. Other enzymes that can cleave DNA are known (eg, S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds), Nucleases , Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

類似地，裂解半結構域可來源於如以上所闡述之需要二聚來達成裂解活性之任何核酸酶或其部分。一般而言，若融合蛋白質包含裂解半結構域，則需要兩個融合蛋白質來進行裂解。替代地，可使用包含兩個裂解半結構域之單一蛋白質。兩個裂解半結構域可來源於相同的內切核酸酶(或其功能片段)，或各裂解半結構域可來源於不同的內切核酸酶(或其功能片段)。另外，兩個融合蛋白質之標靶位點較佳相對於彼此安置，以便該兩個融合蛋白質與其個別標靶位點結合將裂解半結構域置於相對於彼此呈允許裂解半結構域形成功能裂解結構域(例如,藉由二聚)之空間取向。因而，在某些實施例中，標靶位點之近邊邊緣由5至8個核苷酸或由15至18個核苷酸隔開。然而，可在兩個標靶位點之間***任何整數個核苷酸或核苷酸配對(例如，2至50個核苷酸配對以上)。一般而言，裂解位點介於標靶位點之間。Similarly, the cleavage half-domain can be derived from any nuclease or portion thereof that requires dimerization for cleavage activity as described above. In general, if the fusion protein contains a cleavage half-domain, then two fusion proteins are required for cleavage. Alternatively, a single protein comprising both cleavage half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragment thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragment thereof). Additionally, the target sites of the two fusion proteins are preferably positioned relative to each other such that binding of the two fusion proteins to their respective target sites positions the cleavage half-domains relative to each other to allow the cleavage half-domains to form functional cleavage Spatial orientation of domains (eg, by dimerization). Thus, in certain embodiments, the proximal edges of the target sites are separated by 5 to 8 nucleotides or by 15 to 18 nucleotides. However, any integer number of nucleotides or nucleotide pairs (eg, 2 to 50 nucleotide pairs or more) can be inserted between the two target sites. Generally, the cleavage site is between the target sites.

限制內切核酸酶(限制酶)存在於許多物種中且能夠與DNA發生序列特異性結合(在識別位點處)且在結合位點處或附近使DNA裂解。某些限制酶(例如，IIS型)使DNA在自識別位點移除之位點處裂解且具有單獨的結合及裂解結構域。舉例而言，IIS型酶Fok I催化DNA在來自於其一個股鏈上之識別位點之9個核苷酸處及來自於其另一股鏈上之識別位點的13個核苷酸處發生雙鏈裂解。參見例如美國專利第5,356,802號、第5,436,150號及第5,487,994號；以及Li等人,(1992)Proc. Natl. Acad. Sci. USA 89:4275-4279；Li等人, (1993)Proc. Natl. Acad. Sci. USA 90:2764-2768；Kim等人, (1994a)Proc. Natl. Acad. Sci. USA 91:883-887；Kim等人, (1994b)J. Biol. Chem. 269:31,978-31,982。因而，在一個實施例中，融合蛋白質包含來自至少一種IIS型限制酶之裂解結構域(或裂解半結構域)及一或多個鋅指結合結構域，該等結構域可能經工程改造或可能未經工程改造。Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at the recognition site) and cleavage of the DNA at or near the binding site. Certain restriction enzymes (eg, Type IIS) cleave DNA at sites removed from the recognition site and have separate binding and cleavage domains. For example, the Type IIS enzyme Fok I catalyzes DNA at 9 nucleotides from a recognition site on one of its strands and 13 nucleotides from a recognition site on its other strand Double-strand cleavage occurs. See, eg, US Patent Nos. 5,356,802, 5,436,150, and 5,487,994; and Li et al., (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al., (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al, (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al, (1994b) J. Biol. Chem. 269:31,978- 31,982. Thus, in one embodiment, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may be engineered or may be Not engineered.

對於序列選擇性(人工)核酸酶之最佳裂解特異性，需要安排諸多條件以使得中靶結合及活性不飽和。根據定義，在飽和條件下，使用與達成完全中靶活性所必需之量相比過量之核酸酶。此過量不提供中靶益處，但儘管如此仍可增加脫靶位點處之裂解。對於單體核酸酶，可藉由進行簡單劑量反應研究以鑑定並避免滴定曲線上之飽和平線區而容易地避免飽和條件。然而，對於諸如ZFN、TALEN或dCas-Fok之二聚核酸酶而言，若個別單體之結合親和力不相似，則鑑定及避免飽和條件可能更複雜。在此種情況下，使用簡單1:1核酸酶比率之劑量反應研究將僅揭示較弱結合單體之飽和點。在此種情況下，若例如單體親和力相差10倍，則在1:1滴定研究中鑑定之飽和點下，較高親和力單體將以比其需要之濃度高出10倍之濃度存在。較高親和力單體之所得過量又可能導致脫靶活性增加而在預定標靶處之裂解方面不提供任何有益增加，從而可能導致任何指定核酸酶配對之總體特異性降低。For optimal cleavage specificity of sequence-selective (artificial) nucleases, conditions need to be arranged so that on-target binding and activity are not saturated. By definition, under saturating conditions, an excess of nuclease is used compared to the amount necessary to achieve full on-target activity. This excess provides no on-target benefit, but can nonetheless increase cleavage at off-target sites. For monomeric nucleases, saturation conditions can be easily avoided by performing simple dose response studies to identify and avoid saturation plateaus on the titration curve. However, for dimeric nucleases such as ZFNs, TALENs or dCas-Fok, identifying and avoiding saturation conditions can be more complicated if the binding affinities of the individual monomers are not similar. In this case, a dose-response study using a simple 1:1 nuclease ratio would reveal only the saturation point of the weaker binding monomer. In this case, if, for example, the affinity of the monomers differs by a factor of 10, the higher affinity monomer will be present at a concentration 10-fold higher than its required concentration at the saturation point identified in the 1:1 titration study. The resulting excess of higher affinity monomers, in turn, may result in increased off-target activity without providing any beneficial increase in cleavage at the intended target, thereby possibly resulting in a reduction in the overall specificity of any given nuclease pairing.

裂解結構域與結合結構域隔開之例示性IIS型限制酶為Fok I。此特定酶為活性二聚體。Bitinaite等人, (1998)Proc. Natl. Acad. Sci. USA 95:10,570-10,575。相應地，出於本發明之目的，用於所揭示之融合蛋白質中之Fok I酶部分被視為裂解半結構域。因而，對於靶向雙鏈裂解、使用鋅指-Fok I融合物對細胞序列進行靶向置換及靶向雙鏈裂解與使用鋅指-Fok I融合物對細胞序列進行靶向置換而言，可使用兩個融合蛋白質(各包含一個Fok I裂解半結構域)來重構催化活性裂解結構域。替代地，亦可使用含有鋅指結合結構域及兩個Fok I裂解半結構域之單一多肽分子。本發明中在其他部分提供靶向裂解及使用鋅指-Fok I融合物進行靶向序列變化之參數。An exemplary Type IIS restriction enzyme with the cleavage domain separated from the binding domain is Fok I. This particular enzyme is an active dimer. Bitinaite et al., (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Accordingly, for the purposes of the present invention, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-strand cleavage, targeted displacement of cellular sequences using zinc finger- Fok I fusions, and targeted double-strand cleavage and targeted displacement of cellular sequences using zinc finger- Fok I fusions, The catalytically active cleavage domain was reconstituted using two fusion proteins, each containing a Fok I cleavage half-domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence changes using zinc finger- Fok I fusions are provided elsewhere in this invention.

裂解結構域或裂解半結構域可為蛋白質中保留裂解活性或保留多聚(例如二聚)以形成功能裂解結構域之能力的任何部分。A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or retains the ability to multimerize (eg, dimerize) to form a functional cleavage domain.

例示性IIS型限制酶描述於美國專利第7,888,121號中，該專利全文併入本文中。其他限制酶亦含有單獨的結合及裂解結構域，且本發明涵蓋此等限制酶。參見例如Roberts等人, (2003)Nucleic Acids Res. 31:418-420。Exemplary Type IIS restriction enzymes are described in US Patent No. 7,888,121, which is incorporated herein in its entirety. Other restriction enzymes also contain separate binding and cleavage domains, and such restriction enzymes are encompassed by the present invention. See, eg, Roberts et al., (2003) Nucleic Acids Res. 31:418-420.

在某些實施例中，裂解結構域包含一或多個經工程改造之裂解半結構域(亦稱為二聚結構域突變體)，其可減少或防止同源二聚，如例如參見例如美國專利第7,914,796號、第8,034,598號及第8,623,618號中所描述，所有專利之揭示內容以全文引用之方式併入本文中。在Fok I之位置446、447、479、483、484、486、487、490、491、496、498、499、500、531、534、537及538上之胺基酸殘基均為影響Fok I裂解半結構域二聚之標靶，其中編號係相對於具有以下所示之序列的晶體結構1FOK.pdb及2FOK.pdb (參見Wah等人,(1997)Nature 388:97-100)： 野生型FokI裂解半結構域(SEQ ID NO:18) QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFIn certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domains (also known as dimerization domain mutants) that reduce or prevent homodimerization, as described, eg, in the U.S. Described in Patent Nos. 7,914,796, 8,034,598, and 8,623,618, the disclosures of all patents are incorporated herein by reference in their entirety. The amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537 and 538 of Fok I all affect Fok I Targets for dimerization of cleavage half-domains, where numbering is relative to the crystal structures 1FOK.pdb and 2FOK.pdb with the sequences shown below (see Wah et al., (1997) Nature 388:97-100): Wild type FokI cleavage half-domain (SEQ ID NO: 18) QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

Fok I中可形成專性異源二聚體之例示性經工程改造之裂解半結構域包括第一裂解半結構域包括在Fok I之位置490及538上之胺基酸殘基處之突變且第二裂解半結構域包括胺基酸殘基486及499處之突變的配對。Exemplary engineered cleavage half-domains in Fok I that can form obligate heterodimers include the first cleavage half-domain comprising mutations at amino acid residues at positions 490 and 538 of Fok I and The second cleavage half-domain includes a pair of mutations at amino acid residues 486 and 499.

因而，在一個實施例中，490處之突變以Lys (K)置換Glu (E)；538處之突變以Lys (K)置換Ile (I)；486處之突變以Glu (E)置換Gln (Q)；且位置499處之突變以Lys (K)置換Ile (I)。特定言之，藉由使一個裂解半結構域中之位置490 (E→K)及538 (I→K)突變以產生命名為「E490K:I538K」之經工程改造之裂解半結構域且藉由使另一裂解半結構域中之位置486 (Q→E)及499 (I→L)突變以產生命名為「Q486E:I499L」之經工程改造之裂解半結構域來製備本文中所描述之經工程改造之裂解半結構域。本文中所描述之經工程改造之裂解半結構域為專性異源二聚體突變體，其中經由ZFN同源二聚體所致之異常裂解被減至最少或消除。參見例如美國專利公開案第2008/0131962號，該專利之揭示內容出於所有目的而以全文引用之方式併入。在某些實施例中，經工程改造之裂解半結構域包含位置486、499及496處之突變(相對於野生型FokI進行編號)，例如在位置486處以Glu (E)殘基置換野生型Gln (Q)殘基、在位置499處以Leu (L)殘基置換野生型Ile (I)殘基及在位置496處以Asp (D)或Glu (E)殘基置換野生型Asn (N)殘基(分別亦稱為「ELD」及「ELE」結構域)。在其他實施例中，經工程改造之裂解半結構域包含位置490、538及537處之突變(相對於野生型FokI進行編號)，例如在位置490處以Lys (K)殘基置換野生型Glu (E)殘基、在位置538處以Lys (K)殘基置換野生型Ile (I)殘基及在位置537處以Lys (K)或Arg (R)殘基置換野生型His (H)殘基(分別亦稱為「KKK」及「KKR」結構域)。在其他實施例中，經工程改造之裂解半結構域包含位置490及537處之突變(相對於野生型FokI進行編號)，例如在位置490處以Lys (K)殘基置換野生型Glu (E)殘基及在位置537處以Lys (K)或Arg (R)殘基置換野生型His (H)殘基(分別亦稱為「KIK」及「KIR」結構域)。參見例如美國專利第7,914,796號、第8,034,598號及第8,623,618號。在其他實施例中，經工程改造之裂解半結構域包含「Sharkey」、「Sharkey」突變及「Sharkey」與「Sharkey」突變之組合(參見Guo等人, (2010)J. Mol. Biol . 400(1):96-107)。Thus, in one embodiment, the mutation at 490 replaces Glu (E) with Lys (K); the mutation at 538 replaces Ile (I) with Lys (K); the mutation at 486 replaces Gln with Glu (E) ( Q); and the mutation at position 499 replaces Ile (I) with Lys (K). Specifically, by mutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domain to generate an engineered cleavage half-domain designated "E490K:I538K" and by Positions 486 (Q→E) and 499 (I→L) in the other cleavage half-domain were mutated to generate an engineered cleavage half-domain designated "Q486E:I499L" to prepare the cleavage half-domains described herein. Engineered cleavage half-domains. The engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage via ZFN homodimerization is minimized or eliminated. See, eg, US Patent Publication No. 2008/0131962, the disclosure of which is incorporated by reference in its entirety for all purposes. In certain embodiments, the engineered cleavage half-domain comprises mutations at positions 486, 499, and 496 (numbering relative to wild-type FokI), eg, replacing wild-type Gln with a Glu (E) residue at position 486 (Q) residue, replacement of the wild-type Ile (I) residue with a Leu (L) residue at position 499 and replacement of the wild-type Asn (N) residue with an Asp (D) or Glu (E) residue at position 496 (also known as the "ELD" and "ELE" domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490, 538, and 537 (numbered relative to wild-type FokI), such as replacing wild-type Glu with a Lys (K) residue at position 490 ( E) residues, the wild-type Ile (I) residue was replaced by a Lys (K) residue at position 538 and the wild-type His (H) residue was replaced by a Lys (K) or Arg (R) residue at position 537 ( Also known as the "KKK" and "KKR" domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbering relative to wild-type FokI), eg, replacing wild-type Glu (E) with a Lys (K) residue at position 490 residues and the wild-type His (H) residue at position 537 was replaced by a Lys (K) or Arg (R) residue (also referred to as the "KIK" and "KIR" domains, respectively). See, eg, US Patent Nos. 7,914,796, 8,034,598, and 8,623,618. In other embodiments, the engineered cleavage half-domains comprise "Sharkey", "Sharkey" mutations, and combinations of "Sharkey" and "Sharkey" mutations (see Guo et al., (2010) J. Mol. Biol . 400 (1):96-107).

因而，來源於FokI之裂解半結構域可包含如SEQ ID NO:18中所示之一或多個胺基酸殘基處之突變，包括如以上所描述之二聚結構域中之突變、催化結構域中之突變、諸如磷酸酯接觸殘基之其他胺基酸殘基處之突變以及選自二聚結構域中之突變、催化結構域中之突變、諸如磷酸酯接觸殘基之其他胺基酸殘基處之突變的突變的任何組合。突變包括取代(以不同的殘基取代野生型胺基酸)、***(一或多個胺基酸殘基)、缺失(一或多個胺基酸殘基)及選自取代、***及缺失之突變的任何組合。在某些實施例中，使殘基414-426、443-450、467-488、501-502、521-531中之一或多者(相對於SEQ ID NO:18進行編號)及此種殘基之任何組合突變，此係因為在Miller等人((2007)Nat Biotechnol 25:778-784)所描述之ZFN與其標靶位點結合之之分子模型中，此等殘基位於接近DNA主鏈處。在某些實施例中，使位置416、422、447、448及525上之一或多個殘基突變。在某些實施例中，突變包括以不同的殘基，例如絲胺酸(S)殘基取代野生型殘基。在某些實施例中，本文中所描述之核酸酶的FokI裂解結構域包含ELD二聚結構域突變、KKR二聚結構域突變、K525S突變及選自ELD二聚結構域突變或KKR二聚結構域突變及K525S突變之任何組合。Thus, the cleavage half-domain derived from FokI may comprise mutations at one or more amino acid residues as shown in SEQ ID NO: 18, including mutations in the dimerization domain as described above, catalytic Mutations in domains, mutations at other amino acid residues such as phosphate contact residues and selected from mutations in dimerization domains, mutations in catalytic domains, other amino groups such as phosphate contact residues Any combination of mutations at acid residues. Mutations include substitutions (replacement of wild-type amino acids with different residues), insertions (one or more amino acid residues), deletions (one or more amino acid residues) and selected from substitutions, insertions and deletions any combination of mutations. In certain embodiments, one or more of residues 414-426, 443-450, 467-488, 501-502, 521-531 (numbered relative to SEQ ID NO: 18) and such residues are Mutation of any combination of bases because these residues are located close to the DNA backbone in the molecular model of ZFN binding to its target site described by Miller et al. ((2007) Nat Biotechnol 25:778-784). place. In certain embodiments, one or more residues at positions 416, 422, 447, 448, and 525 are mutated. In certain embodiments, the mutation comprises substituting a wild-type residue with a different residue, eg, a serine (S) residue. In certain embodiments, the FokI cleavage domain of the nucleases described herein comprises an ELD dimerization domain mutation, a KKR dimerization domain mutation, a K525S mutation, and is selected from an ELD dimerization domain mutation or a KKR dimerization structure Any combination of domain mutations and K525S mutations.

本文中所描述之經工程改造之裂解結構域可使用任何適合之方法來製備，例如，藉由如美國專利第7,888,121號、第7,914,796號、第8,034,598號及第8,623,618號中所描述之野生型裂解半結構域(Fok I)之定點突變誘發。此外，本文中所描述之裂解結構域可使用任何適合之連接子，包括但不限於美國專利第9,394,531號及第9,567,609號中所描述之連接子與DNA結合結構域(例如，ZFP)融合。The engineered cleavage domains described herein can be prepared using any suitable method, eg, by wild-type cleavage as described in US Pat. Nos. 7,888,121, 7,914,796, 8,034,598, and 8,623,618 Site-directed mutagenesis of the half-domain ( Fok I). Furthermore, the cleavage domains described herein can be fused to a DNA binding domain (eg, ZFP) using any suitable linker, including but not limited to the linkers described in US Pat. Nos. 9,394,531 and 9,567,609.

替代地，可使用所謂的「***酶(split-enzyme)」技術在核酸標靶位點處活體內組裝核酸酶(參見例如美國專利公開案第2009/0068164號)。此種***酶之組件可表現在單獨的表現構建體上，或可連接在一個開放閱讀框中，其中個別組件例如由自裂解2A肽或IRES序列隔開。組件可為個別鋅指結合結構域或兆鹼基核酸酶核酸結合結構域之結構域。Alternatively, so-called "split-enzyme" technology can be used to assemble nucleases in vivo at nucleic acid target sites (see, eg, US Patent Publication No. 2009/0068164). Components of such a cleavage enzyme can be expressed on separate expression constructs, or can be linked in an open reading frame, with the individual components separated, for example, by a self-cleaving 2A peptide or IRES sequence. A component can be an individual zinc finger binding domain or a domain of a meganuclease nucleic acid binding domain.

可在使用前篩檢核酸酶之活性，例如，在如WO 2009/042163及20090068164中所描述之基於酵母之染色體系統中。核酸酶之表現可受組成性啟動子或誘導性啟動子控制，例如在棉子糖、半乳糖及棉子糖與半乳糖之組合存在下得以活化(解壓抑)且在葡萄糖存在下受到壓抑之半乳糖激酶啟動子。Nucleases can be screened for activity prior to use, eg, in yeast-based chromosomal systems as described in WO 2009/042163 and 20090068164. The expression of nucleases can be controlled by constitutive or inducible promoters, such as those that are activated (derepressed) in the presence of raffinose, galactose, and a combination of raffinose and galactose and repressed in the presence of glucose. Galactokinase promoter.

如本文中所描述之核酸酶可在標靶位點中製造一或多個雙鏈切口、一或多個單鏈切口及一或多個雙鏈切口與一或多個單鏈切口之組合。在某些實施例中，核酸酶包含無催化活性裂解結構域(例如FokI、Cas蛋白及FokI與Cas蛋白之組合)。參見例如美國專利第9,200,266號；第8,703,489號；及Guillinger等人, (2014)Nature Biotech. 32(6):577-582。無催化活性裂解結構域可與催化活性結構域組合充當切口酶，以製造單鏈切口。因此，兩個切口酶可組合用於在特定區域中製造雙鏈切口。此項技術中亦已知其他切口酶，例如McCaffery等人, (2016)Nucleic Acids Res. 44(2):e11. doi: 10.1093/nar/gkv878. Epub 2015年10月19日。 標靶位點Nucleases as described herein can make one or more double-stranded nicks, one or more single-stranded nicks, and a combination of one or more double-stranded nicks and one or more single-stranded nicks in a target site. In certain embodiments, the nuclease comprises a catalytically inactive cleavage domain (eg, Fokl, Cas protein, and a combination of Fokl and Cas protein). See, eg, US Patent Nos. 9,200,266; 8,703,489; and Guillinger et al., (2014) Nature Biotech. 32(6):577-582. The catalytically inactive cleavage domain can act as a nickase in combination with the catalytically active domain to make single-strand nicks. Thus, two nickases can be used in combination to make double-stranded nicks in specific regions. Other nickases are also known in the art, eg McCaffery et al., (2016) Nucleic Acids Res. 44(2):e11. doi: 10.1093/nar/gkv878. Epub 2015 Oct 19. target site

如以上詳細描述，DNA結構域可經工程改造以結合任何所選序列。與天然存在之DNA結合結構域相比，經工程改造之DNA結合結構域可具有新穎結合特異性。在某些實施例中，DNA結合結構域結合BCL11A增強子序列內之序列，例如，標靶位點(典型地，9、10、11、12、13、14、15、16、17、18、19、20、21或甚至更多個鹼基對)介於BCL11A之外顯子2與外顯子3之間，包括結合如表1中所示之BCL11A增強子序列中之DNAseI過敏位點(例如+58)內之序列的DNA結合結構域。工程改造方法包括但不限於合理設計及不同的選擇類型。合理設計包括例如使用包含三鏈體(或四鏈體)核苷酸序列及個別鋅指胺基酸序列之資料庫，其中各三鏈體或四鏈體核苷酸序列與可結合特定三鏈體或四鏈體序列之一或多個鋅指胺基酸序列締合。參見例如共有美國專利第6,453,242號及第6,534,261號，該等專利以全文引用之方式併入本文中。亦可進行TAL效應因子結構域之合理設計。參見例如美國專利公開案第2011/0301073號。As described in detail above, the DNA domains can be engineered to bind any sequence of choice. Engineered DNA binding domains can have novel binding specificities compared to naturally occurring DNA binding domains. In certain embodiments, the DNA binding domain binds a sequence within a BCL11A enhancer sequence, eg, a target site (typically, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more base pairs) between BCL11A exon 2 and exon 3, including binding to the DNAseI hypersensitive site in the BCL11A enhancer sequence as shown in Table 1 ( For example, a DNA binding domain of a sequence within +58). Engineering methods include but are not limited to rational design and different selection types. Rational design includes, for example, the use of databases comprising triplex (or quadruplex) nucleotide sequences and individual zinc finger amino acid sequences, where each triplex or quadruplex nucleotide sequence is associated with a specific triplex One or more zinc finger amino acid sequences are associated with one or more of the sequences of the body or quadruplex. See, eg, co-owned US Pat. Nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entirety. Rational design of TAL effector domains can also be performed. See, eg, US Patent Publication No. 2011/0301073.

適用於DNA結合結構域之例示性選擇方法，包括噬菌體呈現及雙雜交體系統，揭示於美國專利第5,789,538號、第5,925,523號、第6,007,988號、第6,013,453號、第6,410,248號、第6,140,466號、第6,200,759號及第6,242,568號以及PCT公開案第WO 98/37186號、第WO 98/53057號、第WO 00/27878號、第WO 01/88197號及英國專利第GB 2,338,237號中。另外，增強對鋅指結合結構域之結合特異性已描述於例如共有WO 02/077227中。Exemplary selection methods suitable for DNA binding domains, including phage display and two-hybrid systems, are disclosed in US Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759 and 6,242,568 and in PCT Publications WO 98/37186, WO 98/53057, WO 00/27878, WO 01/88197 and British Patent No. GB 2,338,237. Additionally, enhancing binding specificity to zinc finger binding domains has been described, for example, in consensus WO 02/077227.

標靶位點之選擇；用於設計及構建融合蛋白質(及編碼其之聚核苷酸)之核酸酶及方法對熟習此項技術者為已知的且詳細描述於美國專利公開案第2005/0064474號及第2006/0188987號中，各案以全文引用之方式併入本文中。Selection of target sites; nucleases and methods for designing and constructing fusion proteins (and polynucleotides encoding the same) are known to those skilled in the art and are described in detail in US Patent Publication No. 2005/ 0064474 and 2006/0188987, each of which is incorporated herein by reference in its entirety.

另外，如此等及其他參考文獻中所揭示，DNA結合結構域(例如，多指型鋅指蛋白質)及DNA結合結構域與功能結構域之融合物可使用任何適合之連接子序列，包括例如具有5個以上胺基酸之連接子連接在一起。美國專利第8,772,453號、第7,888,121號(例如，「ZC」連接子)、第6,479,626號、第6,903,185號及第7,153,949號；美國公開案第2009/0305419號及第2015/0064789號。本文中所描述之蛋白質可包括介於蛋白質之個別DNA結合結構域之間的適合連接子之任何組合。亦參見美國專利第8,586,526號。 供體In addition, as disclosed in these and other references, DNA binding domains (eg, polydactyl zinc finger proteins) and fusions of DNA binding domains and functional domains can use any suitable linker sequence, including, for example, having Linkers of more than 5 amino acids are linked together. US Patent Nos. 8,772,453, 7,888,121 (eg, "ZC" linkers), 6,479,626, 6,903,185, and 7,153,949; US Publication Nos. 2009/0305419 and 2015/0064789. The proteins described herein can include any combination of suitable linkers between the individual DNA binding domains of the protein. See also US Patent No. 8,586,526. donor

在某些實施例中，本發明係關於使用本文中所描述之BCL11A增強子區域結合分子對外源序列進行核酸酶介導之靶向整合至細胞基因組中。如以上所指出，***外源序列(亦稱為「供體序列」或「供體」或「轉殖基因」)例如用於缺失指定區域、用於矯正突變基因、用於缺失指定區域與矯正突變基因之組合或用於增加野生型基因之表現。應顯而易見，供體序列典型地與置放其之基因組序列不一致。供體序列可含有側接兩個具有同源性之區域以便在相關位置上允許有效HDR之非同源序列，或可經由諸如NHEJ之非同源性引導修復機制進行整合。另外，供體序列可包括含有與細胞染色質中之相關區域不同源之序列的載體分子。供體分子可含有若干個與細胞染色質具有同源性之不連續區域，且舉例而言，在用作由本文中所描述之核酸酶之一誘導之DSB之修復受質時導致BCL11A增強子區域(或其片段)缺失。此外，對於正常情況下不存在於相關區域中之序列的靶向***，該等序列可存在於供體核酸分子中且側接與相關區域中之序列具有同源性的區域。In certain embodiments, the invention relates to nuclease-mediated targeted integration of exogenous sequences into the genome of cells using the BCL11A enhancer region binding molecules described herein. As noted above, insertion of exogenous sequences (also referred to as "donor sequences" or "donor" or "transgenic genes"), for example, for deletion of a specified region, for correction of a mutated gene, for deletion of a specified region and correction of Combinations of mutant genes may be used to increase the performance of wild-type genes. It should be apparent that the donor sequence is typically not identical to the genomic sequence in which it is placed. The donor sequence may contain non-homologous sequences flanking two regions of homology to allow efficient HDR at relevant positions, or may be integrated via a non-homology-directed repair mechanism such as NHEJ. Additionally, the donor sequence can include a vector molecule containing sequences that are not homologous to the relevant region in the cellular chromatin. The donor molecule may contain several discrete regions of homology to cellular chromatin and, for example, result in the BCL11A enhancer when used as a repair substrate for DSBs induced by one of the nucleases described herein A region (or fragment thereof) is deleted. Furthermore, for targeted insertion of sequences that are not normally present in the region of interest, such sequences may be present in the donor nucleic acid molecule flanking regions of homology to sequences in the region of interest.

用於***之聚核苷酸亦可稱為「外源」聚核苷酸、「供體」聚核苷酸或分子或「轉殖基因」。供體聚核苷酸可為單鏈或雙鏈DNA或RNA，或可呈線性或環狀形式引入細胞中。參見例如美國專利申請公開案第2010/0047805號及第2011/0207221號。供體序列較佳包含在DNA MC內，其可呈環狀或線性形式引入細胞中。若呈線性形式引入，則可藉由熟習此項技術者已知的方法來保護供體序列之末端(例如，免受外切核酸酶降解)。舉例而言，向線性分子之3'末端添加一或多個雙去氧核苷酸殘基，並且將自互補寡核苷酸視情況連接至一端或兩端。參見例如Chang等人, (1987)Proc. Natl. Acad. Sci. USA 84:4959-4963；Nehls等人, (1996)Science 272:886-889。用於保護外源聚核苷酸免受降解之其他方法包括但不限於添加末端胺基及使用經修飾之核苷酸間鍵聯，舉例而言，諸如硫代磷酸酯、磷醯胺酯及O-甲基核糖或去氧核糖殘基。若呈雙鏈形式引入，則供體可包括一或多個核酸酶標靶位點，例如，側接欲整合至細胞基因組中之轉殖基因的核酸酶標靶位點。參見例如美國專利公開案第2013/0326645號。The polynucleotides used for insertion may also be referred to as "foreign" polynucleotides, "donor" polynucleotides or molecules, or "transgenic genes." Donor polynucleotides can be single- or double-stranded DNA or RNA, or can be introduced into cells in linear or circular form. See, eg, US Patent Application Publication Nos. 2010/0047805 and 2011/0207221. The donor sequence is preferably contained within the DNA MC, which can be introduced into the cell in circular or linear form. If introduced in linear form, the ends of the donor sequence can be protected (eg, from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of a linear molecule, and a self-complementary oligonucleotide is ligated to one or both ends as appropriate. See, eg, Chang et al, (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al, (1996) Science 272:886-889. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amine groups and the use of modified internucleotide linkages, such as, for example, phosphorothioates, phosphoramids, and O-methylribose or deoxyribose residues. If introduced in double-stranded form, the donor may include one or more nuclease target sites, eg, nuclease target sites flanking the transgenic gene to be integrated into the genome of the cell. See, eg, US Patent Publication No. 2013/0326645.

聚核苷酸可作為具有其他序列，舉例而言，諸如複製起點、啟動子及編碼抗生素抗性之基因的載體分子之一部分引入細胞中。此外，供體聚核苷酸可作為裸核酸、作為與諸如脂質體或泊洛沙姆之試劑複合之核酸引入，或可藉由病毒(例如，腺病毒、AAV、疱疹病毒、反轉錄病毒、慢病毒及整合酶缺乏型慢病毒(IDLV))遞送。Polynucleotides can be introduced into cells as part of a vector molecule having other sequences, such as, for example, origins of replication, promoters, and genes encoding antibiotic resistance. In addition, donor polynucleotides can be introduced as naked nucleic acids, as nucleic acids complexed with agents such as liposomes or poloxamers, or can be introduced by viruses (eg, adenoviruses, AAVs, herpesviruses, retroviruses, Lentivirus and integrase deficient lentivirus (IDLV)) delivery.

在某些實施例中，雙鏈供體包括長度超過1 kb，例如介於2 kb與200 kb之間、介於2 kb與10 kb之間(或介於其之間的任何值)的序列(例如，編碼序列，亦稱為轉殖基因)。雙鏈供體亦包括例如至少一個核酸酶標靶位點。在某些實施例中，該供體包括至少2個標靶位點，例如對於ZFN或TALEN配對。典型地，核酸酶標靶位點在轉殖基因序列外，例如，轉殖基因序列之5'及/或3'，以便使轉殖基因裂解。核酸酶裂解位點可針對任何核酸酶。在某些實施例中，雙鏈供體中所含有之核酸酶標靶位點針對用於使經由非同源性依賴性方法將裂解供體整合至其中之內源標靶裂解的相同核酸酶。In certain embodiments, double-stranded donors include sequences in excess of 1 kb in length, such as between 2 kb and 200 kb, between 2 kb and 10 kb (or any value in between) (eg, coding sequences, also known as transgenes). The double-stranded donor also includes, for example, at least one nuclease target site. In certain embodiments, the donor includes at least 2 target sites, eg, for ZFN or TALEN pairings. Typically, the nuclease target site is outside the transgenic gene sequence, eg, 5' and/or 3' of the transgenic gene sequence, in order to cleave the transgenic gene. Nuclease cleavage sites can be for any nuclease. In certain embodiments, the nuclease target site contained in the double-stranded donor is directed to the same nuclease used to cleave the endogenous target into which the cleavage donor was integrated via a non-homology-dependent method .

一般***供體以便在整合位點處由內源啟動子驅動其表現，亦即，驅動已***供體之內源基因(例如球蛋白、AAVS1等)之表現的啟動子。然而，應顯而易見，該供體可包含啟動子、增強子及啟動子與增強子兩者之組合，例如組成性啟動子或者誘導性或組織特異性啟動子。The donor is typically inserted so that its expression is driven by an endogenous promoter at the integration site, ie, a promoter that drives the expression of an endogenous gene (eg, globulin, AAVS1, etc.) into which the donor has been inserted. It should be apparent, however, that the donor may comprise promoters, enhancers, and combinations of both, such as constitutive promoters or inducible or tissue-specific promoters.

可將供體分子***內源基因中，以便表現所有、一些或無一內源基因。在其他實施例中，將轉殖基因(例如，有或無球蛋白編碼序列)整合至任何內源基因座，例如安全港基因座中。參見例如美國專利公開案第2008/0299580號、第2008/0159996號及第2010/00218264號。Donor molecules can be inserted into endogenous genes to express all, some or none of the endogenous genes. In other embodiments, the transgenic gene (eg, with or without globulin coding sequences) is integrated into any endogenous locus, eg, a safe harbor locus. See, eg, US Patent Publication Nos. 2008/0299580, 2008/0159996, and 2010/00218264.

此外，儘管對於表現而言並不需要，但外源序列亦可包括轉錄或轉譯調控序列，例如啟動子、增強子、絕緣子、內部核糖體進入位點、編碼2A肽之序列、聚腺苷酸化信號及其組合。In addition, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosomal entry sites, sequences encoding 2A peptides, polyadenylation signals and their combinations.

本文中所描述之供體序列上所攜帶之轉殖基因可使用此項技術中已知的標準技術，諸如PCR自質體、細胞或其他來源分離。供使用之供體可包括不同類型之拓撲結構，包括環超螺旋、環鬆弛、線性及其類似拓撲結構。替代地，其可使用標準寡核苷酸合成技術進行化學合成。另外，供體可能被甲基化或缺乏甲基化。供體可呈細菌或酵母人工染色體(BAC或YAC)形式。Transgenic genes carried on the donor sequences described herein can be isolated from plastids, cells or other sources using standard techniques known in the art, such as PCR. Donors for use can include different types of topologies, including loop supercoiled, loop relaxed, linear, and the like. Alternatively, it can be chemically synthesized using standard oligonucleotide synthesis techniques. Additionally, the donor may be methylated or lack methylation. Donors can be in the form of bacterial or yeast artificial chromosomes (BAC or YAC).

本文中所描述之雙鏈供體聚核苷酸可包括一或多個非天然鹼基、一或多個主鏈及一或多個非天然鹼基與一或多個主鏈之組合。特定言之，可使用本文中所描述之方法來進行***具有甲基化胞嘧啶之供體分子，以便在相關區域中達成轉錄靜止狀態。The double-stranded donor polynucleotides described herein can include one or more unnatural bases, one or more backbones, and a combination of one or more unnatural bases and one or more backbones. In particular, insertion of a donor molecule with methylated cytosines can be performed using the methods described herein to achieve transcriptional quiescence in the region of interest.

外源(供體)聚核苷酸可包含任何相關序列(外源序列)。例示性外源序列包括但不限於任何多肽編碼序列(例如，cDNA)、啟動子序列、增強子序列、抗原決定基標籤、標記基因、裂解酶識別位點及多種類型之表現構建體。標記基因包括但不限於編碼介導抗生素抗性(例如安比西林抗性、卡那黴素抗性、新黴素抗性、G418抗性、嘌呤黴素抗性、潮黴素抗性、殺稻瘟菌素抗性)之蛋白質的序列、編碼彩色或螢光或發光蛋白質(例如，綠色螢光蛋白、增強型綠色螢光蛋白、紅色螢光蛋白、螢光素酶)以及介導增強之細胞生長、基因擴增(例如，二氫葉酸還原酶)及增強之細胞生長與基因擴增之組合的蛋白質的序列。抗原決定基標籤包括例如FLAG、His、Myc、串聯親和力純化(TAP)、HA、可生物素化肽或任何可偵測胺基酸序列之一或多個副本。The exogenous (donor) polynucleotide may comprise any related sequence (exogenous sequence). Exemplary exogenous sequences include, but are not limited to, any polypeptide coding sequence (eg, cDNA), promoter sequences, enhancer sequences, epitope tags, marker genes, lyase recognition sites, and various types of expression constructs. Marker genes include, but are not limited to, coding for mediating antibiotic resistance (eg, ampicillin resistance, kanamycin resistance, neomycin resistance, G418 resistance, puromycin resistance, hygromycin resistance, rice killing Sequences of proteins encoding blasticidin resistance), encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and cells that mediate enhancement Sequences of proteins for growth, gene amplification (eg, dihydrofolate reductase) and a combination of enhanced cell growth and gene amplification. Epitope tags include, for example, FLAG, His, Myc, tandem affinity purification (TAP), HA, biotinylated peptides, or one or more copies of any detectable amino acid sequence.

在一較佳實施例中，外源序列(轉殖基因)包含編碼需要在細胞中表現之任何多肽，包括但不限於抗體、抗原、酶、受體(細胞表面或細胞核)、激素、淋巴因子、細胞因子、報告多肽、生長因子及以上任一者之功能片段的聚核苷酸。編碼序列可為例如cDNA。In a preferred embodiment, the exogenous sequence (transgenic gene) comprises encoding any polypeptide that needs to be expressed in the cell, including but not limited to antibodies, antigens, enzymes, receptors (cell surface or nucleus), hormones, lymphokines , cytokines, reporter polypeptides, growth factors and polynucleotides of functional fragments of any of the above. The coding sequence can be, for example, a cDNA.

舉例而言，外源序列可包含編碼患有遺傳性疾病之個體中缺乏或無功能之多肽的序列，該遺傳性疾病包括但不限於以下遺傳性疾病中之任一種：軟骨發育不全、色盲、酸性麥芽糖酶缺乏症、腺苷脫胺酶缺乏症(OMIM第102700號)、腎上腺腦白質失養症、艾卡爾迪症候群(aicardi syndrome)、α-1抗胰蛋白酶缺乏症、α型地中海型貧血、雄性激素失敏症候群、亞伯氏症(apert syndrome)、致心律失常型右心室發育不良(arrhythmogenic right ventricular dysplasia)、共濟失調性微血管擴張、巴氏症候群(barth syndrome)、β型地中海型貧血、藍色橡皮泡樣痣症候群(blue rubber bleb nevus syndrome)、卡納萬病(canavan disease)、慢性肉芽腫病(CGD)、貓哭症候群(cri du chat syndrome)、囊性纖維化、德爾肯氏病(dercum’s disease)、外胚層發育不良、範科尼氏貧血症(fanconi anemia)、進行性肌肉骨化症、X染色體脆折症候群、半乳糖血症、高雪氏病(Gaucher’s disease)、全身性神經節苷脂貯積症(例如GM1)、血色素沉著症、β球蛋白第6個密碼子中之血紅素C突變(HbC)、血友病、亨丁頓氏病(Huntington's disease)、胡爾勒症候群(Hurler Syndrome)、低磷酸酯酶症、柯林菲特氏症候群(Klinefleter syndrome)、克拉畢氏病(Krabbes Disease)、蘭-吉二氏症候群(Langer-Giedion Syndrome)、白血球黏附缺乏症(LAD，OMIM第116920號)、腦白質失養症、長QT症候群、馬凡氏症候群(Marfan syndrome)、牟比士症候群(Moebius syndrome)、黏多醣症(MPS)、指甲髕骨症候群、腎性尿崩症、神經纖維瘤、尼曼-匹克病(Neimann-Pick disease)、骨發生不全、卟啉症、普瑞德-威利症候群(Prader-Willi syndrome)、早老症、普洛提斯症候群(Proteus syndrome)、視網膜母細胞瘤、瑞特氏症候群(Rett syndrome)、魯賓斯坦-泰比症候群(Rubinstein-Taybi syndrome)、聖菲利柏氏症候群(Sanfilippo syndrome)、嚴重複合型免疫缺乏症(SCID)、許旺氏症候群(Shwachman syndrome)、鐮形血球病(鐮形血球貧血症)、史密斯-馬格尼斯症候群(Smith-Magenis syndrome)、史蒂克勒症候群(Stickler syndrome)、戴薩克斯病(Tay-Sachs disease)、血小板減少-橈骨缺失(TAR)症候群、崔契爾柯林斯症候群(Treacher Collins syndrome)、三染色體症、結節性硬化症、特納氏症候群(Turner’s syndrome)、尿素循環障礙、逢希伯-林道病(von Hippel-Landau disease)、瓦登伯格氏症候群(Waardenburg syndrome)、威廉氏症候群(Williams syndrome)、威爾森氏病(Wilson’s disease)、威斯科特-奧爾德里奇二氏症候群(Wiskott-Aldrich syndrome)、X性聯淋巴增生症候群(XLP，OMIM第308240號)。For example, the exogenous sequence may comprise a sequence encoding a polypeptide that is deficient or nonfunctional in an individual suffering from a genetic disorder including, but not limited to, any of the following genetic disorders: achondroplasia, color blindness, Acid maltase deficiency, adenosine deaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardi syndrome, alpha-1 antitrypsin deficiency, alpha thalassemia , Androgen insensitivity syndrome, Abert syndrome (apert syndrome), arrhythmogenic right ventricular dysplasia (arrhythmogenic right ventricular dysplasia), ataxia microvascular dilatation, Barth syndrome (barth syndrome), beta Mediterranean Anemia, blue rubber bleb nevus syndrome, canavan disease, chronic granulomatous disease (CGD), cri du chat syndrome, cystic fibrosis, del Dercum's disease, ectodermal dysplasia, fanconi anemia, musculoskeletal ossification progressive, X-fragile syndrome, galactosemia, Gaucher's disease , systemic gangliosidosis (eg GM1), hemochromatosis, heme C mutation in beta globulin codon 6 (HbC), hemophilia, Huntington's disease , Hurler Syndrome, Hypophosphatasia, Klinefleter Syndrome, Krabbes Disease, Langer-Giedion Syndrome, Leukocyte Adhesion deficiency (LAD, OMIM No. 116920), leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), nail patella syndrome , nephrogenic diabetes insipidus, neurofibromatosis, Neimann-Pick disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome, progeria, progeria Proteus syndrome, Retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome ), Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sickle cell anemia), Smith-Magnes syndrome ( Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease, thrombocytopenia-absence of the radius (TAR) syndrome, Treacher Collins syndrome, trisomy , tuberous sclerosis complex, Turner's syndrome, urea cycle disorder, von Hippel-Landau disease, Waardenburg syndrome, Williams syndrome ), Wilson's disease, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP, OMIM No. 308240).

可藉由靶向整合加以治療之其他例示性疾病包括後天性免疫缺乏症、溶酶體貯積病(例如，高雪氏病、GM1、法布瑞氏病(Fabry disease)及戴薩克斯病)、黏多醣症(例如，韓特氏病(Hunter’s disease)、賀勒氏病(Hurler’s disease))、血紅素病(例如，鐮形血球病、HbC、α型地中海型貧血、β型地中海型貧血)及血友病。Other exemplary diseases that can be treated by targeted integration include acquired immunodeficiency, lysosomal storage diseases (eg, Gaucher's disease, GM1, Fabry disease, and Dessachs disease) , mucopolysaccharidosis (eg, Hunter's disease, Hurler's disease), heme disease (eg, sickle cell disease, HbC, alpha thalassemia, beta thalassemia ) and hemophilia.

在某些實施例中，外源序列可包含允許選擇已經歷靶向整合之細胞的標記基因(以上所描述)及編碼其他功能性之連接序列。標記基因之非限制性實例包括GFP、藥物選擇標記物及其類似物。In certain embodiments, the exogenous sequence may comprise marker genes (described above) that allow selection of cells that have undergone targeted integration, and linker sequences encoding additional functionality. Non-limiting examples of marker genes include GFP, drug selectable markers, and analogs thereof.

可***之其他基因序列可包括例如用於置換突變序列之野生型基因。舉例而言，可將野生型因子IX基因序列***該基因之內源副本已突變之幹細胞的基因組中。可將野生型副本***在內源基因座處，或可替代地靶向安全港基因座。Other gene sequences that can be inserted can include, for example, wild-type genes for replacement of mutated sequences. For example, a wild-type factor IX gene sequence can be inserted into the genome of a stem cell in which the endogenous copy of the gene has been mutated. The wild-type copy can be inserted at the endogenous locus, or alternatively targeted to the safe harbor locus.

遵循本發明說明書之教示內容來構建此種表現卡匣利用了分子生物學技術中眾所周知的方法(參見例如Ausubel或Maniatis)。在使用表現卡匣來產生轉殖基因動物之前，可藉由將表現卡匣引入適合之細胞株(例如，原代細胞、經轉型細胞或永生化細胞株)中來測試表現卡匣對與所選控制元件相關之壓力誘導因子的反應性。Construction of such expression cassettes following the teachings of the present specification utilizes methods well known in the art of molecular biology (see eg, Ausubel or Maniatis). Before using the expression cassette to generate transgenic animals, the expression cassette pair can be tested against all cell lines by introducing the expression cassette into a suitable cell line (eg, primary, transformed or immortalized cell lines). The responsiveness of stress-inducible factors associated with selected control elements.

此外，儘管對於表現而言並不需要，但外源序列亦可包括轉錄或轉譯調控序列，例如啟動子、增強子、絕緣子、內部核糖體進入位點、編碼2A肽之序列、聚腺苷酸化信號及2A多肽與聚腺苷酸化信號之組合。此外，可將相關基因之控制元件可操作地連接至報告基因以產生嵌合基因(例如，報告基因表現卡匣)。In addition, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosomal entry sites, sequences encoding 2A peptides, polyadenylation Combination of signal and 2A polypeptide with polyadenylation signal. In addition, control elements of related genes can be operably linked to reporter genes to generate chimeric genes (eg, reporter gene expression cassettes).

亦可達成非編碼核酸序列之靶向***。亦可將編碼反義RNA、RNAi、shRNA及微型RNA (miRNA)之序列用於靶向***。Targeted insertion of non-coding nucleic acid sequences can also be achieved. Sequences encoding antisense RNA, RNAi, shRNA, and microRNA (miRNA) can also be used for targeted insertion.

在其他實施例中，供體核酸可包含非編碼序列作為其他核酸酶設計之特定標靶位點。隨後，可在細胞中表現其他核酸酶，使得原始供體分子裂解且藉由***另一相關供體分子加以修飾。以此方式，可產生供體分子之反復整合，從而允許特定相關基因座處或安全港基因座處之特質堆疊。 遞送In other embodiments, the donor nucleic acid may contain non-coding sequences as specific target sites for other nuclease designs. Subsequently, other nucleases can be expressed in the cell such that the original donor molecule is cleaved and modified by insertion of another related donor molecule. In this way, iterative integration of donor molecules can be generated, allowing for stacking of traits at specific relevant loci or at safe harbor loci. deliver

如本文中(表1)所描述之核酸酶、編碼此等核酸酶之聚核苷酸、供體聚核苷酸及包含本文中所描述之蛋白質、聚核苷酸及蛋白質與聚核苷酸之組合的組合物可藉由任何適合之手段活體內或擬體內遞送至任何細胞類型中。Nucleases as described herein (Table 1), polynucleotides encoding such nucleases, donor polynucleotides and proteins, polynucleotides and proteins and polynucleotides comprising the herein described The combined compositions can be delivered to any cell type in vivo or in vivo by any suitable means.

適合之細胞包括真核(例如動物)及原核細胞以及真核及原核細胞株。此種細胞或由此種細胞產生之細胞株的非限制性實例包括COS、CHO (例如，CHO-S、CHO-K1、CHO-DG44、CHO-DUXB11、CHO-DUKX、CHOK1SV)、VERO、MDCK、WI38、V79、B14AF28-G3、BHK、HaK、NS0、SP2/0-Ag14、HeLa、HEK293 (例如，HEK293-F、HEK293-H、HEK293-T)及perC6細胞以及昆蟲細胞，諸如草地貪夜蛾(Sf)，或真菌細胞，諸如釀酒酵母、畢赤酵母及裂殖酵母。在某些實施例中，該細胞株為CHO、MDCK或HEK293細胞株。適合之細胞亦包括幹細胞，舉例而言，諸如胚胎幹細胞、誘導多能幹細胞、造血幹細胞、神經元幹細胞及間質幹細胞。Suitable cells include eukaryotic (eg, animal) and prokaryotic cells as well as eukaryotic and prokaryotic cell strains. Non-limiting examples of such cells or cell lines derived from such cells include COS, CHO (eg, CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK , WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (eg, HEK293-F, HEK293-H, HEK293-T) and perC6 cells as well as insect cells such as Moth (Sf), or fungal cells such as Saccharomyces cerevisiae, Pichia and Schizosaccharomyces cerevisiae. In certain embodiments, the cell line is a CHO, MDCK or HEK293 cell line. Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.

遞送如本文中所描述之核酸酶的方法描述於例如美國專利第6,453,242號、第6,503,717號、第6,534,261號、第6,599,692號、第6,607,882號、第6,689,558號、第6,824,978號、第6,933,113號、第6,979,539號、第7,013,219號及第7,163,824號，所有專利之揭示內容以全文引用之方式併入本文中。Methods of delivering nucleases as described herein are described, for example, in US Pat. Nos. 7,013,219 and 7,163,824, the disclosures of all patents are incorporated herein by reference in their entirety.

亦可使用含有編碼一或多個如本文中所描述之ZFN的序列的載體來遞送如本文中所描述之核酸酶、供體構建體及核酸酶與供體構建體之組合。可使用任何載體系統，包括但不限於質體載體、反轉錄病毒載體、慢病毒載體、腺病毒載體、痘病毒載體、疱疹病毒載體及腺相關病毒載體等。亦參見美國專利第6,534,261號、第6,607,882號、第6,824,978號、第6,933,113號、第6,979,539號、第7,013,219號及第7,163,824號，該等專利以全文引用之方式併入本文中。此外，應顯而易見，此等載體中之任一者均可包含一或多個治療所需之序列。因而，當將一或多種核酸酶及供體構建體引入細胞中時，該等核酸酶、供體聚核苷酸及核酸酶與供體聚核苷酸之組合可攜帶在相同的載體上或在不同的載體上(DNA MC)。當使用多個載體時，各載體可包含編碼一或多個核酸酶、一或多個供體構建體及一或多個核酸酶與一或多個供體構建體之組合的序列。可使用習知基於病毒及非病毒之基因轉移方法將編碼核酸酶、供體構建體及核酸酶與供體構建體之組合的核酸引入細胞(例如，哺乳動物細胞)及靶組織中。非病毒載體遞送系統包括DNA或RNA質體、DNA MC、裸核酸及與諸如脂質體或泊洛沙姆之遞送媒劑複合之核酸。適合之非病毒載體包括奈米趨向性載體，包括可購自InCellArt (France)之載體。病毒載體遞送系統包括在遞送至細胞之後具有附加體型或整合型基因組之DNA及RNA病毒。關於經工程改造之DNA結合蛋白及包含此等結合蛋白之融合蛋白之活體內遞送的綜述，參見例如Rebar (2004)Expert Opinion Invest. Drugs 13(7):829-839；Rossi等人, (2007)Nature Biotech. 25(12):1444-1454；以及一般基因遞送參考文獻，諸如Anderson,Science 256:808-813 (1992)；Nabel及Felgner,TIBTECH 11:211-217 (1993)；Mitani及Caskey,TIBTECH 11:162-166 (1993)；Dillon,TIBTECH 11:167-175 (1993)；Miller,Nature 357:455-460 (1992)；Van Brunt,Biotechnology 6(10):1149-1154 (1988)；Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995)；Kremer及Perricaudet,British Medical Bulletin 51(1):31-44 (1995)；Haddada等人,Current Topics in Microbiology and Immunology ,Doerfler及Böhm (編) (1995)；及Yu等人,Gene Therapy 1:13-26 (1994)。Nucleases, donor constructs, and combinations of nuclease and donor constructs as described herein can also be delivered using vectors containing sequences encoding one or more ZFNs as described herein. Any vector system can be used, including but not limited to plastid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors, herpes virus vectors, adeno-associated virus vectors, and the like. See also US Patent Nos. 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, which are incorporated herein by reference in their entirety. Furthermore, it should be apparent that any of these vectors may contain one or more sequences required for therapy. Thus, when one or more nucleases and donor constructs are introduced into a cell, the nucleases, donor polynucleotides and combinations of nucleases and donor polynucleotides can be carried on the same vector or on a different vector (DNA MC). When multiple vectors are used, each vector may comprise sequences encoding one or more nucleases, one or more donor constructs, and a combination of one or more nucleases and one or more donor constructs. Nucleic acids encoding nucleases, donor constructs, and combinations of nucleases and donor constructs can be introduced into cells (eg, mammalian cells) and target tissues using conventional viral and non-viral-based gene transfer methods. Non-viral vector delivery systems include DNA or RNA plastids, DNA MCs, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Suitable non-viral vectors include nanotropism vectors, including those available from InCellArt (France). Viral vector delivery systems include DNA and RNA viruses with episomal or integrated genomes after delivery to cells. For a review of the in vivo delivery of engineered DNA-binding proteins and fusion proteins comprising such binding proteins, see, eg, Rebar (2004) Expert Opinion Invest. Drugs 13(7):829-839; Rossi et al., (2007 ) Nature Biotech. 25(12):1444-1454; and general gene delivery references such as Anderson, Science 256:808-813 (1992); Nabel and Felgner, TIBTECH 11:211-217 (1993); Mitani and Caskey , TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988) Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., Current Topics in Microbiology and Immunology , Doerfler and Böhm ( ed) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

核酸之非病毒遞送方法包括電穿孔、脂質轉染、顯微注射、生物彈道、病毒體、脂質體、免疫脂質體、聚陽離子或脂質:核酸結合物、裸DNA、人工病毒體、膜變形及試劑增強DNA吸收。亦可將使用例如Sonitron 2000系統(Rich-Mar)之音波穿孔用於遞送核酸。Non-viral delivery methods of nucleic acids include electroporation, lipofection, microinjection, bioballistics, virions, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, membrane deformation and Reagents enhance DNA uptake. Sonic perforation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids.

其他例示性核酸遞送系統包括由Amaxa Biosystems (Cologne，Germany)、Maxcyte, Inc. (Rockville，Maryland)、BTX Molecular Delivery Systems (Holliston，MA)及Copernicus Therapeutics Inc.提供之彼等核酸遞送系統(參見例如US6008336)。脂質轉染描述於例如美國專利第5,049,386號、第4,946,787號及第4,897,355號中，且脂質轉染試劑可購自市面(例如Transfectam™及Lipofectin™)。適用於聚核苷酸之有效受體識別脂質轉染的陽離子及中性脂質包括Felgner、WO 91/17424、WO 91/16024之彼等脂質。Other exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA), and Copernicus Therapeutics Inc. (see e.g. US6008336). Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (eg, Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those from Felgner, WO 91/17424, WO 91/16024.

脂質:核酸複合物，包括靶向脂質體，諸如免疫脂質複合物之製備對熟習此項技術者為眾所周知的(參見例如Crystal,Science 270:404-410 (1995)；Blaese等人, Cancer Gene Ther. 2:291-297 (1995)；Behr等人, Bioconjugate Chem . 5:382-389 (1994)；Remy等人, Bioconjugate Chem. 5:647-654 (1994)；Gao等人, Gene Therapy 2:710-722 (1995)；Ahmad等人, Cancer Res. 52:4817-4820 (1992)；美國專利第4,186,183號、第4,217,344號、第4,235,871號、第4,261,975號、第4,485,054號、第4,501,728號、第4,774,085號、第4,837,028號及第4,946,787號)。其他脂質:核酸複合物包括包含新穎陽離子脂質、新穎聚乙二醇化脂質及新穎陽離子脂質與新穎聚乙二醇化脂質之組合的彼等脂質:核酸複合物(參見例如美國臨時專利申請案第62/432,042號及第62/458,373號)。The preparation of lipid:nucleic acid complexes, including targeted liposomes, such as immunolipid complexes, is well known to those skilled in the art (see, eg, Crystal, Science 270:404-410 (1995); Blaese et al ., Cancer Gene Ther 2:291-297 (1995) ; Behr et al , Bioconjugate Chem . 5:382-389 (1994); Remy et al , Bioconjugate Chem . 5:647-654 (1994); Gao et al , Gene Therapy 2: 710-722 (1995); Ahmad et al ., Cancer Res. 52:4817-4820 (1992); U.S. Pat. 4,774,085, 4,837,028 and 4,946,787). Other lipid:nucleic acid complexes include those lipid:nucleic acid complexes comprising novel cationic lipids, novel pegylated lipids, and combinations of novel cationic lipids and novel pegylated lipids (see, e.g., U.S. Provisional Patent Application No. 62/ 432,042 and 62/458,373).

其他遞送方法包括使用將欲遞送之核酸包封至EnGeneIC遞送媒劑(EDV)中。使用抗體之一個臂對靶組織具有特異性且另一個臂對EDV具有特異性的雙特異性抗體將此等EDV特異性遞送至靶組織。該抗體將EDV帶至靶細胞表面，接著藉由細胞吞噬作用將EDV帶入細胞中。一旦在細胞中，便釋放內含物(參見MacDiarmid等人,(2009)Nature Biotechnology 27(7):643)。Other delivery methods include the use of encapsulation of the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). These EDVs are specifically delivered to target tissues using bispecific antibodies in which one arm of the antibody is specific for the target tissue and the other arm is specific for EDV. The antibody brings EDV to the surface of target cells and then into cells by phagocytosis. Once in the cell, the contents are released (see MacDiarmid et al., (2009) Nature Biotechnology 27(7):643).

使用基於RNA或DNA病毒之系統遞送編碼經工程改造之ZFP、TALE及CRISPR/Cas系統之核酸利用高度演化方法使病毒靶向體內之特定細胞並且將病毒有效負載運送至細胞核。可將病毒載體直接投與患者(活體內)，或可在試管內使用其處理細胞並且將經修飾之細胞投與患者(擬體內)。用於遞送ZFP之習知基於病毒之系統包括但不限於用於基因轉移之反轉錄病毒、慢病毒、腺病毒、腺相關病毒、牛痘及單純疱疹病毒載體。有可能利用反轉錄病毒、慢病毒及腺相關病毒基因轉移法整合在宿主基因組中，通常引起所***之轉殖基因的長期表現。另外，已在許多不同的細胞類型及靶組織中觀測到高轉導效率。The use of RNA or DNA virus-based systems to deliver nucleic acids encoding engineered ZFP, TALE and CRISPR/Cas systems utilizes a highly evolved approach to target viruses to specific cells in the body and deliver viral payloads to the nucleus. The viral vector can be administered directly to a patient (in vivo), or it can be used in vitro to treat cells and administer the modified cells to the patient (in vivo). Known virus-based systems for delivering ZFPs include, but are not limited to, retroviral, lentiviral, adenovirus, adeno-associated virus, vaccinia, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible using retroviral, lentiviral and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgenic gene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

可藉由併入外來包被蛋白，從而擴增靶細胞之潛在靶群體來改變反轉錄病毒之趨向性。慢病毒載體為能夠轉導或感染非***細胞且典型地產生高病毒效價的反轉錄病毒載體。反轉錄病毒基因轉移系統之選擇視靶組織而定。反轉錄病毒載體由具有至多6-10 kb外來序列之包封容量的順式作用長末端重複序列構成。最小順式作用LTR足以複製並包封載體，接著用於將治療基因整合至靶細胞中以提供永久性轉殖基因表現。廣泛使用之反轉錄病毒載體包括基於鼠類白血病病毒(MuLV)、長臂猿白血病病毒(GaLV)、猿猴免疫缺陷病毒(SIV)、人類免疫缺陷病毒(HIV)及其組合之彼等反轉錄病毒載體(參見例如Buchscher等人,J. Virol . 66:2731-2739 (1992)；Johann等人, J. Virol. 66:1635-1640 (1992)；Sommerfelt等人,Virol. 176:58-59 (1990)；Wilson等人,J. Virol. 63:2374-2378 (1989)；Miller等人,J. Virol. 65:2220-2224 (1991)；PCT/US94/05700)。Retroviral tropism can be altered by the incorporation of foreign coat proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with an encapsulation capacity of up to 6-10 kb of foreign sequences. The minimal cis-acting LTR is sufficient to replicate and encapsulate the vector, which is then used to integrate the therapeutic gene into target cells to provide permanent transgenic gene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof ( See, eg, Buchscher et al, J. Virol . 66:2731-2739 (1992); Johann et al , J. Virol. 66:1635-1640 (1992); Sommerfelt et al, Virol. 176:58-59 (1990) ; Wilson et al, J. Virol. 63:2374-2378 (1989); Miller et al, J. Virol. 65:2220-2224 (1991); PCT/US94/05700).

在瞬時表現較佳之應用中，可使用基於腺病毒之系統。基於腺病毒之載體能夠在許多細胞類型中具有極高轉導效率且不需要細胞***。利用此種載體，已獲得高效價及高表現水準。可在相對簡單之系統中大量產生此載體。腺相關病毒(「AAV」)載體亦用於用標靶核酸轉導細胞，例如，用於核酸及肽之試管內產生，以及用於活體內及擬體內基因療法程序(參見例如West等人,Virology 160:38-47 (1987)；美國專利第4,797,368號；WO 93/24641；Kotin,Human Gene Therapy 5:793-801 (1994)；Muzyczka,J. Clin. Invest. 94:1351 (1994)。重組AAV載體之構建描述於許多出版物中，包括美國專利第5,173,414號；Tratschin等人,Mol. Cell. Biol. 5:3251-3260 (1985)；Tratschin等人,Mol. Cell. Biol. 4:2072-2081 (1984)；Hermonat及Muzyczka,PNAS 81:6466-6470 (1984)；及Samulski等人,J. Virol. 63:03822-3828 (1989)。In applications where transient performance is better, adenovirus-based systems can be used. Adenovirus-based vectors are capable of extremely high transduction efficiencies in many cell types and do not require cell division. Using this carrier, high titers and high performance levels have been achieved. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, e.g., for in vitro production of nucleic acids and peptides, and for in vivo and in vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); US Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). The construction of recombinant AAV vectors is described in numerous publications, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4: 2072-2081 (1984); Hermonat and Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al, J. Virol. 63:03822-3828 (1989).

當前有至少六種病毒載體方法可用於在臨床試驗中進行基因轉移，該等臨床試驗利用涉及由***輔助細胞株中之基因補助缺陷載體以產生轉導劑之方法。There are currently at least six viral vector methods available for gene transfer in clinical trials utilizing methods that involve complementation of a defective vector by a gene inserted into a helper cell line to generate a transducing agent.

pLASN及MFG-S為已用於臨床試驗中之反轉錄病毒載體之實例(Dunbar等人, Blood 85:3048-305 (1995)；Kohn等人, Nat. Med . 1:1017-102 (1995)；Malech等人, PNAS 94:22 12133-12138 (1997))。PA317/pLASN為用於基因療法試驗之第一治療載體。(Blaese等人, Science 270:475-480 (1995))。針對MFG-S包封載體已觀測到50%以上之轉導效率。(Ellem等人, Immunol Immunother . 44(1):10-20 (1997)；Dranoff等人, Hum. Gene Ther. 1:111-2 (1997)。pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al ., Blood 85:3048-305 (1995); Kohn et al ., Nat. Med . 1:1017-102 (1995) ; Malech et al ., PNAS 94:22 12133-12138 (1997)). PA317/pLASN is the first therapeutic vector for gene therapy trials. (Blaese et al ., Science 270:475-480 (1995)). Transduction efficiencies of over 50% have been observed for MFG-S encapsulated vectors. (Ellem et al ., Immunol Immunother . 44(1):10-20 (1997); Dranoff et al ., Hum. Gene Ther. 1:111-2 (1997).

重組腺相關病毒載體(rAAV)為有望替代基於缺陷性非致病性小病毒腺相關2型病毒之基因遞送系統。所有載體均來源於僅保留側接轉殖基因表現卡匣之AAV 145 bp反向末端重複序列的質體。由於整合至經轉導細胞之基因組中而達成有效基因轉移及穩定轉殖基因遞送為此載體系統之關鍵特徵。(Wagner等人, Lancet 351:9117 1702-3 (1998)；Kearns等人, Gene Ther. 9:748-55 (1996))。根據本發明，亦可使用其他AAV血清型，包括AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9及AAVrh.10以及任何新穎AAV血清型。Recombinant adeno-associated virus vectors (rAAV) are promising alternatives to gene delivery systems based on defective non-pathogenic adeno-associated virus type 2 viruses. All vectors were derived from plastids retaining only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgenic gene delivery due to integration into the genome of transduced cells are key features of this vector system. (Wagner et al , Lancet 351:9117 1702-3 (1998); Kearns et al , Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh.10, as well as any novel AAV serotypes, may also be used in accordance with the present invention.

複製缺陷型重組腺病毒載體(Ad)可在高效價下產生且容易感染許多不同的細胞類型。大部分腺病毒載體經工程改造以使得轉殖基因置換Ad E1a、E1b或E3基因；隨後，複製缺陷型載體在反式提供已刪除之基因功能之人類293細胞中增殖。Ad載體可在活體內轉導多種類型之組織，包括不***之已分化細胞，諸如在肝臟、腎臟及肌肉中所發現之彼等細胞。習知Ad載體具有較大攜帶容量。臨床試驗中使用之Ad載體之實例涉及利用肌肉內注射進行抗腫瘤免疫之聚核苷酸療法( Sterman等人, Hum. Gene Ther. 7:1083-9 (1998))。臨床試驗中用於基因轉移之腺病毒載體的其他實例包括Rosenecker等人, Infection 24:1 5-10 (1996)；Sterman等人, Hum. Gene Ther. 9:7 1083-1089 (1998)；Welsh等人, Hum. Gene Ther. 2:205-18 (1995)；Alvarez等人, Hum. Gene Ther . 5:597-613 (1997)；Topf等人, Gene Ther . 5:507-513 (1998)；Sterman等人, Hum. Gene Ther. 7:1083-1089 (1998)。Replication-deficient recombinant adenoviral vectors (Ads) can be produced at high titers and readily infect many different cell types. Most adenoviral vectors are engineered so that the transgenic gene replaces the Ad E1a, E1b, or E3 gene; replication-deficient vectors are then propagated in human 293 cells that provide the function of the deleted gene in trans. Ad vectors can transduce various types of tissues in vivo, including differentiated cells that do not divide, such as those found in liver, kidney, and muscle. Conventional Ad vectors have large carrying capacity. An example of an Ad vector used in clinical trials involves polynucleotide therapy for antitumor immunity using intramuscular injection (Sterman et al ., Hum. Gene Ther. 7:1083-9 (1998)). Other examples of adenoviral vectors used for gene transfer in clinical trials include Rosenecker et al ., Infection 24:1 5-10 (1996); Sterman et al ., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al , Hum. Gene Ther. 2:205-18 (1995); Alvarez et al , Hum. Gene Ther . 5:597-613 (1997); Topf et al , Gene Ther . 5:507-513 (1998) ; Sterman et al ., Hum. Gene Ther. 7:1083-1089 (1998).

使用包封細胞來形成能夠感染宿主細胞之病毒粒子。此種細胞包括可用於包封AAV及腺病毒之HEK293及Sf9細胞，以及包封反轉錄病毒之ψ2細胞或PA317細胞。用於基因療法之病毒載體通常由將核酸載體包封至病毒粒子中之生產者細胞株產生。載體典型地含有包封及隨後整合至宿主中(若適用)所需之最小病毒序列，其他病毒序列被編碼欲表現之蛋白質的表現卡匣置換。由包封細胞株反式提供缺失之病毒功能。舉例而言，基因療法中所使用之AAV載體典型地僅具有需要包封並整合至宿主基因組中之來自AAV基因組之反向末端重複(ITR)序列。病毒DNA被包封在細胞株中，其含有編碼其他AAV基因(亦即，rep 及cap )但缺乏ITR序列之輔助質體。該細胞株亦感染有腺病毒作為輔助因子。該輔助病毒促進輔助質體之AAV載體複製及AAV基因表現。輔助質體由於缺乏ITR序列而未大量包封。腺病毒污染可藉由例如腺病毒比AAV更敏感之熱處理而減少。在一些實施例中，使用桿狀病毒表現系統來產生AAV (參見例如美國專利第6,723,551號及第7,271,002號)。Encapsulated cells are used to form viral particles capable of infecting host cells. Such cells include HEK293 and Sf9 cells that can be used to encapsulate AAV and adenovirus, and ψ2 cells or PA317 cells that encapsulate retrovirus. Viral vectors for gene therapy are typically produced from producer cell lines that encapsulate the nucleic acid vector into virions. Vectors typically contain the minimal viral sequences required for encapsulation and subsequent integration into the host (if applicable), other viral sequences are replaced by expression cassettes encoding the proteins to be expressed. The missing viral function is provided in trans by the encapsulating cell line. For example, AAV vectors used in gene therapy typically only have inverted terminal repeat (ITR) sequences from the AAV genome that need to be encapsulated and integrated into the host genome. Viral DNA is encapsulated in cell lines that contain helper plastids encoding other AAV genes (ie, rep and cap ) but lacking ITR sequences. This cell line was also infected with adenovirus as a cofactor. The helper virus promotes AAV vector replication and AAV gene expression in helper plastids. Helper plastids were not heavily encapsulated due to the lack of ITR sequences. Adenovirus contamination can be reduced by, for example, heat treatment where adenovirus is more sensitive than AAV. In some embodiments, AAV is produced using a baculovirus expression system (see, eg, US Pat. Nos. 6,723,551 and 7,271,002).

自293或桿狀病毒系統中純化出AAV粒子典型地包括使產生病毒之細胞生長，繼而自細胞上清液收集病毒粒子或將細胞溶解並且自粗溶解產物收集病毒。接著藉由此項技術中已知的方法來純化AAV，包括離子交換層析(例如參見美國專利第7,419,817號及第6,989,264號)、離子交換層析及CsCl密度離心(例如PCT公開案WO2011094198A10)、免疫親和力層析(例如WO2016128408)或使用AVB瓊脂糖進行純化(例如GE Healthcare Life Sciences)。Purification of AAV particles from a 293 or baculovirus system typically involves growing virus-producing cells followed by collection of virus particles from cell supernatants or lysing cells and collecting virus from crude lysates. The AAV is then purified by methods known in the art, including ion exchange chromatography (eg, see US Pat. Nos. 7,419,817 and 6,989,264), ion exchange chromatography, and CsCl density centrifugation (eg, PCT publication WO2011094198A10), Immunoaffinity chromatography (eg WO2016128408) or purification using AVB Sepharose (eg GE Healthcare Life Sciences).

在許多基因療法應用中，將基因療法載體以高度特異性遞送至特定組織類型為理想的。相應地，可藉由將配位體表現為與病毒外表面上之病毒外殼蛋白的融合蛋白質來修飾病毒載體，以便對指定細胞類型具有特異性。選擇對已知存在於相關細胞類型上之受體具有親和力的配位體。舉例而言，據Han等人,Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995)報告，莫洛尼鼠類白血病毒可經修飾以表現與gp70融合之人類調蛋白(heregulin)，且該重組病毒感染表現人類表皮生長因子受體之某些人類乳癌細胞。此原理可延伸至其他病毒-靶細胞配對，其中該靶細胞表現受體且該病毒表現包含細胞表面受體配位體之融合蛋白質。舉例而言，絲狀噬菌體可經工程改造以呈現實際上對任何所選細胞受體均具有特定結合親和力的抗體片段(例如，FAB或Fv)。儘管以上描述主要適用於病毒載體，但相同的原理可適用於非病毒載體。此種載體可經工程改造以包含有利於被特定靶細胞吸收之特定吸收序列。In many gene therapy applications, it is desirable to deliver gene therapy vectors with a high degree of specificity to specific tissue types. Accordingly, viral vectors can be modified to be specific for a given cell type by expressing the ligand as a fusion protein with the viral coat protein on the outer surface of the virus. Ligands are selected that have affinity for receptors known to be present on relevant cell types. For example, as reported by Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995), Moloney murine leukemia virus can be modified to express a human heregulin fused to gp70 , and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs in which the target cell expresses the receptor and the virus expresses a fusion protein comprising a cell surface receptor ligand. For example, filamentous bacteriophages can be engineered to present antibody fragments (eg, FAB or Fv) with specific binding affinity for virtually any cellular receptor of choice. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

可藉由投與個別患者，典型地藉由如以下所描述之全身投與(例如，靜脈內、腹膜內、肌肉內、皮下或顱內輸注)或局部施用而在活體內遞送基因療法載體。替代地，可將載體擬體內遞送至細胞，諸如來自個別患者之外植細胞(例如淋巴細胞、骨髓吸出物、組織活檢)或萬能供血者造血幹細胞，繼而通常在選擇已併入載體之細胞之後將細胞再植入患者中。Gene therapy vectors can be delivered in vivo by administration to individual patients, typically by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical administration as described below. Alternatively, the vector can be delivered to cells in vivo, such as explanted cells from an individual patient (eg, lymphocytes, bone marrow aspirate, tissue biopsy) or hematopoietic stem cells from pluripotent donors, followed by selection, typically after selection of cells that have incorporated the vector. The cells are reimplanted into the patient.

含有核酸酶、供體構建體及核酸酶與供體構建體之組合的載體(例如反轉錄病毒、腺病毒、脂質體等)亦可直接投與生物體以便在活體內轉導細胞。替代地，可投與裸DNA。投與係藉由正常情況下用於引入分子最終與血液或組織細胞接觸之任何途徑，包括但不限於注射、輸注、局部施用及電穿孔。投與此種核酸之適合方法對熟習此項技術者為可利用且眾所周知的，且儘管可使用超過一種途徑來投與特定組合物，但特定途徑通常可提供比另一途徑更即時且更有效之反應。Vectors (eg, retroviruses, adenoviruses, liposomes, etc.) containing nucleases, donor constructs, and combinations of nucleases and donor constructs can also be administered directly to organisms for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any route normally used to introduce the molecule into final contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. Suitable methods of administering such nucleic acids are available and well known to those skilled in the art, and although more than one route may be used to administer a particular composition, a particular route may generally provide a more immediate and more effective approach than another route. reaction.

適合於引入本文中所描述之聚核苷酸(例如，編碼核酸酶之聚核苷酸、雙鏈供體及編碼核酸酶之聚核苷酸與雙鏈供體之組合)的載體包括非整合性慢病毒載體(IDLV)。參見例如Ory等人, (1996)Proc. Natl. Acad. Sci. USA 93:11382-11388；Dull等人, (1998)J. Virol. 72:8463-8471；Zuffery等人, (1998)J. Virol. 72:9873-9880；Follenzi等人, (2000)Nature Genetics 25:217-222；美國專利公開案第2009/0117617號。Vectors suitable for introduction of the polynucleotides described herein (eg, nuclease-encoding polynucleotides, double-stranded donors, and combinations of nuclease-encoding polynucleotides and double-stranded donors) include non-integrating Sexual lentiviral vector (IDLV). See, eg, Ory et al, (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al, (1998) J. Virol. 72:8463-8471; Zuffery et al, (1998) J. Virol. 72:9873-9880; Follenzi et al., (2000) Nature Genetics 25:217-222; US Patent Publication No. 2009/0117617.

醫藥學上可接受之載劑部分由所投與之特定組合物以及由用於投與該組合物之特定方法決定。相應地，醫藥組合物有多種適合之調配物可利用，如以下所描述(參見例如Remington's Pharmaceutical Sciences , 第17版, 1989)。The pharmaceutically acceptable carrier is determined in part by the particular composition to be administered and by the particular method used to administer the composition. Accordingly, pharmaceutical compositions are available in a variety of suitable formulations, as described below (see, eg, Remington's Pharmaceutical Sciences , 17th ed., 1989).

應顯而易見，可使用相同或不同的系統來遞送核酸酶編碼序列及供體構建體。舉例而言，可由相同的DNA MC攜帶核酸酶及供體。替代地，可由MC攜帶供體聚核苷酸，而可由標準質體或AAV載體攜帶一或多個核酸酶。此外，可藉由相同或不同的途徑(肌肉內注射、尾靜脈注射、其他靜脈內注射、腹膜內投與或肌肉內注射)投與不同的載體。可同時或按任何相繼順序遞送載體。It should be apparent that the same or different systems can be used to deliver the nuclease coding sequence and the donor construct. For example, the nuclease and donor can be carried by the same DNA MC. Alternatively, the donor polynucleotide may be carried by the MC and one or more nucleases may be carried by a standard plastid or AAV vector. In addition, different vectors can be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injections, intraperitoneal administration or intramuscular injection). The vectors can be delivered simultaneously or in any sequential order.

因而，本發明包括活體內或擬體內治療順從***編碼治療蛋白質之轉殖基因的疾病及病狀。將組合物以在血清或靶器官或細胞中獲得治療多肽之所要濃度的有效量投與人類患者。投與可藉由將聚核苷酸遞送至所要靶細胞之任何手段來進行。舉例而言，涵蓋活體內及擬體內方法。靜脈內注射至門靜脈為較佳投與方法。其他活體內投與模式包括例如直接注射至肝葉或膽管中及遠端靜脈內注射至肝臟，包括通過肝動脈、直接注射至肝實質、經由肝動脈注射及通過膽道之逆行注射。擬體內投與模式包括對已切除肝細胞或其他肝臟細胞進行試管內轉導，繼而將經轉導之已切除輸注回人類患者之門靜脈血管系、肝實質或膽道中，參見例如Grossman等人, (1994)Nature Genetics , 6:335-341。Thus, the present invention includes in vivo or in vivo treatment of diseases and conditions amenable to insertion of a transgenic gene encoding a therapeutic protein. The composition is administered to a human patient in an amount effective to achieve the desired concentration of the therapeutic polypeptide in serum or target organs or cells. Administration can be by any means of delivering polynucleotides to the desired target cells. For example, in vivo and in vivo methods are contemplated. Intravenous injection into the portal vein is the preferred method of administration. Other modes of in vivo administration include, for example, direct injection into the liver lobe or bile duct and distal intravenous injection into the liver, including via the hepatic artery, direct injection into the liver parenchyma, injection via the hepatic artery, and retrograde injection via the biliary tract. In vivo modes of administration include in vitro transduction of resected hepatocytes or other liver cells followed by infusion of the transduced resected back into the portal vasculature, liver parenchyma or biliary tract of a human patient, see e.g. Grossman et al., (1994) Nature Genetics , 6:335-341.

欲投與之核酸酶及供體之有效量將因患者及根據相關治療多肽而各異。相應地，最佳由投與組合物之醫師來決定有效量，且可由一般熟習此項技術者容易地確定適當劑量。在允許足以整合及表現的時間(舉例而言，典型地4至15天)之後，分析治療多肽之血清或其他組織水準及與投與之前的初始水準相比較將確定所投與之量是過低、在正確範圍內或是或高。適用於初始及後續投與之方案亦為可變的，但以初始投與繼之以在必要時進行後續投與較為典型。後續投與可能以介於每日至每年至每若干年之範圍內的可變間隔投與。熟習此項技術者應瞭解，可推薦適當免疫抑制技術以避免因遞送載體之免疫抑制而抑制或妨礙轉導，參見例如Vilquin等人, (1995)Human Gene Ther ., 6:1391-1401。The effective amounts of nuclease and donor to be administered will vary from patient to patient and according to the relevant therapeutic polypeptide. Accordingly, the effective amount is best determined by the physician administering the composition, and the appropriate dose can be readily determined by one of ordinary skill in the art. After allowing time sufficient for integration and expression (eg, typically 4 to 15 days), analysis of serum or other tissue levels of therapeutic polypeptide and comparison to initial levels prior to administration will determine whether the amount administered is excessive low, within the correct range, or high. Applicable to the initial and subsequent investment programs are also variable, but an initial investment followed by a subsequent investment as necessary is more typical. Subsequent administrations may be administered at variable intervals ranging from daily to annual to every several years. Those skilled in the art will appreciate that appropriate immunosuppressive techniques can be recommended to avoid inhibiting or hindering transduction due to immunosuppression of the delivery vehicle, see eg, Vilquin et al., (1995) Human Gene Ther ., 6:1391-1401.

用於擬體內及活體內投與之調配物包括處於液體或已乳化液體中之懸浮液。通常將活性成分與醫藥學上可接受且同該活性成分相容之賦形劑混合。適合之賦形劑包括例如水、生理食鹽水、葡萄糖、甘油、乙醇或其類似物及其組合。另外，該組合物可含有微量輔助物質，諸如潤濕或乳化劑、pH值緩衝劑、穩定劑或可增強醫藥組合物之有效性的其他試劑。 細胞Formulations for in vivo and in vivo administration include suspensions in liquids or emulsified liquids. The active ingredient is usually mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Additionally, the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, or other agents that may enhance the effectiveness of the pharmaceutical composition. cell

本文中亦描述內源BCL11A增強子序列受本文中(表1)所描述之核酸酶修飾之細胞及細胞株。該修飾可為例如與細胞之野生型序列相比。對於該修飾而言，該細胞或細胞株可為異型接合或同型接合的。對BCL11A序列之修飾可包含***缺失。Also described herein are cells and cell lines in which the endogenous BCL11A enhancer sequence is modified with the nucleases described herein (Table 1). The modification can be, for example, compared to the wild-type sequence of the cell. For this modification, the cell or cell line may be heterozygous or homozygous. Modifications to the BCL11A sequence may include indels.

修飾較佳在核酸酶結合位點、裂解位點及結合位點與裂解位點之組合處或附近，例如，在裂解位點上游或下游1-300 (或介於其之間的任何值)個鹼基對內，更佳在結合位點、裂解位點或結合位點及裂解位點任一側之1-100個鹼基對(或介於其之間的任何值)內，甚至更佳在結合位點、裂解位點或結合位點及裂解位點任一側上之1-50個鹼基對(或介於其之間的任何值)內。在某些實施例中，該修飾處於BCL11A增強子之「+58」區域處或附近，例如，處於表1第一行中之任一者中所示之核酸酶結合位點處或附近。Modifications are preferably at or near nuclease binding sites, cleavage sites, and combinations of binding and cleavage sites, e.g., 1-300 upstream or downstream of the cleavage site (or any value in between) within 1 base pairs, more preferably within 1-100 base pairs (or any value in between) of the binding site, cleavage site or either side of the binding site and the cleavage site, or even more Preferably within 1-50 base pairs (or any value in between) on either side of the binding site, the cleavage site, or both the binding site and the cleavage site. In certain embodiments, the modification is at or near the "+58" region of the BCL11A enhancer, eg, at or near the nuclease binding site shown in any one of the first row of Table 1.

任何細胞或細胞株均可經修飾，例如幹細胞，例如胚胎幹細胞、誘導型多能幹細胞、造血幹細胞、神經元幹細胞及間質幹細胞。如本文中所描述之細胞的其他非限制性實例包括T細胞(例如CD4+、CD3+、CD8+等)；樹突狀細胞；B細胞。亦提供幹細胞之子代，包括部分或完全分化之細胞(例如RBC或RBC前驅細胞)。包括經修飾之BCL11A序列的其他細胞株的非限制性實例包括COS、CHO (例如，CHO-S、CHO-K1、CHO-DG44、CHO-DUXB11、CHO-DUKX、CHOK1SV)、VERO、MDCK、WI38、V79、B14AF28-G3、BHK、HaK、NS0、SP2/0-Ag14、HeLa、HEK293 (例如，HEK293-F、HEK293-H、HEK293-T)及perC6細胞以及昆蟲細胞，諸如草地貪夜蛾(Sf)，或真菌細胞，諸如釀酒酵母、畢赤酵母及裂殖酵母。Any cell or cell line can be modified, such as stem cells such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. Other non-limiting examples of cells as described herein include T cells (eg, CD4+, CD3+, CD8+, etc.); dendritic cells; B cells. Progeny of stem cells are also provided, including partially or fully differentiated cells (eg, RBCs or RBC precursor cells). Non-limiting examples of other cell lines that include modified BCL11A sequences include COS, CHO (eg, CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38 , V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (eg, HEK293-F, HEK293-H, HEK293-T) and perC6 cells as well as insect cells such as Spodoptera frugiperda ( Sf), or fungal cells such as Saccharomyces cerevisiae, Pichia and Schizosaccharomyces cerevisiae.

如本文中所描述之細胞適用於例如藉由擬體內療法來治療或預防病症。可擴增經核酸酶修飾之細胞，接著使用標準技術再引入患者體內。參見例如Tebas等人,(2014)New Eng J Med 370(10):901。在幹細胞之情況下，在輸注至個體中之後，亦發生此等前驅物活體內分化成表現功能轉殖基因之細胞。亦提供包含如本文中所描述之細胞的醫藥組合物。另外，該等細胞在投與患者之前可低溫保存。Cells as described herein are suitable for the treatment or prevention of disorders, eg, by in vivo therapy. Nuclease-modified cells can be expanded and then reintroduced into a patient using standard techniques. See, eg, Tebas et al., (2014) New Eng J Med 370(10):901. In the case of stem cells, following infusion into an individual, in vivo differentiation of these precursors into cells expressing functionally transgenic genes also occurs. Pharmaceutical compositions comprising cells as described herein are also provided. Additionally, the cells can be cryopreserved prior to administration to a patient.

本文中所揭示之任何經修飾之細胞或細胞株均可顯示增加之γ球蛋白表現。亦提供包含如本文中所描述之經基因修飾之細胞的組合物，諸如醫藥組合物。 應用Any of the modified cells or cell lines disclosed herein can exhibit increased gamma globulin expression. Compositions, such as pharmaceutical compositions, comprising genetically modified cells as described herein are also provided. application

本文中所揭示之方法及組合物係用於對蛋白質之表現進行調節或對編碼遺傳性疾病，諸如鐮形血球病或地中海型貧血中所表現之蛋白質的異常基因序列進行矯正。因而，提供該等方法及組合物以用於治療或預防此種遺傳性疾病。基因組編輯，例如幹細胞之基因組編輯，可用於矯正異常基因，***野生型基因，或改變內源基因之表現。作為非限制性實例，可將例如編碼至少一種球蛋白(例如，α球蛋白、γ球蛋白、β球蛋白及其組合)之野生型基因***細胞中(例如，使用一或多種如本文中所描述之核酸酶***內源BCL11A增強子序列中)，以提供該細胞中缺乏之球蛋白且藉此治療由錯誤球蛋白表現導致之遺傳性疾病，例如血紅素病。替代地或另外，在投與或未投與適當供體之情況下進行基因組編輯可矯正錯誤內源基因，例如矯正α血紅素或β血紅素中之點突變，以恢復該基因之表現或治療遺傳性疾病，例如鐮形血球病、任何直接或間接球蛋白調控基因之敲除或變化(過度表現或壓抑) (例如使γ球蛋白調控基因BCL11A或 BCL11A調控因子KLF1不活化)。特定言之，本發明之方法及組合物在治療或預防血紅素病方面有用。The methods and compositions disclosed herein are used to modulate the expression of proteins or to correct abnormal gene sequences encoding proteins expressed in genetic diseases, such as sickle cell disease or thalassemia. Accordingly, such methods and compositions are provided for use in the treatment or prevention of such genetic diseases. Genome editing, such as that of stem cells, can be used to correct abnormal genes, insert wild-type genes, or alter the expression of endogenous genes. As a non-limiting example, a wild-type gene encoding, eg, at least one globulin (eg, alpha globulin, gamma globulin, beta globulin, and combinations thereof) can be inserted into a cell (eg, using one or more of the herein described The described nucleases are inserted into the endogenous BCL11A enhancer sequence) to provide globulins that are deficient in the cells and thereby treat genetic diseases caused by misglobulin expression, such as heme diseases. Alternatively or additionally, genome editing, with or without the administration of an appropriate donor, can correct an erroneous endogenous gene, such as correcting a point mutation in alpha heme or beta heme, to restore the expression or treatment of the gene Genetic diseases such as sickle cell disease, knockout or alteration (overexpression or repression) of any direct or indirect globulin regulated gene (eg inactivation of the gamma globulin regulated gene BCL11A or the BCL11A regulator KLF1). In particular, the methods and compositions of the present invention are useful in the treatment or prevention of heme disorders.

本發明之核酸酶靶向已知為紅血球生成期間表現BCL11A所需及由此為下調γ球蛋白表現所需之BCL11A增強子區。對此增強子區進行修飾可導致紅血球隨γ球蛋白表現增加，且因而可能有助於治療或預防鐮形血球病或β型地中海型貧血。The nucleases of the present invention target the BCL11A enhancer region known to be required for the expression of BCL11A during erythropoiesis and thus for the downregulation of gamma globulin expression. Modification of this enhancer region can result in increased red blood cell expression with gamma globulin and thus may be useful in the treatment or prevention of sickle cell disease or beta thalassemia.

以下實例係關於本發明之例示性實施例，其中核酸酶包含鋅指核酸酶(ZFN)。應瞭解，此僅出於例示目的，而且可使用其他核酸酶，例如TtAgo及CRISPR/Cas系統；具有經工程改造之DNA結合結構域之歸巢內切核酸酶(兆鹼基核酸酶)；天然存在之經工程改造之歸巢內切核酸酶(兆鹼基核酸酶) DNA結合結構域之融合物，包括具有經工程改造之DNA結合結構域之歸巢內切核酸酶(兆鹼基核酸酶)之組合及天然存在之經工程改造之歸巢內切核酸酶(兆鹼基核酸酶) DNA結合結構域與異源裂解域之融合物、兆鹼基核酸酶與TALE蛋白之融合物，包括異源裂解域之組合及兆鹼基核酸酶與TALE蛋白之融合物。 實例 實例1：鋅指核酸酶之組裝The following examples relate to illustrative embodiments of the invention wherein the nucleases comprise zinc finger nucleases (ZFNs). It will be appreciated that this is for illustrative purposes only and that other nucleases such as TtAgo and CRISPR/Cas systems may be used; homing endonucleases (meganucleases) with engineered DNA binding domains; natural Fusions of engineered homing endonucleases (meganucleases) DNA-binding domains exist, including homing endonucleases (meganucleases) with engineered DNA-binding domains. ) and naturally-occurring engineered homing endonucleases (meganucleases) fusions of DNA binding domains and heterologous cleavage domains, fusions of meganucleases and TALE proteins, including Combinations of heterologous cleavage domains and fusions of meganucleases and TALE proteins. EXAMPLES Example 1: Assembly of Zinc Finger Nucleases

針對人類BCL11A基因組裝ZFN，且藉由如以下所描述對自經轉染細胞分離之DNA進行深度定序分析來測試活性。如所描述來製造對增強子區域之+58區域具有特異性之ZFN。先前已描ZFN配對51857/51949 (參見WO 2016/183298)。 實例2：脫靶分析ZFNs were assembled against the human BCL11A gene and tested for activity by deep sequencing analysis of DNA isolated from transfected cells as described below. ZFNs specific for the +58 region of the enhancer region were made as described. ZFN pairing 51857/51949 has been previously described (see WO 2016/183298). Example 2: Off-target analysis

為了分析由ZFN配對所致之脫靶裂解，進行兩階段無偏特異性分析。在第一階段中，(圖1)，經由寡核苷酸雙鏈體整合位點分析法，使用與Tsai等人((2015),Nat Biotechnol 33(2):187-197. doi: 10.1038/nbt.3117)所描述之程序類似的程序來鑑定各ZFN之候選脫靶位點。To analyze off-target cleavage due to ZFN pairing, a two-stage unbiased specificity analysis was performed. In the first stage, (Fig. 1), via oligonucleotide duplex integration site analysis, using the same method as Tsai et al. ((2015), Nat Biotechnol 33(2): 187-197. doi: 10.1038/ nbt.3117) to identify candidate off-target sites for each ZFN.

寡核苷酸雙鏈體整合位點分析係基於觀測到將核酸酶及雙鏈體DNA之短區段共引入靶細胞中在經由NHEJ DNA修復途徑修復一小部分基因組裂解事件期間導致雙鏈體整合(Orlando等人, (2010),Nucleic Acids Res , 38(15) e152. doi: 10.1093/nar/gkq512；Gabriel等人, (2011),Nat Biotechnol . 2011 Aug 7;29(9):816-23. doi: 10.1038/nbt.1948；Tsai等人,如上 )。在整合後，該雙鏈體提供裂解事件之永久標籤。接著，經由將寡核苷酸銜接子與經剪接之基因組DNA結紮，繼而進行2輪各25個循環之巢式PCR並且對所得供體-基因組接合點進行深度定序來鑑定整合位點。此分析允許評估基因組內之所有潛在整合位點。Oligonucleotide duplex integration site analysis is based on the observation that co-introduction of nucleases and short segments of duplex DNA into target cells results in duplexes during repair of a small fraction of genome cleavage events via the NHEJ DNA repair pathway Integration (Orlando et al, (2010), Nucleic Acids Res , 38(15) e152. doi: 10.1093/nar/gkq512; Gabriel et al, (2011), Nat Biotechnol . 2011 Aug 7;29(9):816- 23. doi: 10.1038/ nbt.1948 ; Tsai et al., supra). After integration, the duplex provides a permanent tag for the cleavage event. Next, integration sites were identified by ligating oligonucleotide adaptors to the spliced genomic DNA, followed by 2 rounds of 25 cycles of nested PCR and deep sequencing of the resulting donor-genomic junctions. This analysis allows assessment of all potential integration sites within the genome.

在K562細胞中進行整合位點分析以使供體遞送、ZFN表現及供體整合最大化。此外，在K562細胞快速***時(倍增時間大約24小時)，預期其對ZFN使細胞標靶裂解之能力施加最低限度之表觀基因限制。利用0.47 μg寡核苷酸雙鏈體供體及400 ng各ZFN編碼mRNA，使用Amaxa shuttle及針對ZFN之最大中靶活性進行最佳化之設定對細胞(2×105 )進行電穿孔。針對寡核苷酸與mRNA之各組合製備四份重複樣品。在轉染後第7天，對各樣品分離基因組DNA (Qiagen DNeasy血液及組織套組)，且使用400 ng (133000個單倍體基因組)作為輸入以用於圖1中概述之擴增方案。接著基本上如(Tsai等人,如上 )所描述來處理樣品。彙集最終產物，定量，並且在MiSeq儀器(Illumina)上使用v2 300循環定序套組以配對末端150 bp讀數及8 bp/16 bp雙指數讀數偵測擴增子各末端上之樣品條碼來進行定序。Integration site analysis was performed in K562 cells to maximize donor delivery, ZFN expression and donor integration. Furthermore, when K562 cells divide rapidly (doubling time of approximately 24 hours), it is expected to impose minimal epigenetic constraints on the ability of ZFNs to lyse cellular targets. Cells (2 x 105 ) were electroporated with 0.47 μg of oligonucleotide duplex donor and 400 ng of each ZFN encoding mRNA using the Amaxa shuttle with settings optimized for maximal on-target activity of the ZFNs. Four replicate samples were prepared for each combination of oligonucleotide and mRNA. On day 7 post-transfection, genomic DNA (Qiagen DNeasy blood and tissue panel) was isolated for each sample and 400 ng (133000 haploid genomes) was used as input for the amplification protocol outlined in Figure 1 . The samples were then processed essentially as described (Tsai et al., supra ). Final products were pooled, quantified, and performed on a MiSeq instrument (Illumina) using the v2 300-cycle sequencing kit with paired-end 150 bp reads and 8 bp/16 bp double exponential reads to detect sample barcodes on each end of the amplicon Sequencing.

為了產生候選脫靶位點清單，針對正確引發序列過濾定序資料，繼而修剪銜接子序列並且映射至基因組。接下來，映射接合點座標，並且使用雙鏈體-基因組接合點以及由DNA剪切所引起之斷裂的位置來鑑定獨特整合事件。接著處理整合事件以鑑定基因組內緊密鄰近之整合簇(最少4個獨特整合事件彼此在100 bp內，取所有重複實驗之和)。將存在於hg38組裝體中不可映射之片段重疊群(亦即，hg38中之chrUn)上的簇自進一步分析中移除。亦移除映射至重複基因座之群簇(群簇中所有序列之基因組的三個以上命中之中值)，因為先前經歷已顯示此等為擴增人為因素。若其餘群簇來源於至少2個重複經ZFN處理樣品(總計4個)且經ZFN處理之樣品相對於對照組展現≥5倍整合事件過量，則將其評為候選ZFN裂解位點。依據經ZFN處理之樣品中的獨特整合之總數對候選裂解位點進行分級。對於ZFN配對51857/51949，經由此分析鑑定之候選基因座提供於圖2中，依據整合體計數進行分級。 實例3：ZFN之最佳化To generate a list of candidate off-target sites, the sequencing data was filtered for correct priming sequences, then adaptor sequences were trimmed and mapped to the genome. Next, the junction coordinates are mapped, and the position of the duplex-genome junction and the breaks caused by DNA cleavage are used to identify unique integration events. Integration events were then processed to identify clusters of integrations in close proximity within the genome (minimum 4 unique integration events within 100 bp of each other, summed over all replicates). Clusters present on unmappable fragment contigs in the hg38 assembly (ie, chrUn in hg38) were removed from further analysis. Clusters that map to duplicate loci (median of three or more hits to the genome for all sequences in the cluster) were also removed, as previous experience has shown these to be amplification artifacts. The remaining clusters were rated as candidate ZFN cleavage sites if they were derived from at least 2 replicate ZFN-treated samples (4 in total) and the ZFN-treated samples exhibited >5-fold excess of integration events relative to the control group. Candidate cleavage sites were ranked according to the total number of unique integrations in ZFN-treated samples. For ZFN pair 51857/51949, candidate loci identified by this analysis are provided in Figure 2, ranked according to integrity counts. Example 3: Optimization of ZFNs

為了減少脫靶裂解，採用核酸酶最佳化策略，其中選擇性地移除非特異性磷酸酯接觸以引起對脫靶裂解之全面抑制(Guilinger等人, (2014)Nat Methods . 11(4):429-35. doi: 10.1038/nmeth.2845；Kleinstiver等人, (2016)Nature 529(7587):490-5. doi: 10.1038/nature16526；Slaymaker等人, (2016)Science ) 351(6268):84-8. doi: 10.1126/science.aad5227) (參見美國臨時申請案第62/443,981號及第62/378,978號)。在鋅指構架內與DNA之磷酸酯主鏈相互作用之關鍵位置上(Pavletich及Pabo, (1991)Science 252(5007):809-17；Elrod-Erickson等人, (1996)Structure 4(10):1171-80) (圖3A至圖3B)以及右側ZFN FokI結構域中之預計亦會發生磷酸酯接觸之單一位置上(圖3C)進行胺基酸取代。To reduce off-target cleavage, a nuclease optimization strategy was employed in which non-specific phosphate contacts were selectively removed to cause global inhibition of off-target cleavage (Guilinger et al., (2014) Nat Methods . 11(4):429 -35. doi: 10.1038/nmeth.2845; Kleinstiver et al, (2016) Nature 529(7587):490-5. doi: 10.1038/nature16526; Slaymaker et al, (2016) Science ) 351(6268):84- 8. doi: 10.1126/science.aad5227) (see US Provisional Application Nos. 62/443,981 and 62/378,978). At key positions for interaction with the phosphate backbone of DNA within the zinc finger framework (Pavletich and Pabo, (1991) Science 252(5007):809-17; Elrod-Erickson et al., (1996) Structure 4(10) : 1171-80) (Figures 3A-3B) and amino acid substitutions at a single position in the right ZFN FokI domain where phosphate contacts are also expected (Figure 3C).

藉由允許自兩個單獨mRNA獨立地表現各ZFN來進一步改良特異性，由此使得能夠使遞送比率最佳化。此等工作所產生之最佳化ZFN配對與原始ZFN配對高度相關，不同之處在於存在能降低與DNA磷酸酯主鏈相互作用之能力但最低限度地影響或不影響序列特異性鹼基識別之取代。與此相符的是，對於原始51857/51949配對之潛在ZFN裂解標靶，整合位點分析產生455個基因座。對於最佳化配對，鑑定出少得多的基因座數目以用於藉由此分析進一步檢查潛在ZFN裂解標靶(總計72個)。對於兩種配對，BCL11A增強子內之預定標靶為最佳基因座。此外，在最佳化配對之BCL11A增強子處注意到相當大比例之整合事件，與其更大特異性相符。Specificity was further improved by allowing each ZFN to be expressed independently from two separate mRNAs, thereby enabling the delivery ratio to be optimized. The optimized ZFN pairings resulting from such work are highly correlated with the original ZFN pairings, except that there are analytes that reduce the ability to interact with the DNA phosphate backbone with minimal or no effect on sequence-specific base recognition. replace. Consistent with this, integration site analysis yielded 455 loci for the potential ZFN cleavage targets of the original 51857/51949 pair. For optimal pairing, a much smaller number of loci were identified for further examination of potential ZFN cleavage targets by this analysis (72 in total). For both pairs, the predetermined target within the BCL11A enhancer was the optimal locus. Furthermore, a substantial proportion of integration events were noted at the optimally paired BCL11A enhancer, consistent with its greater specificity.

重要的是應注意，在定義序列資料處理管線時，保守地選擇關鍵參數，以便包括儘可能多的候選脫靶基因座而不是將其過濾掉。進行此舉以確保將鑑定可表示最佳化ZFN之真實裂解位點的每一個基因座並且在追蹤性***缺失研究中加以測試，即使以接受相當大之數字以致將變成假陽性為代價。預期分析之第一階段將針對各ZFN配對產生一大組候選基因座，其中大多數(尤其對於最佳化ZFN)不表示真實脫靶裂解位點，而是將在追蹤性***缺失研究中被證明對裂解呈陰性之背景事件。It is important to note that when defining the sequence data processing pipeline, key parameters were chosen conservatively in order to include as many candidate off-target loci as possible rather than filter them out. This was done to ensure that every locus that could represent the true cleavage site of the optimized ZFNs would be identified and tested in tracking indel studies, even at the expense of accepting such large numbers that would become false positives. It is expected that the first phase of the analysis will generate a large set of candidate loci for each ZFN pairing, most of which (especially for optimized ZFNs) do not represent true off-target cleavage sites, but will be demonstrated in traceable indel studies Background events negative for lysis.

在分析之第二階段，針對經ZFN處理之CD34+ HSPC中的修飾證據(例如存在***缺失)對經由整合位點分析鑑定之候選脫靶基因座進行篩檢。In the second stage of the analysis, candidate off-target loci identified by integration site analysis were screened for evidence of modification (eg, presence of indels) in ZFN-treated CD34+ HSPCs.

特定言之，使用RNA轉染之臨床規模及臨床條件(120 µg/mL mRNA用於原始ZFN配對及100 µg/mL mRNA用於最佳化配對)，利用原始及最佳化ZFN配對處理來源於經動員外周血之人類CD34+ HSPC。在轉染後2天分離基因組DNA，繼而對候選脫靶基因座進行PCR擴增並且進行深度定序，以便對***缺失水準進行定量。對於原始及最佳化ZFN配對，在此步驟篩檢同一組之137個候選脫靶基因座以及利用原始ZFN之先前研究中已經由其他方法鑑定之較少數目之候選脫靶位點。Specifically, using the clinical scale and clinical conditions of RNA transfection (120 µg/mL mRNA for the original ZFN pairing and 100 µg/mL mRNA for the optimized pairing), treatment with the original and optimized ZFN pairings was derived from Human CD34+ HSPCs mobilized from peripheral blood. Genomic DNA was isolated 2 days post-transfection, followed by PCR amplification of candidate off-target loci and deep sequencing to quantify indel levels. For the original and optimized ZFN pairings, the same set of 137 candidate off-target loci and a smaller number of candidate off-target sites that had been identified by other methods in previous studies using the original ZFNs were screened at this step.

結果顯示最佳化ZFN與原始配對相比顯然更具特異性。此不僅由因ZFN裂解證據而被評為陽性之基因座的數目(原始配對之52相對於最佳化配對之3)而且由所觀測之***缺失水準(對最佳化配對而言，其低得多)顯而易見。圖4顯示在此研究中展現ZFN裂解證據之每一個基因座之***缺失值的圖(注意y軸為對數尺度)。所有此種基因座之聚集脫靶***缺失指示脫靶活性降低300倍(原始配對之46.5%聚集脫靶***缺失相對於最佳化配對之0.15%脫靶***缺失)。達成此脫靶活性降低而未對預定靶位點處之活性造成任何損失(原始配對之72.5%***缺失相對於最佳化ZFN之81.9%)。在此等研究中，原始配對(或親本配對)為51857/51949，而最佳化ZFN配對為63014/65722 (參見下文)。The results show that the optimized ZFNs are significantly more specific than the original pairings. This is determined not only by the number of loci rated positive for evidence of ZFN cleavage (52 for the original pair versus 3 for the optimized pair) but also by the level of indels observed (low for the optimized pair) much more) obvious. Figure 4 shows a graph of indel values for each locus exhibiting evidence of ZFN cleavage in this study (note that the y-axis is on a log scale). Clustered off-target indels for all such loci indicated a 300-fold reduction in off-target activity (46.5% clustered off-target indels for the original pair versus 0.15% off-target indels for the optimized pair). This reduction in off-target activity was achieved without any loss of activity at the intended target site (72.5% indels for original pairings versus 81.9% for optimized ZFNs). In these studies, the original pairing (or parental pairing) was 51857/51949 and the optimized ZFN pairing was 63014/65722 (see below).

核酸酶設計顯示於以下表1中：表 1 ：對 +58 BCL11A 增強子區域具特異性之 ZFN 配對

Figure 106129809-A0304-0001
Nuclease designs are shown in Table 1 below: Table 1 : ZFN pairings specific for the +58 BCL11A enhancer region
Figure 106129809-A0304-0001

表1顯示關於各ZFN之表徵資訊。自左側起，呈現SBS編號(例如51857)，其中ZFN結合之DNA標靶呈現在SBS編號下方。接下來顯示指1-6或1-5之胺基酸識別螺旋設計(表1之細分行2)。表1中在適當螺旋設計下亦顯示對所指示之指的ZFP主鏈序列所做之突變，如美國臨時專利申請案第62/378,978號及第62/443,981號中所描述。在表1所使用之記法中，「Qm5」意謂在所指示之指的位置-5 處(相對於編號-1至+6之螺旋)，此位置上之精胺酸已置換為麩醯胺酸(Q )，而「Qm14」意謂正常情況下存在於位置-14 處之精胺酸(R)已置換為麩醯胺酸(Q )。「無」指示識別螺旋區外無變化。因而，舉例而言，SBS第63014號包括指1、3及5中之Qm5突變，而指2、4及6不具有對鋅指主鏈(例如，識別螺旋區外之鋅指序列)之突變。Table 1 shows the characterization information for each ZFN. From the left, SBS numbers are presented (eg, 51857), with ZFN-bound DNA targets presented below the SBS numbers. The amino acid recognition helix designs referring to 1-6 or 1-5 are shown next (subsection row 2 of Table 1). Mutations made to the indicated ZFP backbone sequences are also shown in Table 1 under appropriate helical design, as described in US Provisional Patent Application Nos. 62/378,978 and 62/443,981. In the notation used in Table 1, "Qm5" means that the arginine at this position has been replaced by glutamine at position -5 of the indicated finger (relative to the helix numbered -1 to +6) acid ( Q ), and "Qm14" means that the arginine (R) normally present at position -14 has been replaced by glutamic acid ( Q ). "None" indicates no change outside the recognition helix. Thus, for example, SBS No. 63014 includes the Qm5 mutation in fingers 1, 3, and 5, while fingers 2, 4, and 6 do not have mutations to the zinc finger backbone (eg, recognizing zinc finger sequences outside the helical region) .

最後，表1之最右行中顯示用於連接DNA結合結構域與FokI裂解結構域之連接子(例如，「L7c5」(LRGSISRARPLNPHP (SEQ ID NO:5)，如例如美國專利第9,567,609號中所描述)被呈現在該行之頭一列，其中FokI磷酸酯接觸突變及二聚突變之位點示於連接子名稱下方之方框中。特定言之，在Fok突變體框之頭一列指示二聚結構域中所發現之突變類型(例如ELD或KKR，如例如美國專利第8,962,281號中所描述)。在二聚突變體名稱下顯示底部所示之FokI結構域中所存在之為了移除非特異性磷酸酯接觸而做之任何突變(例如K525S或R416S，其中胺基酸位置525或416上之絲胺酸殘基已取代為離胺酸或精胺酸，分別如美國臨時專利申請案第62/378,978號及第62/443,981號中所描述)。因而，舉例而言，在SBS第63014號中，連接子為L7c5連接子且FokI裂解結構域包括ELD二聚突變體且不存在磷酸酯接觸突變。此外，對於SBS第65722號，連接子為L0連接子(LRGSQLVKS (SEQ ID NO:6)，亦稱為『標準』連接子，參見美國專利第9,567,609號)且FokI裂解結構域包括KKR二聚突變及K525S FokI磷酸酯接觸突變。Finally, the linker used to connect the DNA binding domain to the FokI cleavage domain (eg, "L7c5" (LRGSISRARPLNPHP (SEQ ID NO: 5), as described, eg, in US Pat. No. 9,567,609) is shown in the far right row of Table 1 description) is presented in the first column of the row, where the sites of the FokI phosphate contact mutation and the dimerization mutation are shown in the box below the linker name. In particular, dimerization is indicated in the first column of the Fok mutant box The type of mutation found in the domain (eg, ELD or KKR, as described, eg, in US Pat. No. 8,962,281). The presence of the FokI domain shown at the bottom is shown under the dimerization mutant name in order to remove non-specific Any mutation made by contact with a sex phosphate (such as K525S or R416S, where the serine residue at amino acid position 525 or 416 has been substituted with lysine or arginine, respectively, as described in U.S. Provisional Patent Application No. 62 /378,978 and 62/443,981). Thus, for example, in SBS No. 63014, the linker is the L7c5 linker and the FokI cleavage domain includes an ELD dimerization mutant and no phosphate contacts are present In addition, for SBS No. 65722, the linker is the L0 linker (LRGSQLVKS (SEQ ID NO: 6), also known as the "standard" linker, see US Pat. No. 9,567,609) and the FokI cleavage domain includes KKR two Polymutation and K525S FokI phosphate contact mutation.

測試所有ZFN之功能性(裂解活性，如藉由如以下實例4中所描述來分析***缺失所測定)且發現具有活性。All ZFNs were tested for functionality (cleavage activity, as determined by analyzing indels as described in Example 4 below) and found to be active.

此外，為了確定最具特異性之ZFN設計，在經ZFN處理之CD34+ HSPC中對由原始ZFN配對所致之脫靶裂解的已知位點進行***缺失分析。為了實現此目的，使用臨床條件及mRNA濃度(120 µg/mL用於原始ZFN配對及100 µg/mL用於最佳化配對)，利用原始及最佳化ZFN配對處理來源於經動員外周血之人類CD34+ HSPC。在轉染後2天自此等細胞及未經處理之對照物分離基因組DNA，繼而對各候選基因座進行PCR擴增並且進行深度定序，以便對***缺失水準進行定量。Furthermore, to determine the most specific ZFN designs, indel analysis was performed in ZFN-treated CD34+ HSPCs for known sites of off-target cleavage by the original ZFN pairing. To achieve this, using clinical conditions and mRNA concentrations (120 µg/mL for original ZFN pairing and 100 µg/mL for optimized pairing), mobilized peripheral blood-derived Human CD34+ HSPCs. Genomic DNA was isolated from these cells and untreated controls 2 days post-transfection, followed by PCR amplification and deep sequencing of each candidate locus to quantify indel levels.

藉由在Illumina MiSeq上使用300次循環藥筒進行配對末端深度定序來測定各基因座處之修飾水準。將配對序列合併，針對所有鹼基之品質評分≥15對經由SeqPrep加以修剪之銜接子進行過濾，接著映射至人類基因組(hg38組裝體)。擯棄映射至不正確基因座之序列。將比野生型擴增子短＞70 bp或＞70%之序列移除以便減少引子二聚體產物。在標靶擴增子與各MiSeq讀數之間進行Needleman-Wunsch比對(Needleman及Wunsch, (1970),J Mol Biol 48(3):443-53))以映射***缺失。如Gabriel等人, 2011 (如上 )中所描述來定義所比對之序列中的***缺失，但亦接受1 bp長之***缺失以避免低估真實事件。注意，有一部分基因座未擴增或未定序，或由於高背景(對照樣品中存在＞1%修飾)或不充足定序深度(＜10000個讀數)而被自分析中排除。以下表2中提供此分析之結果及與『親本』51857/51949 ZFN配對之比較。表 2 ：脫靶裂解分析

Figure 106129809-A0304-0002
實例 4 ： ZFN 在人類 CD34+ 細胞中之活性 The level of modification at each locus was determined by paired-end deep sequencing on an Illumina MiSeq using a 300-cycle cartridge. Paired sequences were pooled, adapters trimmed via SeqPrep were filtered for quality scores > 15 for all bases, and then mapped to the human genome (hg38 assembly). Sequences that map to incorrect loci are discarded. Sequences >70 bp or >70% shorter than the wild-type amplicon were removed in order to reduce primer dimer products. Needleman-Wunsch alignments (Needleman and Wunsch, (1970), J Mol Biol 48(3):443-53)) were performed between target amplicons and individual MiSeq reads to map indels. Indels in the aligned sequences were defined as described in Gabriel et al., 2011 ( supra ), but 1 bp long indels were also accepted to avoid underestimation of true events. Note that a subset of loci were not amplified or sequenced, or were excluded from analysis due to high background (>1% modification in control samples) or insufficient sequencing depth (<10000 reads). The results of this analysis and comparison with the "parental" 51857/51949 ZFN pair are provided in Table 2 below. Table 2 : Off-target cleavage analysis
Figure 106129809-A0304-0002
 Example 4 : Activity of ZFNs in Human CD34+ Cells

對於擬體內測試，在CD34+細胞中測試核酸酶。ZFN作為mRNA提供，其中在試管內如下製造mRNA：使包含編碼ZFN之基因的質體線性化且用於使用mMessage mMachine® T7 Ultra套組(Ambion/Applied Biosystems)進行的試管內mRNA轉錄。接著使用RNeasy®微型套組(Qiagen)來純化mRNA。For pseudo-in vivo testing, nucleases were tested in CD34+ cells. ZFNs were provided as mRNAs, which were produced in vitro by linearizing plastids containing the gene encoding the ZFNs and used for in vitro mRNA transcription using the mMessage mMachine® T7 Ultra Kit (Ambion/Applied Biosystems). The mRNA was then purified using the RNeasy® Mini Kit (Qiagen).

自經動員之外周血分離CD34+細胞且維持在補充有青黴素、鏈黴素及麩醯胺酸以及StemSpan CC110之X-VIVO 10培養基中，並且在37℃及5% CO2 下培育。在分離後或解凍後48小時轉染細胞。將小等分試樣與錐蟲藍之PBS溶液0.4% (w/v) (Corning) 1:1混合，並且在TC20自動化細胞計數器(Bio-Rad)上測定細胞數目。CD34+ cells were isolated from mobilized peripheral blood and maintained in X-VIVO 10 medium supplemented with penicillin, streptomycin and glutamic acid and StemSpan CC110 and incubated at 37°C and 5% CO2 . Cells were transfected 48 hours after isolation or after thawing. Small aliquots were mixed 1:1 with trypan blue in PBS 0.4% (w/v) (Corning) and cell numbers were determined on a TC20 automated cell counter (Bio-Rad).

對於大規模轉染，用MaxCyte電穿孔緩衝液(Maxcyte)洗滌細胞，且以3×107 至5×107 個細胞/mL再懸浮於100 µL電穿孔緩衝液中。典型地，使用介於60 µg/mL與120 µg/mL之間的mRNA濃度來篩檢候選ZFN集合。接著使細胞在30℃下以3×106 個細胞/mL在生長培養基中生長18小時，接著稀釋至1×106 個細胞/mL後在37℃下再持續24小時。為了測定裂解活性，在轉染後2至3天分離基因組DNA，且經由在MiSeq定序器(Illumina)上進行深度定序來量測BCL11A增強子基因座處之基因修飾水準。For large scale transfections, cells were washed with MaxCyte Electroporation Buffer (Maxcyte) and resuspended in 100 μL of Electroporation Buffer at 3×10 7 to 5×10 7 cells/mL. Typically, mRNA concentrations between 60 µg/mL and 120 µg/mL are used to screen candidate ZFN sets. Cells were then grown in growth medium at 3 x 106 cells/mL for 18 hours at 30°C, followed by dilution to 1 x 106 cells/mL for a further 24 hours at 37°C. To determine lytic activity, genomic DNA was isolated 2 to 3 days post-transfection and the level of gene modification at the BCL11A enhancer locus was measured via deep sequencing on the MiSeq sequencer (Illumina).

在CD34+細胞中測試表1之ZFN配對，且活性結果示於以下表3中。表 3 ： ZFN 配對針對 BCL11A 標靶之活性

Figure 106129809-A0304-0003
The ZFN pairings of Table 1 were tested in CD34+ cells and the activity results are shown in Table 3 below. Table 3 : Activity of ZFN pairings against the BCL11A target
Figure 106129809-A0304-0003

除了分析紅血球分化之前在CD34+細胞中之核酸酶活性以外，亦使經編輯之細胞在試管內分化成紅血球細胞。所遵循之方案係基於Giarratana等人, ((2011)Blood 120 (15):2945-53)。簡言之，遵循以下方案： 第0天至第7天：將4×104 個CD34+細胞以2×104 個/mL之密度在分化培養基(EDM) (伊斯科夫氏改良杜爾貝科氏培養基[Iscove's Modified Dulbecco's Medium，IMDM]，330 µg/mL轉鐵蛋白、10 µg/mL人類胰島素、2 U/mL肝素鈉、5%人類AB+血漿)中在10-6 M氫化皮質酮、100 ng/mL幹細胞因子(SCF)、5 ng/mL IL 3及3 IU/mL紅血球生成素(EPO)存在下進行培養。 第4天：將細胞再懸浮於含有SCF、IL-3、EPO及氫化皮質酮之新鮮EDM中。 第7天至第11天：將細胞以1.5×105 個細胞/mL之密度再懸浮於補充有SCF及EPO之新鮮EDM中。 第11天至第21天：在第11天，將細胞以1×106 個/mL重新接種於補充有EPO之新鮮EDM中。隨後在第14天將細胞以5×106 個/mL重新接種於此相同培養基中。此時間段期間之生長平頂期介於第14天至第18天之間，此時細胞活力開始下降，直至第21天時終止培養。In addition to assaying for nuclease activity in CD34+ cells prior to erythrocyte differentiation, edited cells were also differentiated into erythrocytes in vitro. The protocol followed is based on Giarratana et al., ((2011) Blood 120(15):2945-53). Briefly, the following protocol was followed: Day 0 to Day 7: 4 x 104 CD34+ cells were grown at a density of 2 x 104/mL in Differentiation Medium (EDM) (Iskov's Modified Dulbey) Iscove's Modified Dulbecco's Medium [IMDM], 330 µg/mL transferrin, 10 µg/mL human insulin, 2 U/mL sodium heparin, 5% human AB+ plasma) in 10 -6 M corticosterone, Cultures were performed in the presence of 100 ng/mL stem cell factor (SCF), 5 ng/mL IL 3 and 3 IU/mL erythropoietin (EPO). Day 4: Cells were resuspended in fresh EDM containing SCF, IL-3, EPO and corticosterone. Days 7-11: Cells were resuspended in fresh EDM supplemented with SCF and EPO at a density of 1.5 x 105 cells/mL. Days 11 to 21: On day 11, cells were re-seeded in fresh EDM supplemented with EPO at 1 x 106 cells/mL. Cells were then re-seeded in this same medium on day 14 at 5 x 106 cells/mL. The growth plateau during this time period was between day 14 and day 18, when cell viability began to decline until the culture was terminated at day 21.

在接種時及貫穿分化期間藉由使用Nexcelom Bioscience Cellometer K2以AOPI紅血球分析模式量測吖啶橙陽性及碘化丙啶排斥(AOPI)來獲取細胞計數，其中螢光通道1 (AO)設定至700毫秒且螢光通道2 (PI)設定至5000毫秒。Cell counts were obtained at seeding and throughout differentiation by measuring acridine orange positivity and propidium iodide exclusion (AOPI) using the Nexcelom Bioscience Cellometer K2 in AOPI erythrocyte analysis mode with fluorescence channel 1 (AO) set to 700 ms and fluorescence channel 2 (PI) was set to 5000 ms.

在分化之第21天使用以下方案來測定無核細胞之百分比。未經轉染之對照物與經ZFN轉染之樣品間的除核率相當，此兩組之百分比為59%至63%： 1. 細胞計數 2. 100,000個細胞在450×g下室溫向下自旋5 min。 3. 再懸浮於50 µL PBS-BSA+1 µL GlyA-FITC (DAKO)中。 4. 在冰箱中染色15分鐘。 5. 添加1 mL PBS-BSA，渦旋，向下自旋。 6. 再懸浮於250 µL PBS-BSA-NucRed (2滴NucRed/mL)中。 7. 在FACS Canto上使用APC通道獲取NucRed。 8. 有核紅血球細胞將處於GlyA陽性NucRed陰性/下部級分中，且紅血球母細胞將處於GlyA-NucRed雙陽性級分中。The following protocol was used to determine the percentage of anucleated cells on day 21 of differentiation. Nucleation rates were comparable between the untransfected control and ZFN-transfected samples, with percentages ranging from 59% to 63% for both groups: 1. Cell count 2. 100,000 cells were incubated at room temperature at 450 x g. Spin down for 5 min. 3. Resuspend in 50 µL PBS-BSA+1 µL GlyA-FITC (DAKO). 4. Stain in the refrigerator for 15 minutes. 5. Add 1 mL of PBS-BSA, vortex, spin down. 6. Resuspend in 250 µL PBS-BSA-NucRed (2 drops NucRed/mL). 7. Use the APC channel to acquire NucRed on the FACS Canto. 8. Nucleated erythrocytes will be in the GlyA positive NucRed negative/lower fraction and erythroblasts will be in the GlyA-NucRed double positive fraction.

藉由在a)電穿孔之後48小時、b)對細胞進行解凍當天開始試管內分化時及c) 試管內紅血球分化第14天時收集之DNA樣品中進行MiSeq深度定序來量測BCL11A基因修飾。儘管分化進行了21天，但選擇DNA分析之第14天時間點，此係因為其在對大部分紅血球細胞進行除核，由此造成DNA回收損失之前。在BCL11A增強子處所觀測之修飾百分比連同轉染條件之細節一起列出於表4中。表 4 ：藉由 MiSeq 分析獲得之 BCL11A 基因修飾水準

Figure 106129809-A0304-0004
BCL11A genetic modification was measured by MiSeq deep sequencing in DNA samples collected a) 48 hours after electroporation, b) when cells were thawed on the day of in vitro differentiation, and c) at day 14 of in vitro erythrocyte differentiation . Although differentiation took place for 21 days, the day 14 time point for DNA analysis was chosen because it precedes the denucleation of the majority of red blood cells, resulting in loss of DNA recovery. The percent modification observed at the BCL11A enhancer is listed in Table 4 along with details of the transfection conditions. Table 4 : BCL11A gene modification levels obtained by MiSeq analysis
Figure 106129809-A0304-0004

此等資料顯示利用最佳化配對63014與65722 mRNA進行CD34+細胞轉染在BCL11A增強子標靶位點處引起非常有效之基因修飾(＞75%之對偶基因經修飾)，且在對細胞進行冷凍及解凍之後及紅血球分化之後，修飾得到非常良好之維持(保留＞90%之修飾)。These data show that transfection of CD34+ cells with optimized pairing of 63014 and 65722 mRNA results in very efficient genetic modification at the BCL11A enhancer target site (>75% of the paired genes are modified), and the cells are frozen And after thawing and after erythrocyte differentiation, the modification was very well maintained (>90% modification retained).

選擇ZFN配對63014/65722來進行進一步分析。此等ZFN之胺基酸序列顯示如下，其中各胺基酸序列包含核定位信號(NLS，Kalderon等人,(1984)Cell 39 (3 Pt 2):499-509)及增強中靶ZFN活性之親水性肽(Hopp等人,(1988)Nat Biotechnol 6:1204-10)，兩者皆與N末端編碼序列融合。因而，ZFN之mRNA及胺基酸序列如下： 63014 mRNA (1725 nt) 5'gggagacaagcuuugaauuacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgaauucgccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaugacgaugacaagauggcccccaagaagaagaggaaggucggcauccacgggguacccgccgcuauggcugagaggcccuuccagugucgaaucugcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccgcaacuucucccugaccaugcauaccaagauacacacgggcagccaaaagcccuuccagugucgaaucugcaugcagaacuucaguuccaccggcaaccugaccaaccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugccaccuccggcucccugacccgccauaccaagauacacacgcacccgcgcgccccgaucccgaagcccuuccagugucgaaucugcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugccgcccaguguugucuguuccaccauaccaagauacaccugcggggauccaucagcagagccagaccacugaacccgcacccggagcuggaggagaagaaguccgagcugcggcacaagcugaaguacgugccccacgaguacaucgagcugaucgagaucgccaggaacagcacccaggaccgcauccuggagaugaaggugauggaguucuucaugaagguguacggcuacaggggaaagcaccugggcggaagcagaaagccugacggcgccaucuauacagugggcagccccaucgauuacggcgugaucguggacacaaaggccuacagcggcggcuacaaucugccuaucggccaggccgacgagauggagagauacguggaggagaaccagacccgggauaagcaccucaaccccaacgagugguggaagguguacccuagcagcgugaccgaguucaaguuccuguucgugagcggccacuucaagggcaacuacaaggcccagcugaccaggcugaaccacaucaccaacugcaauggcgccgugcugagcguggaggagcugcugaucggcggcgagaugaucaaagccggcacccugacacuggaggaggugcggcgcaaguucaacaacggcgagaucaacuucagaucuugauaacucgagucuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgcuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgggacauucuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag (SEQ ID NO:28)。ZFN pair 63014/65722 was selected for further analysis. The amino acid sequences of these ZFNs are shown below, where each amino acid sequence comprises a nuclear localization signal (NLS, Kalderon et al., (1984) Cell 39(3 Pt 2):499-509) and a function that enhances the activity of the on-target ZFNs. Hydrophilic peptides (Hopp et al., (1988) Nat Biotechnol 6:1204-10), both fused to the N-terminal coding sequence. Thus, mRNA ZFN and the amino acid sequence as follows: 63014 mRNA (1725 nt) 5'gggagacaagcuuugaauuacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgaauucgccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaugacgaugacaagauggcccccaagaagaagaggaaggucggcauccacgggguacccgccgcuauggcugagaggcccuuccagugucgaaucugcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccgcaacuucucccugaccaugcauaccaagauacacacgggcagccaaaagcccuuccagugucgaaucugcaugcagaacuucaguuccaccggcaaccugaccaaccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugccaccuccggcucccugacccgccauaccaagauacacacgcacccgcgcgccccgaucccgaagcccuuccagugucgaaucugcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugccgcccaguguugucuguuccaccauaccaagauacaccugcggggauccaucagcagagccagaccacugaacccgcacccggagcuggaggagaagaaguccgagcugcggcacaagcugaaguacgugccccacgaguacaucgagcugaucgagaucgccaggaacagcacccaggaccgcauccuggagaugaaggugauggaguucuucaugaagguguacggcuacaggggaaagcaccugggcggaagcagaaagccu gacggcgccaucuauacagugggcagccccaucgauuacggcgugaucguggacacaaaggccuacagcggcggcuacaaucugccuaucggccaggccgacgagauggagagauacguggaggagaaccagacccgggauaagcaccucaaccccaacgagugguggaagguguacccuagcagcgugaccgaguucaaguuccuguucgugagcggccacuucaagggcaacuacaaggcccagcugaccaggcugaaccacaucaccaacugcaauggcgccgugcugagcguggaggagcugcugaucggcggcgagaugaucaaagccggcacccugacacuggaggaggugcggcgcaaguucaacaacggcgagaucaacuucagaucuugauaacucgagucuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgcuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgggacauucuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag (SEQ ID NO: 28).

63014胺基酸序列(識別螺旋區加下劃線；連接子以斜體大寫字母顯示；對指1、3及5主鏈殘基之突變以雙下劃線顯示；二聚結構域突變(ELD)以粗斜體顯示；親水性肽以小寫字母文字指示；且核定位信號(NLS)以斜體小寫字母顯示)：MdykdhdgdykdhdidykddddkMApkkkrkv GIHGVPAAMAERPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFARNFSLTMHTKIHTGSQKPFQCRICMQNFSSTGNLTNHIRTHTGEKPFACDICGRKFATSGSLTRHTKIHTHPRAPIPKPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFAAQCCLFHHTKIHLRGSISRARPLNPHP ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEM E RYVEENQTR D KH L NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS (SEQ ID NO:29)。 65722 mRNA (1680個核苷酸)： 5'gggagacaagcuugaauacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgaauucgccuagagaucuggcggcggagagggcagaggaagucuucuaaccugcggugacguggaggagaaucccggcccuaggaccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaugacgaugacaagauggcccccaagaagaagaggaaggucggcauucaugggguacccgccgcuauggcugagaggcccuuccagugucgaaucugcaugcagaaguuugcccgcaacgaccaccgcaccacccauaccaagauacacacgggcgagaagcccuuccagugucgaaucugcaugcagaacuucagucagaaggcccaccugauccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccagaagggcacccugggcgagcauaccaagauacacacgggaucucagaagcccuuccagugucgaaucugcaugcagaacuucagucgcggccgcgaccugucccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccgccgcgacaaccugcacucccauaccaagauacaccugcggggaucccagcuggugaagagcgagcuggaggagaagaaguccgagcugcggcacaagcugaaguacgugccccacgaguacaucgagcugaucgagaucgccaggaacagcacccaggaccgcauccuggagaugaaggugauggaguucuucaugaagguguacggcuacaggggaaagcaccugggcggaagcagaaagccugacggcgccaucuauacagugggcagccccaucgauuacggcgugaucguggacacaaaggccuacagcggcggcuacaaucugccuaucggccaggccgacgagaugcagagauacgugaaggagaaccagacccggaauaagcacaucaaccccaacgagugguggaagguguacccuagcagcgugaccgaguucaaguuccuguucgugagcggccacuucagcggcaacuacaaggcccagcugaccaggcugaaccgcaaaaccaacugcaauggcgccgugcugagcguggaggagcugcugaucggcggcgagaugaucaaagccggcacccugacacuggaggaggugcggcgcaaguucaacaacggcgagaucaacuucugauaacucgagucuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgcuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgggacauucuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag (SEQ ID NO:30)。63014 amino acid sequence (recognition helical region is underlined; linkers are shown in italic capital letters; mutations to finger 1, 3 and 5 backbone residues are double underlined; dimerization domain mutations (ELD) are shown in bold italics a display body; hydrophilic peptide lowercase letter display; and the nuclear localization signal (NLS) in italic lowercase letters): MdykdhdgdykdhdidykddddkMA pkkkrkv GIHGVPAAMAERPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFARNFSLTMHTKIHTGSQKPFQCRICMQNFSSTGNLTNHIRTHTGEKPFACDICGRKFATSGSLTRHTKIHTHPRAPIPKPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFAAQCCLFHHTKIH LRGSISRARPLNPHP ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEM E RYVEENQTR D KH L NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS (SEQ ID NO: 29). 65722 mRNA (1680 nucleotides): 5'gggagacaagcuugaauacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgaauucgccuagagaucuggcggcggagagggcagaggaagucuucuaaccugcggugacguggaggagaaucccggcccuaggaccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaugacgaugacaagauggcccccaagaagaagaggaaggucggcauucaugggguacccgccgcuauggcugagaggcccuuccagugucgaaucugcaugcagaaguuugcccgcaacgaccaccgcaccacccauaccaagauacacacgggcgagaagcccuuccagugucgaaucugcaugcagaacuucagucagaaggcccaccugauccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccagaagggcacccugggcgagcauaccaagauacacacgggaucucagaagcccuuccagugucgaaucugcaugcagaacuucagucgcggccgcgaccugucccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccgccgcgacaaccugcacucccauaccaagauacaccugcggggaucccagcuggugaagagcgagcuggaggagaagaaguccgagcugcggcacaagcugaaguacgugccccacgaguacaucgagcugaucgagaucgccaggaacagcacccaggaccgcauccuggagaugaaggugauggaguucuucaugaagguguacggcuacaggggaaagcaccugggcggaagcagaaagccugacggcgccaucuauacagugggcagccccaucgauuacggcgugaucguggacacaaag gccuacagcggcggcuacaaucugccuaucggccaggccgacgagaugcagagauacgugaaggagaaccagacccggaauaagcacaucaaccccaacgagugguggaagguguacccuagcagcgugaccgaguucaaguuccuguucgugagcggccacuucagcggcaacuacaaggcccagcugaccaggcugaaccgcaaaaccaacugcaauggcgccgugcugagcguggaggagcugcugaucggcggcgagaugaucaaagccggcacccugacacuggaggaggugcggcgcaaguucaacaacggcgagaucaacuucugauaacucgagucuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgcuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgggacauucuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag (SEQ ID NO: 30).

65722胺基酸序列(識別螺旋區加下劃線；連接子以斜體大寫字母顯示；親水性肽以小寫字母表示；核酸酶定位信號以斜體小寫字母表示；對指1、2及4主鏈殘基之突變以雙下劃線顯示；二聚結構域突變(ELD)以粗斜體顯示；且FokI磷酸酯接觸突變以波狀下劃線顯示)： MdykdhdgdykdhdidykddddkMApkkkrkv GIHGVPAAMAERPFQCRICMQKFARNDHRTTHTKIHTGEKPFQCRICMQNFSQKAHLIRHIRTHTGEKPFACDICGRKFAQKGTLGEHTKIHTGSQKPFQCRICMQNFSRGRDLSRHIRTHTGEKPFACDICGRKFARRDNLHSHTKIHLRGSQLVKS ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYV K ENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFSGNYKAQLTRLN RK TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO:31)。實例 5 ：評定紅血球後代中之球蛋白水準 65722 Amino acid sequence (identification helical regions are underlined; linkers are shown in italic capital letters; hydrophilic peptides are shown in lower case letters; nuclease localization signals are shown in italic lower case letters; pairs refer to 1, 2, and 4 backbone residues group of mutated are shown in double underline; dimerization domain mutants (ELD) are shown in bold italics; FokI and the mutants are shown in contact with a phosphate wavy underline): MdykdhdgdykdhdidykddddkMA pkkkrkv GIHGVPAAMAERPFQCRICMQKFARNDHRTTHTKIHTGEKPFQCRICMQNFSQKAHLIRHIRTHTGEKPFACDICGRKFAQKGTLGEHTKIHTGSQKPFQCRICMQNFSRGRDLSRHIRTHTGEKPFACDICGRKFARRDNLHSHTKIH LRGSQLVKS ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYV K ENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFSGNYKAQLTRLN RK TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 31 ). Example 5 : Assessment of globulin levels in erythrocyte progeny

針對以上表4中所示之兩種細胞製劑，藉由RT-qPCR來測定在分化第14天(在除核及紅血球成熟過程中發生總mRNA水準大幅下降之前)分離之細胞mRNA中的α球蛋白mRNA、β球蛋白mRNA及γ球蛋白mRNA之水準。所示γ球蛋白mRNA值相對於得自相同樣品之β球蛋白mRNA (圖5A)或相對於α球蛋白mRNA值(圖5B)進行正規化(基於未經轉染之RT-PCR標準物中之比率定義為1，使用任意單位)。For the two cell preparations shown in Table 4 above, alpha spheres in mRNA of cells isolated at day 14 of differentiation (before a substantial drop in total mRNA levels during denucleation and erythrocyte maturation) were determined by RT-qPCR Levels of protein mRNA, beta globulin mRNA and gamma globulin mRNA. The indicated gamma globulin mRNA values were normalized to either beta globulin mRNA (FIG. 5A) or to alpha globulin mRNA values (FIG. 5B) from the same samples (based on untransfected RT-PCR standards). The ratio is defined as 1, using arbitrary units).

使用在第21天(紅血球分化之終點)分離之蛋白質樣品的逆相HPLC來確定ZFN介導之對BCL11A紅血球增強子之修飾在蛋白質層面上是否使胎兒血紅素升高。測定γ球蛋白(Aγ及Gγ峰之總和)與α球蛋白比率以及γ球蛋白(Aγ及Gγ峰之總和)/總β樣球蛋白比率(Aγ、Gγ、β及δ球蛋白峰之總和)，並且示於圖 6中。Reverse phase HPLC of protein samples isolated at day 21 (end point of erythrocyte differentiation) was used to determine whether ZFN-mediated modification of the BCL11A erythrocyte enhancer increases fetal heme at the protein level. The ratio of γ globulin (sum of Aγ and Gγ peaks) to α globulin and the ratio of γ globulin (sum of Aγ and Gγ peaks)/total β-like globulin (sum of Aγ, Gγ, β and δ globulin peaks) were determined and shown in Figure 6.

在此實驗中，在63014/65722介導之BCL11A增強子破環後，在HSPC之紅血球後代中觀測到胎兒球蛋白百分比升高大約3至4倍，達至約15%-20%之水準。實例 6 ：在 NSG 小鼠中植入經編輯之細胞 In this experiment, following 63014/65722-mediated disruption of the BCL11A enhancer, an approximately 3- to 4-fold increase in percent fetal globulin was observed in the erythrocyte progeny of HSPC, to a level of approximately 15%-20%. Example 6 : Engraftment of edited cells in NSG mice

接著將經編輯之人類CD34+細胞注入NSG小鼠中以評定植入。在移植後8週及12週、16週及20週時收集之外周血中以及12週及20週時收集之骨髓中使用螢光活化細胞分選(FACS)來量測人類嵌合程度(亦即，人類CD45+細胞之百分比)。另外，為了測試經ZFN修飾之細胞的植入水準，藉由對ZFN標靶基因座進行直接高通量定序來評估BCL11A增強子基因座處之基因破環水準，並且與輸入材料中所量測之標靶基因修飾水準相比較。The edited human CD34+ cells were then injected into NSG mice to assess engraftment. Human chimerism was measured using fluorescence-activated cell sorting (FACS) in peripheral blood collected at 8 and 12 weeks, 16 weeks and 20 weeks, and bone marrow collected at 12 and 20 weeks after transplantation (also That is, the percentage of human CD45+ cells). Additionally, to test the level of engraftment in ZFN-modified cells, the level of gene disruption at the BCL11A enhancer locus was assessed by direct high-throughput sequencing of the ZFN target locus, and compared with the amount of the input material. The level of modification of the detected target gene is compared.

利用G-SCF及普樂沙福(Plerixafor)對得自兩名健康供體之HSPC (稱為PB-MR-003及PB-MR-004)進行動員，並且如Yannaki等人((2012)Mol Ther 20(1):230-8. doi: 10.1038/mt.2011)中所描述加以純化。使用Fresenius-Kabi Lovo裝置對白血球透析術產物進行血小板消耗，隨後使用Miltenyi Biotech CliniMACS Plus儀器使其富含CD34+細胞。接著將經純化之細胞接種於培養基中以進行轉染。HSPCs (referred to as PB-MR-003 and PB-MR-004) obtained from two healthy donors were mobilized using G-SCF and Plerixafor, and as described in Yannaki et al. ((2012) Mol Purified as described in Ther 20(1):230-8. doi: 10.1038/mt.2011). Leukocyte dialysis products were subjected to platelet depletion using the Fresenius-Kabi Lovo device and subsequently enriched for CD34+ cells using the Miltenyi Biotech CliniMACS Plus instrument. The purified cells are then seeded in culture medium for transfection.

在CD34+細胞純化之後兩天，在120 µg/mL編碼親本ZFN配對之單一mRNA 63014/65722或最佳量之編碼最佳化ZFN配對之兩種單獨mRNA 80 µg/mL 63014及20 µg/mL 65722存在下使用Maxcyte儀器對細胞進行電穿孔。在轉染之前，留出細胞之等分試樣作為未經轉染之對照物。自PB-MR-003轉染9500萬個細胞，且自PB-MR-004轉染12000萬個細胞。Two days after CD34+ cell purification, at 120 µg/mL of a single mRNA encoding the parental ZFN pair 63014/65722 or optimal amounts of two separate mRNAs 80 µg/mL 63014 and 20 µg/mL encoding the optimized ZFN pair Cells were electroporated using a Maxcyte instrument in the presence of 65722. Aliquots of cells were set aside as untransfected controls prior to transfection. 95 million cells were transfected from PB-MR-003 and 120 million cells were transfected from PB-MR-004.

電穿孔後，在30℃下進行短暫隔夜培養，接著在37℃下將細胞再培養24小時。電穿孔後兩天，獲取用於DNA分析之細胞等分試樣並且收集其餘細胞，低溫保存並儲存在液氮中。After electroporation, cells were incubated briefly overnight at 30°C, followed by an additional 24 hours at 37°C. Two days after electroporation, cell aliquots were taken for DNA analysis and the remaining cells were collected, cryopreserved and stored in liquid nitrogen.

處理：在照射之前用10 mg/kg/天拜有利(Baytril)水將小鼠處理1至2天，且在移植之前16至24小時在300 RAD下進行亞致死照射。經由尾靜脈注射進行移植(參見下文)。接著使小鼠接受新鮮拜有利水。一週後替換拜有利水，且在移植之後14天中斷拜有利水添加。Treatment: Mice were treated with 10 mg/kg/day Baytril water for 1 to 2 days prior to irradiation and sublethal irradiation at 300 RAD 16 to 24 hours prior to transplantation. Transplantation was performed via tail vein injection (see below). Mice were then given fresh Bayerisque water. Bailiff water was replaced after one week, and Bailiff water addition was discontinued 14 days after transplantation.

移植：移植當天，在37℃下對X-Vivo 10/1% PSG+3細胞因子混合液(重組人類幹細胞因子(SCF)、重組人類促血小板生成素(TPO)及重組人類Flt-3配位體(Flt-3L))進行預升溫，在環境溫度下製備新鮮PBS/0.1% BSA (無菌/過濾)。在37℃下將低溫保存細胞解凍，球粒化，再懸浮於預升溫X-Vivo培養基中，再次球粒化，再懸浮於PBS/0.1% BSA中並且計數。再一次球粒化之後，使細胞球粒再懸浮於每隻小鼠550 µL之PBS/0.1%BSA中(基於細胞計數，2×106 個細胞/mL)。接著在室溫下利用25號針將細胞注入小鼠尾靜脈中。研究組示於以下表5中。表 5 ：植入經編輯之 hCD34+ 細胞之給藥組

Figure 106129809-A0304-0005
Transplantation: On the day of transplantation, the X-Vivo 10/1% PSG+3 cytokine mixture (recombinant human stem cell factor (SCF), recombinant human thrombopoietin (TPO) and recombinant human Flt-3) was coordinated at 37°C (Flt-3L)) was pre-warmed and fresh PBS/0.1% BSA (sterile/filtered) was prepared at ambient temperature. Cryopreserved cells were thawed at 37°C, pelleted, resuspended in pre-warmed X-Vivo medium, pelleted again, resuspended in PBS/0.1% BSA and counted. After one more pelletization, the cell pellets were resuspended in 550 µL of PBS/0.1% BSA per mouse ( 2 x 106 cells/mL based on cell count). Cells were then injected into the tail vein of mice using a 25-gauge needle at room temperature. The study groups are shown in Table 5 below. Table 5 : Dosing groups implanted with edited hCD34+ cells
Figure 106129809-A0304-0005

每日觀測動物之一般健康狀況，並且在前2週每日稱重，且此後每兩週稱重。在移植後8週、12週、16週及20週時自下頜下靜脈(100 µL)或對於已處死之動物在移植後12週及20週時經由心臟穿刺(1 mL)收集外周血。在移植後12週時對各組中之半數動物(5隻小鼠/組)施以安樂死，並且收集骨髓及末梢血液以供分析。在移植後20週時將各組中之其餘動物(5隻小鼠/組)處死。Animals were observed daily for general health and weighed daily for the first 2 weeks and biweekly thereafter. Peripheral blood was collected from the submandibular vein (100 µL) at 8, 12, 16 and 20 weeks post-transplantation or via cardiac puncture (1 mL) for sacrificed animals at 12 and 20 weeks post-transplantation. Half of the animals in each group (5 mice/group) were euthanized at 12 weeks post-transplantation, and bone marrow and peripheral blood were collected for analysis. The remaining animals in each group (5 mice/group) were sacrificed 20 weeks after transplantation.

血液收集、細胞收集及處理：經由下頜下靜脈或心臟穿刺收集外周血至EDTA管中，並且在500×g下離心5分鐘以移除血漿。進行磷酸鹽緩衝生理食鹽水(PBS)牛血清白蛋白(BSA)洗滌及離心後，向球粒添加10倍體積之溶血緩衝液，並且在37℃下將混合物培育15分鐘，離心並且再次洗滌。在1 mL PBS BSA中復原球粒化級分；移出等分試樣並且在1,000×g下離心5分鐘，保存所得球粒以用於基因定型。將上清液級分用於FACS分析。Blood collection, cell collection and processing: Peripheral blood was collected via submandibular vein or cardiac puncture into EDTA tubes and centrifuged at 500 xg for 5 minutes to remove plasma. After phosphate buffered saline (PBS) bovine serum albumin (BSA) washing and centrifugation, 10 volumes of hemolysis buffer was added to the pellet, and the mixture was incubated at 37°C for 15 minutes, centrifuged and washed again. The pelleted fractions were reconstituted in 1 mL of PBS BSA; aliquots were removed and centrifuged at 1,000 xg for 5 minutes and the resulting pellets were saved for genotyping. The supernatant fractions were used for FACS analysis.

將骨髓、股骨、脛骨及髖骨收集在含有胎牛血清(FCS)之伊斯科夫氏改良杜爾貝科氏培養基(IMDM)中；將總骨髓快速傾入PBS BSA溶液中並且使用70 µm耐綸過濾器進行過濾。用PBS BSA將體積調節至10 mL，並且將等分試樣用於細胞計數(Cellometer)。Bone marrow, femur, tibia and hip were collected in Iskov's modified Dulbecco's medium (IMDM) containing fetal calf serum (FCS); total bone marrow was quickly poured into PBS BSA solution and 70 µm Nylon filter for filtration. The volume was adjusted to 10 mL with PBS BSA and aliquots were used for cell counting (Cellometer).

使用MiSeq深度定序來分析ZFN活性。簡言之，自注射後8週及12週時獲得之血液樣品或自注射後12週時獲得之骨髓分離注射未經轉染之對照CD34+ HSPC或經靶向ZFN mRNA之增強子轉染之CD34+ HSPC的小鼠的基因組DNA。對相關區域( BCL11A基因座內含有ZFN結合位點)進行PCR擴增，並且藉由在Illumina平台(MiSeq)上進行配對末端深度定序來測定修飾水準。ZFN activity was analyzed using MiSeq deep sequencing. Briefly, untransfected control CD34+ HSPCs or CD34+ transfected with enhancers targeting ZFN mRNA were injected from blood samples obtained at 8 and 12 weeks post-injection or bone marrow isolation obtained at 12 weeks post-injection Genomic DNA of HSPC mice. The region of interest (containing the ZFN binding site within the BCL11A locus) was PCR amplified and the level of modification determined by paired-end deep sequencing on the Illumina platform (MiSeq).

為了產生與Illumina MiSeq定序平台相容之文庫，使用兩組融合引子在連續PCR中將銜接子、條碼及流動細胞結合劑(短DNA序列)附接至標靶特異性擴增子。在對小鼠血液及骨髓樣品中之人類BCL11A增強子修飾進行MiSeq評估時，由於此等樣品中之靶DNA量較低，必須調整方案。To generate a library compatible with the Illumina MiSeq sequencing platform, two sets of fusion primers were used to attach adapters, barcodes, and flow cell binding agents (short DNA sequences) to target-specific amplicons in sequential PCR. In the MiSeq assessment of human BCL11A enhancer modifications in mouse blood and bone marrow samples, the protocol had to be adjusted due to the low amount of target DNA in these samples.

將以下引子用於MiSeq Adaptor PCR：PRJIYLFN-f2： ACA CGA CGC TCT TCC GAT CTN NNN AGT CCT CTT CTA CCC CAC CCA (SEQ ID NO:32)及 PRJIYLFN-r4： GAC GTG TGC TCT TCC GAT CTC TAC TCT TAG ACA TAA CAC ACC AGG G (SEQ ID NO:33)。The following primers were used for MiSeq Adaptor PCR: PRJIYLFN-f2: ACA CGA CGC TCT TCC GAT CTN NNN AGT CCT CTT CTA CCC CAC CCA (SEQ ID NO: 32) and PRJIYLFN-r4: GAC GTG TGC TCT TCC GAT CTC TAC TCT TAG ACA TAA CAC ACC AGG G (SEQ ID NO: 33).

為了進行分析，藉由DNeasy自小鼠骨髓樣品分離DNA，並且將大約100 ng DNA用於各PCR反應。藉由Tissue XS自小鼠血液樣品分離DNA，並且所分離之15 µL DNA中之10 µL用於各反應。除DNA以外，向各MiSeq PCR反應中添加以下各物：25 µL HotStar Taq混合物(Qiagen)、0.5 µL以上所列出之各BCL11A增強子引子(以100 nM之濃度)及達到50 µL總反應體積之水。典型MiSeq PCR條件為：95℃變性15分鐘，及94℃下30秒、62℃下30秒及72℃下40秒之循環30次，繼而72℃下10分鐘伸長。在MiSeq PCR之後，用水對PCR產物進行介於1:50與1:200之間的稀釋，或對於具有極低起始細胞數目之樣品，則不加稀釋。利用1 µL如以上所描述加以稀釋之MiSeq PCR產物、25 µL HotStar Taq混合物、1 µL前置條碼引子、1 µL反置條碼引子(兩者皆處於10 nM濃度下)及達到50 µL總反應體積之水來進行條碼PCR。條碼 PCR條件為：95℃變性15分鐘，及94℃下30秒、60℃下30秒及72℃下30秒之循環18次，繼而在72℃下10分鐘伸長。彙集條碼PCR產物並且在Illumina MiSeq定序器上定序。結果示於以上表5中。For analysis, DNA was isolated from mouse bone marrow samples by DNeasy, and approximately 100 ng of DNA was used in each PCR reaction. DNA was isolated from mouse blood samples by Tissue XS, and 10 μL of the 15 μL DNA isolated was used for each reaction. In addition to DNA, add the following to each MiSeq PCR reaction: 25 µL of HotStar Taq mix (Qiagen), 0.5 µL of each of the BCL11A enhancer primers listed above (at 100 nM concentration) and bring to a total reaction volume of 50 µL of water. Typical MiSeq PCR conditions are: denaturation at 95°C for 15 minutes, and 30 cycles of 30 seconds at 94°C, 30 seconds at 62°C, and 40 seconds at 72°C, followed by elongation at 72°C for 10 minutes. Following MiSeq PCR, PCR products were diluted between 1:50 and 1:200 in water, or undiluted for samples with very low starting cell numbers. Use 1 µL of MiSeq PCR product diluted as described above, 25 µL of HotStar Taq mix, 1 µL of pre-barcode primers, 1 µL of reverse barcoded primers (both at 10 nM concentration) and to a total reaction volume of 50 µL water for barcode PCR. The barcode PCR conditions were: denaturation at 95°C for 15 minutes, and 18 cycles of 30 seconds at 94°C, 30 seconds at 60°C and 30 seconds at 72°C, followed by elongation at 72°C for 10 minutes. Barcoded PCR products were pooled and sequenced on an Illumina MiSeq sequencer. The results are shown in Table 5 above.

用於嵌合及細胞譜系測定之FACS分析。為了評定人類嵌合度，分別利用hCD45-APC Cy7 (Biolegend)及hCD45-BV510 (BD Biosciences)抗體對外周血(植入後8週、12週、16週及20週時)及骨髓(植入後12週及20週時)中之細胞級分進行染色，並且進行FACS分析。另外，藉由用以下表6中所描述之特定抗體將骨髓細胞染色來進行造血譜系分析：表 6 ：細胞標記物之抗體源

Figure 106129809-A0304-0006
FACS analysis for chimerism and cell lineage determination. To assess human chimerism, hCD45-APC Cy7 (Biolegend) and hCD45-BV510 (BD Biosciences) antibodies were used in peripheral blood (8, 12, 16, and 20 weeks post-implantation) and bone marrow (post-implantation), respectively. Cell fractions at 12 and 20 weeks) were stained and subjected to FACS analysis. Additionally, hematopoietic lineage analysis was performed by staining bone marrow cells with specific antibodies described in Table 6 below: Table 6 : Antibody Sources for Cell Markers
Figure 106129809-A0304-0006

另外，為了純化並分選HSPC群體，吾等使用了利用磁性細胞分離(MACS)之富集/耗竭策略。首先用CD19-生物素、CD3-生物素、B220-生物素、TER119-生物素及m-ckit-生物素(BD Biosciences)將骨髓細胞染色，接著與抗生物素珠粒(Miltenyi Biotec)一起培育。使用置於MACS之磁場中的LS管柱(Miltenyi Biotec)來分離陽性級分及耗竭級分。分離之後，用鏈黴親和素-APC、CD3-FITC、CD19-PE、CD45-BV510 (BD Biosciences)將陽性級分染色且用CD34-FITC (BD Biosciences)、Gly-A-PE (DAKO)、CD19-APC (BD)、Lin-APC (Biolegend)、鏈黴親和素-APC、CD45-BV510、CD33-PE-CF594 (BD)及CD38-PECy-7 (Biolegend)將耗竭級分染色。Additionally, to purify and sort the HSPC population, we used an enrichment/depletion strategy using magnetic cell separation (MACS). Bone marrow cells were first stained with CD19-biotin, CD3-biotin, B220-biotin, TER119-biotin and m-ckit-biotin (BD Biosciences), followed by incubation with anti-biotin beads (Miltenyi Biotec) . Positive and depleted fractions were separated using LS columns (Miltenyi Biotec) placed in the magnetic field of MACS. After isolation, positive fractions were stained with streptavidin-APC, CD3-FITC, CD19-PE, CD45-BV510 (BD Biosciences) and stained with CD34-FITC (BD Biosciences), Gly-A-PE (DAKO), Depleted fractions were stained for CD19-APC (BD), Lin-APC (Biolegend), Streptavidin-APC, CD45-BV510, CD33-PE-CF594 (BD) and CD38-PECy-7 (Biolegend).

使用如以上所描述之標準程序將未經轉染之HSPC及經63014/65722轉染之HSPC植入NSG小鼠中。藉由使用FACS量測hCD45陽性細胞之分數來評定植入後此等小鼠中之人類嵌合度。Untransfected HSPCs and 63014/65722 transfected HSPCs were implanted into NSG mice using standard procedures as described above. Human chimerism in these mice after implantation was assessed by measuring the fraction of hCD45 positive cells using FACS.

圖7顯示移植後8週及12週時收集之外周血中之人類CD45+細胞之百分比，且圖8顯示顯示第12週時收集之骨髓中的百分比。如所示，此研究中之植入水準為植入未經轉染之對照物及經63014/65722轉染之HSPC後存在相當之人類嵌合。分佈在各組中之60隻小鼠中僅有3隻不具有CD45+細胞，指示植入失敗。Figure 7 shows the percentage of human CD45+ cells in peripheral blood collected at 8 and 12 weeks post-transplantation, and Figure 8 shows the percentage in bone marrow collected at week 12. As indicated, the level of engraftment in this study was comparable human chimerism after engraftment of untransfected controls and HSPCs transfected with 63014/65722. Only 3 of the 60 mice distributed in each group had no CD45+ cells, indicating engraftment failure.

藉由使用標準程序對第12週時獲得之具有抗體識別譜系特異性細胞表面標記物之骨髓細胞進行FACS分析來測試多種造血細胞譜系之重構。如圖9中所示，觀測到經BCL11A特異性ZFN編碼mRNA處理之CD34+細胞後代與未經轉染之細胞之後代之間在注射後第12週時所分析之所有骨髓中人類造血譜系的表現相當。分離在植入後第12週時被處死之小鼠的骨髓，且藉由FACS使用識別所指示之譜系標記物的抗體來分析多種造血譜系之分佈。所有數值均提供為所指示譜系標記物之陽性染色細胞之比率相對於人類CD45陽性細胞之百分比，但圖9C中之表現紅血球標記物Cd71+ (Ter119)之細胞提供為整個群體中陽性染色細胞之百分比，此係因為紅血球細胞並非CD45陽性的。Reconstitution of various hematopoietic cell lineages was tested by FACS analysis of bone marrow cells obtained at week 12 with antibodies recognizing lineage-specific cell surface markers using standard procedures. As shown in Figure 9, the performance of the human hematopoietic lineage in all bone marrow analyzed at week 12 post-injection was observed between the progeny of CD34+ cells treated with mRNA encoding a BCL11A-specific ZFN and the progeny of non-transfected cells quite. Bone marrow from mice sacrificed at 12 weeks post-implantation was isolated and the distribution of the various hematopoietic lineages was analyzed by FACS using antibodies recognizing the indicated lineage markers. All values are provided as the ratio of positively stained cells for the indicated lineage markers relative to the percentage of human CD45 positive cells, except that the cells in Figure 9C expressing the red blood cell marker Cd71+ (Ter119) are provided as the percentage of positively stained cells in the entire population , this is because the red blood cells are not CD45 positive.

藉由使用如以上所描述之MiSeq定序平台對ZFN標靶區域進行深度定序來評定BCL11A紅血球增強子處之基因修飾水準(具有***及缺失[***缺失]之對偶基因的%)。在圖10中顯示第8週及第12週血液樣品之資料且在圖11中顯示經63014/65722處理之細胞的第12週骨髓樣品及來源於第12週骨髓細胞樣品之經分選譜系的資料。為了進行比較，在轉染之後2天量測之***缺失百分比(如表5中所列出)亦示於圖10及圖11之圖上。The level of gene modification at the BCL11A erythrocyte enhancer (% of paired genes with indels and deletions [indels]) was assessed by deep sequencing of the ZFN target region using the MiSeq sequencing platform as described above. Data for week 8 and week 12 blood samples are shown in Figure 10 and week 12 bone marrow samples of 63014/65722 treated cells and sorted lineages derived from week 12 bone marrow cell samples are shown in Figure 11 material. For comparison, the percentage of indels (as listed in Table 5) measured 2 days after transfection are also shown on the graphs of Figures 10 and 11 .

另外，對於兩個經63014/65722處理之HSPC供體集合，在不同的時間點及不同的譜系中發現BCL11A紅血球增強子處之基因修飾得以良好保留。在BCL11A依賴性( B細胞，『CD19』；原始祖細胞，『CD38H』)及非BCL11A依賴性(骨髓『CD33』)譜系兩者中觀測到相當之修飾。儘管PB-MR-003供體樣品中之輸入基因修飾水準高於PB-MR-004供體樣品中，但與來源於PB-MR-003之彼等修飾水準相比，PB-MR-004衍生細胞在小鼠中始終顯示較高修飾水準，亦即，較佳修飾保留。In addition, gene modifications at the BCL11A erythrocyte enhancer were found to be well preserved at different time points and in different lineages for the two 63014/65722-treated HSPC donor pools. Comparable modifications were observed in both BCL11A-dependent (B cells, "CD19"; primitive progenitors, "CD38H") and BCL11A-independent (myeloid "CD33") lineages. Although the input gene modification levels in the PB-MR-003 donor samples were higher than those in the PB-MR-004 donor samples, PB-MR-004 derived PB-MR-004 derived Cells consistently showed higher levels of modification in mice, ie, better retention of modifications.

總體而言，在小鼠中觀測之BCL11A紅血球增強子處之基因修飾保留與在使用眾多靶向多種基因標靶之ZFN的先前小鼠實驗中所觀測的一致。Overall, the genetic modification at the BCL11A erythrocyte enhancer observed in mice remains consistent with that observed in previous mouse experiments using numerous ZFNs targeting multiple gene targets.

此外，因為人類紅血球祖細胞在小鼠中不能分化，故為了測定此等細胞中發生之BCL11A靶向基因修飾之量，自小鼠移出骨髓細胞且在試管內分化。在此等實驗中，如以上所描述在植入後第12週時自處死之小鼠移出骨髓衍生人類細胞並且在試管內分化。藉由在分化第14天對自細胞分離之DNA進行高通量Miseq定序來量測BCL11A標靶基因修飾。In addition, because human erythrocyte progenitor cells do not differentiate in mice, to determine the amount of BCL11A-targeted genetic modification occurring in these cells, bone marrow cells were removed from mice and differentiated in vitro. In these experiments, bone marrow-derived human cells were removed from sacrificed mice at week 12 post-implantation and differentiated in vitro as described above. BCL11A target gene modifications were measured by high-throughput Miseq sequencing of DNA isolated from cells at day 14 of differentiation.

修飾資料(***缺失)提供於圖12中，該圖顯示紅血球分化第14天之修飾水準。由自一隻小鼠分離之細胞產生的各培養物在試管內分化第14天時之***缺失百分比顯著不同，從而體現在此等條件下獲得之擴增的寡細胞性質。資料指示由63014/65722 ZFN介導之BCL11A增強子修飾在紅血球分化期間不顯著變化。如在血液及骨髓樣品中所觀測，PB-MR-004衍生細胞之紅血球後代樣品顯示比PB-MR-003衍生細胞之紅血球後代更高的平均修飾水準。Modification data (indels) are provided in Figure 12, which shows modification levels at day 14 of erythrocyte differentiation. The percentage of indels at day 14 of in vitro differentiation between cultures derived from cells isolated from one mouse varied significantly, reflecting the oligocellular nature of the expansion obtained under these conditions. The data indicate that BCL11A enhancer modification mediated by 63014/65722 ZFNs does not change significantly during erythrocyte differentiation. As observed in blood and bone marrow samples, samples of erythrocyte progeny of PB-MR-004 derived cells showed higher mean modification levels than erythrocyte progeny of PB-MR-003 derived cells.

藉由對在試管內紅血球分化第14天時自細胞分離之RNA進行RT-PCR分析來測定多種球蛋白mRNA之相對水準，並且將資料提供於圖13A中，其中對來自各組之5種紅血球培養物求取相對γ球蛋白與β球蛋白mRNA及γ球蛋白與α球蛋白mRNA比率(圖13B)。在未經轉染及經63014/65722處理之樣品中，γ球蛋白與β球蛋白或γ球蛋白與α球蛋白mRNA比率在相同組之個別小鼠的紅血球後代之間廣泛不同。供體PB-MR-004衍生培養物所顯示之γ球蛋白比率平均低於得自供體PB-MR-003之彼等γ球蛋白比率，與針對PB-MR-004衍生樣品觀測到更佳成熟一致。然而，儘管存在此變異性，但經ZFN處理之樣品平均顯示γ球蛋白mRNA水準與其個別未經轉染之對應物相比存在約1.5至2倍增加。Relative levels of various globulin mRNAs were determined by RT-PCR analysis of RNA isolated from cells at day 14 of in vitro erythrocyte differentiation, and the data are provided in Figure 13A, in which 5 erythrocytes from each group were analyzed. The cultures were determined for relative gamma to beta mRNA and gamma to alpha mRNA ratios (FIG. 13B). In untransfected and 63014/65722-treated samples, the gamma to beta or gamma to alpha mRNA ratios varied widely among the erythrocyte progeny of individual mice in the same group. Donor PB-MR-004 derived cultures exhibited gamma globulin ratios that were on average lower than those from donor PB-MR-003, with better maturation observed for PB-MR-004 derived samples Consistent. However, despite this variability, ZFN-treated samples showed, on average, about a 1.5- to 2-fold increase in gamma globulin mRNA levels compared to their individual non-transfected counterparts.

藉由HPLC分析來評定球蛋白水準。圖14顯示在分化第16天時收集之樣品的球蛋白分析。測定γ球蛋白(Aγ及Gγ峰之總和)與α球蛋白比率，以及γ球蛋白(Aγ及Gγ峰之總和)/總β樣球蛋白比率(Aγ、Gγ、β及δ球蛋白峰之總和)，並且將各組之平均值示於各柱條上方。與PB-MR-003衍生樣品之不良紅血球分化一致，來源於此供體之未經轉染細胞中之γ球蛋白水準極高(約30%)，且因此ZFN處理僅引起γ球蛋白水準增加1.2倍。PB-MR-004顯示更典型之未經轉染水準(約9%)，且在由小鼠進行12週傳代之後展現γ球蛋白水準增加約2倍。Globulin levels were assessed by HPLC analysis. Figure 14 shows globulin analysis of samples collected at day 16 of differentiation. Determination of gamma globulin (sum of Aγ and Gγ peaks) to alpha globulin ratio, and gamma globulin (sum of Aγ and Gγ peaks)/total β-like globulin ratio (sum of Aγ, Gγ, β and delta globulin peaks), and The mean of each group is shown above each bar. Consistent with poor erythrocyte differentiation in PB-MR-003 derived samples, gamma globulin levels in untransfected cells derived from this donor were extremely high (about 30%), and thus ZFN treatment caused only an increase in gamma globulin levels 1.2 times. PB-MR-004 showed more typical untransfected levels (approximately 9%) and exhibited an approximately 2-fold increase in gamma globulin levels after 12 weeks of passage from mice.

認為具有＞8.6% γ球蛋白之患者與γ球蛋白水準＜8.6%之患者相比本質上有一定優勢(Platt等人, (1994)N Engl J Med , 330:1639-44)。事實上，藉由植入經編輯之細胞而達成非鐮形血球RBC之嵌合10%至20%百分比可能引起臨床改良(Chang等人,(2017)Mol Ther Methods Clin Dev 4:137-148. doi 10.1016/j.omtm.2016.12.009)。因而，儘管必須經歷試管內紅血球分化過程，但所偵測之嵌合細胞百分比及γ球蛋白水準指示治療效力。Patients with >8.6% gamma globulin are considered to be inherently superior to those with gamma globulin levels <8.6% (Platt et al., (1994) N Engl J Med , 330:1639-44). In fact, achieving 10% to 20% chimerism of non-sickle cell RBCs by engrafting edited cells may lead to clinical improvement (Chang et al., (2017) Mol Ther Methods Clin Dev 4:137-148. doi 10.1016/j.omtm.2016.12.009). Thus, the percentage of chimeric cells and gamma globulin levels detected are indicative of therapeutic efficacy despite having to undergo an in vitro erythrocyte differentiation process.

本文中所提及之所有專利、專利申請案及出版物均以全文引用之方式併入本文中。All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

雖然已出於清楚理解之目的以說明及實例之方式相當詳細地提供了揭示內容，但熟習此項技術者應顯而易見，可在不背離本發明之精神或範疇的情況下實施多種變化及修改。相應地，以上描述及實例不應被視為具有限制性。Although the disclosure has been presented in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. Accordingly, the above description and examples should not be regarded as limiting.

圖1A及圖1B為描繪寡核苷酸雙鏈體整合位點分析之概述的示意圖。圖1顯示該分析之第一步驟，其中在補充寡核苷酸雙鏈體DNA之存在下用ZFN處理細胞，該補充寡核苷酸雙鏈體DNA被俘獲至所得裂解事件部分中。圖1B顯示次順序步驟，其中將細胞培養七天，分離基因組DNA，且使用引子經由銜接子介導之PCR將基因組側接供體整合位點之區段擴增至經整合之寡核苷酸雙鏈體。接著對擴增子進行定序以揭示候選裂解位點。1A and 1B are schematic diagrams depicting an overview of oligonucleotide duplex integration site analysis. Figure 1 shows the first step of the assay, in which cells are treated with ZFNs in the presence of complementary oligonucleotide duplex DNA, which is captured into the resulting fraction of cleavage events. Figure 1B shows sub-sequential steps in which cells were cultured for seven days, genomic DNA was isolated, and primers were used to amplify segments of the genome flanking the donor integration site into integrated oligonucleotide duplexes via adaptor-mediated PCR. chain body. The amplicons were then sequenced to reveal candidate cleavage sites.

圖2描繪藉由寡核苷酸雙鏈體整合位點分析法，使用51857/51949原始或親本ZFN配對鑑定之455個潛在脫靶基因座之位置。上部63個基因座以灰色突出顯示且在追蹤性***缺失分析中加以分析。各基因座係藉由指示最可能之裂解位置的染色體及鹼基數目以及指示在該基因座處偵測之整合體的數目來鑑定。Figure 2 depicts the locations of 455 potential off-target loci identified by oligonucleotide duplex integration site analysis using 51857/51949 original or parental ZFN pairings. The upper 63 loci are highlighted in grey and analyzed in a traceable indel analysis. Each locus was identified by the number of chromosomes and bases indicating the most likely cleavage location and the number of integrants detected at that locus.

圖3A至圖3C描繪鋅指支架內之例示性磷酸酯接觸殘基。圖3A描繪與DNA分子之鋅指相互作用，且顯示野生型精胺酸側鏈之位置及其如何與DNA分子之磷酸酯主鏈相互作用。圖3B (SEQ ID No:7至17)描繪例示性ZFN序列，顯示此精胺酸位於各鋅指之主要序列中(經由粗箭頭指示之『R』殘基豎列)，而且亦突出顯示ZFP主鏈中經麩醯胺酸取代以消除相應磷酸酯接觸(個別加框『R』殘基)之彼等精胺酸。該等序列亦顯示識別螺旋區(其中將殘基-1至+3、+5及+6加框且加陰影)以及介於C末端鋅指結構域與裂解結構域(裂解結構域未圖示)之間的連接子部分。圖3C進一步描繪FokI裂解結構域中所存在之另一潛在主鏈接觸離胺酸側鏈的空間位置，其在特異性最佳化期間可取代成絲胺酸。3A-3C depict exemplary phosphate contact residues within zinc finger scaffolds. Figure 3A depicts zinc finger interactions with DNA molecules and shows the location of the wild-type arginine side chain and how it interacts with the phosphate backbone of the DNA molecule. Figure 3B (SEQ ID Nos: 7-17) depicts exemplary ZFN sequences showing that this arginine is located in the main sequence of each zinc finger (vertical column of "R" residues indicated by thick arrows), and also highlights the ZFP These arginines were substituted in the backbone with glutamic acids to eliminate the corresponding phosphate contacts (individually boxed "R" residues). The sequences also show the recognition helical region (where residues -1 to +3, +5 and +6 are boxed and shaded) and between the C-terminal zinc finger domain and the cleavage domain (cleavage domain not shown) ) between the connecting subsections. Figure 3C further depicts the steric position of another potential backbone present in the FokI cleavage domain that contacts the lysine side chain, which can be substituted to serine during specificity optimization.

圖4為經由在不同的測試基因座處用原始51857/51949配對或用最佳化63014/65722配對處理CD34細胞來比較修飾水準之圖。在x軸底部或x軸上顯示被鑑定為潛在裂解標靶之基因座，其中各位點之***缺失百分比示於垂直軸或y軸上。注意，y軸以對數尺度顯示。暗灰色柱條顯示被51857/51949配對裂解之基因座及所偵測之裂解量，而淺灰色柱條為被最佳化配對裂解之彼等基因座，其中幾乎所有量測之裂解均之靶向BCL11a靶序列處。Figure 4 is a graph comparing modification levels by treating CD34 cells with the original 51857/51949 pairing or with the optimized 63014/65722 pairing at different tested loci. Loci identified as potential cleavage targets are shown at the bottom of the x-axis or on the x-axis, with the percentage of indels at each site shown on the vertical or y-axis. Note that the y-axis is displayed on a log scale. The dark grey bars show the loci cleaved by the 51857/51949 pair and the amount of cleavage detected, while the light grey bars are those loci that were cleaved by the optimized pair, where nearly all measured cleavage was the target to the BCL11a target sequence.

圖5A及圖5B為描繪在用BCL11A特異性ZFN進行治療及紅血球分化後在hCD34+細胞中產生之球蛋白mRNA之相對比率的圖。用ZFN配對處理或不處理來源於兩個健康人類供體之CD34+細胞(PB-MR-003或PB-MR-004)，接著分析α、β及γ球蛋白表現。用於在ZFN處理後測定所發現之γ球蛋白mRNA之量的最佳方法為將表現變化表示為γ球蛋白與β球蛋白之比率(圖5A)或γ球蛋白與α球蛋白mRNA之比率(圖 5B)。Figures 5A and 5B are graphs depicting the relative ratios of globulin mRNA produced in hCD34+ cells following treatment with BCL11A-specific ZFNs and erythrocyte differentiation. CD34+ cells derived from two healthy human donors (PB-MR-003 or PB-MR-004) were either treated or not paired with ZFNs and then analyzed for alpha, beta and gamma globulin expression. The best method for determining the amount of gamma globulin mRNA found after ZFN treatment is to express changes in performance as the ratio of gamma globulin to beta globulin (Figure 5A) or the ratio of gamma globulin to alpha globulin mRNA (Fig. 5B).

圖6為描繪經處理之CD34+細胞中所產生之γ球蛋白之相對量的圖。如上，使用來源於健康人類供體之兩個CD34+細胞批次(PB-MR-003及PB-MR-004)。在此實驗中，兩個供體批次中在63014/65722介導之BCL11A增強子破環後，在HSPC之紅血球後代中均觀測到胎兒球蛋白百分比升高大約3至4倍，達至約15%-20%之水準。Figure 6 is a graph depicting the relative amounts of gamma globulin produced in treated CD34+ cells. As above, two CD34+ cell batches (PB-MR-003 and PB-MR-004) derived from healthy human donors were used. In this experiment, an approximately 3- to 4-fold increase in percent fetal globulin was observed in erythrocyte progeny of HSPCs following 63014/65722-mediated disruption of the BCL11A enhancer in both donor batches, to approximately 15%-20% level.

圖7為指示植入有如以上所描述之經63014/65722處理之供體批次(「+ZFN」)的小鼠中的相關人類嵌合的圖。藉由使用FACS偵測在其表面上攜帶hCD45標記物之細胞來量測人類嵌合。指示移植後8或12週時收集之外周血中的人類hCD45+細胞之百分比。資料顯示此研究中之良好植入水準，在植入未經轉染之對照物(「(-)」)及經ZFN轉染之HSPC (「+ZFN」)後具有相當之人類嵌合。空心圓形及三角形表示個別動物。Figure 7 is a graph indicating the relevant human chimerism in mice engrafted with the 63014/65722-treated donor batch ("+ZFN") as described above. Human chimerism was measured by using FACS to detect cells carrying the hCD45 marker on their surface. The percentage of human hCD45+ cells in peripheral blood collected at 8 or 12 weeks post-transplantation is indicated. The data show a good level of engraftment in this study, with comparable human chimerism following engraftment of untransfected controls ("(-)") and ZFN-transfected HSPCs ("+ZFN"). Open circles and triangles represent individual animals.

圖8顯示在植入小鼠之骨髓中偵測之嵌合百分比，其中人類細胞係藉由其細胞表面上存在hCD45而加以鑑定。在植入後12週時分析樣品。Figure 8 shows the percentage of chimerism detected in bone marrow implanted in mice in which human cell lines were identified by the presence of hCD45 on their cell surfaces. Samples were analyzed at 12 weeks post-implantation.

圖9A至圖9D為描繪藉由對第12週時獲得之具有抗體識別譜系特異性細胞表面標記物之植入小鼠中骨髓細胞進行FACS分析而測試之多種造血細胞譜系之重構的圖。資料顯示經BCL11A特異性ZFN mRNA處理之CD34+細胞後代(「『14/』22」)與未經轉染之細胞之後代(「(-)」)之間在注射後第12週時所分析之所有骨髓中人類造血譜系之呈現相當。顯示得自來源於兩個供體(「003」及「004」)之細胞之淋巴細胞、骨髓細胞、紅血球及HSPC的資料(分別圖9A至圖9D)。資料顯示經Bcl11A ZFN mRNA處理之CD34+細胞後代與未經轉染之細胞之後代之間在注射後第12週時所分析之所有骨髓中人類造血譜系的表現相當。9A-9D are graphs depicting remodeling of various hematopoietic cell lineages tested by FACS analysis of bone marrow cells obtained at week 12 with antibodies recognizing lineage-specific cell surface markers in engrafted mice. Data show the difference between progeny of CD34+ cells treated with BCL11A-specific ZFN mRNA ("'14/'22") and progeny of untransfected cells ("(-)") analyzed at week 12 post-injection The presentation of the human hematopoietic lineage was comparable in all bone marrow. Data are shown for lymphocytes, bone marrow cells, erythrocytes and HSPCs derived from cells derived from two donors ("003" and "004") (FIGS. 9A-9D, respectively). The data show comparable performance of the human hematopoietic lineage in all bone marrow analyzed at week 12 post-injection between progeny of CD34+ cells treated with Bcl11A ZFN mRNA and progeny of untransfected cells.

圖10為描繪藉由深度定序分析之自植入小鼠外周血分離之DNA中在BCL11A標靶處之基因修飾水準的圖。顯示輸入細胞(ZFN轉染後2天，(「+」))及植入後8或12週之血細胞的資料，且顯示基因修飾之良好保留。在處理列中以「(-)」表示未經轉染之細胞。Figure 10 is a graph depicting the level of gene modification at the BCL11A target in DNA isolated from peripheral blood of engrafted mice by deep sequencing analysis. Data are shown for infused cells (2 days post-ZFN transfection, ("+")) and blood cells 8 or 12 weeks post-implantation and show good retention of the genetic modification. Untransfected cells are indicated by "(-)" in the treatment column.

圖11為描繪植入經ZFN處理之細胞(「+」)後骨髓細胞樣品在BCL11A標靶處之基因修飾之量的圖。在處理列中以「(-)」表示未經處理之細胞。在BCL11A依賴性譜系(B細胞，表現CD19標記物；原始祖細胞，表現CD45及高水準之CD38)及非BCL11A依賴性(骨髓)譜系中觀測到相當之修飾。儘管PB-MR-003供體樣品中之輸入基因修飾水準高於PB-MR-004供體樣品中，但與來源於PB-MR-003之彼等修飾水準相比，PB-MR-004衍生細胞在小鼠中始終顯示較高修飾水準，亦即，較佳修飾保留。Figure 11 is a graph depicting the amount of genetic modification at the BCL11A target in bone marrow cell samples following implantation of ZFN-treated cells ("+"). Untreated cells are indicated by "(-)" in the treated column. Comparable modifications were observed in BCL11A-dependent lineages (B cells, expressing markers of CD19; primitive progenitors, expressing CD45 and high levels of CD38) and BCL11A-independent (myeloid) lineages. Although the input gene modification levels in the PB-MR-003 donor samples were higher than those in the PB-MR-004 donor samples, PB-MR-004 derived PB-MR-004 derived Cells consistently showed higher levels of modification in mice, ie, better retention of modifications.

圖12為描繪試管內分化14天後來源於第12週骨髓細胞之紅血球細胞中之基因修飾之量的圖。資料來自於最初植入以上所描述之兩個不同的供體的小鼠，且顯示由ZFN處理(「+ ZFN」)介導之BCL11A修飾在紅血球分化期間不顯著變化。以「(-) ZFN」指示未經ZFN處理之細胞。Figure 12 is a graph depicting the amount of gene modification in erythrocytes derived from bone marrow cells at week 12 after 14 days of in vitro differentiation. Data are from mice originally implanted with two different donors as described above, and show that BCL11A modification mediated by ZFN treatment ("+ZFN") does not change significantly during erythrocyte differentiation. Cells not treated with ZFN are indicated by "(-) ZFN".

圖13A及圖13B為描繪γ球蛋白編碼mRNA之相對量的圖，其中γ球蛋白mRNA之濃度描繪為γ球蛋白/β球蛋白mRNA之比率(圖9A)或為γ球蛋白/α球蛋白mRNA之比率 (圖9B)。在未經轉染之樣品(「(-) ZFN」)及經ZFN處理之樣品(「+ ZFN」)中，γ球蛋白與β球蛋白或γ球蛋白與α球蛋白mRNA比率在來自同一群組之個別小鼠之紅血球後代之間廣泛不同。然而，儘管存在此變異性及藉由使用兩個不同的人類供體而引入之變異性，但經63014/65722處理之樣品平均值顯示在γ球蛋白mRNA水準方面與其個別未經轉染之對應物相比增加約1.5至2倍。Figures 13A and 13B are graphs depicting the relative amounts of gamma globulin-encoding mRNA, where the concentration of gamma globulin mRNA is plotted as the ratio of gamma globulin/beta globulin mRNA (FIG. 9A) or as gamma globulin/alpha globulin ratio of mRNA (FIG. 9B). In untransfected samples ("(-) ZFNs") and ZFN-treated samples ("+ ZFNs"), the gamma to beta globulin or gamma to alpha globulin mRNA ratios were significantly different from those from the same population The erythrocyte progeny of the individual mice of the groups varied widely. However, despite this variability and the variability introduced by using two different human donors, the mean values of the 63014/65722-treated samples showed their respective untransfected counterparts in terms of gamma globulin mRNA levels It is about 1.5 to 2 times more than that of other substances.

圖14為描繪得自植入小鼠之骨髓衍生細胞中在γ球蛋白之量(表示為γ球蛋白/α球蛋白或γ球蛋白/總β樣蛋白之比率)方面之差異的圖，其中將骨髓細胞提交至試管內分化方案。進入分化16天時量測蛋白質水準。測定γ (Gamma)球蛋白(Aγ與Gγ峰之總和)與α (alpha)球蛋白比率，以及γ球蛋白(Aγ與Gγ峰之總和)/總β樣球蛋白比率(Aγ、Gγ、β及δ球蛋白峰之總和)。與PB-MR-003衍生樣品之不良紅血球分化一致，來源於此供體之未經轉染之細胞中的γ球蛋白水準非常高(~30%)，且因此，ZFN處理(「+ ZFN」)引起γ球蛋白水準與未經處理之細胞(「(-) ZFN」)相比僅增加1.2倍。PB-MR-004顯示更典型之未經轉染水準(約9%)，且在由小鼠進行12週傳代之後展現γ球蛋白水準增加約2倍。Figure 14 is a graph depicting differences in the amount of gamma globulin (expressed as the ratio of gamma globulin/alpha globulin or gamma globulin/total beta-like protein) in bone marrow-derived cells implanted in mice, wherein Submit myeloid cells to the in vitro differentiation protocol. Protein levels were measured 16 days into differentiation. Determination of γ (Gamma) globulin (sum of Aγ and Gγ peaks) to α (alpha) globulin ratio, and γ globulin (sum of Aγ and Gγ peaks)/total β-like globulin ratio (Aγ, Gγ, β and δ globulins) sum of protein peaks). Consistent with the poor erythrocyte differentiation of PB-MR-003 derived samples, gamma globulin levels were very high (~30%) in untransfected cells from this donor, and thus, ZFN treatment ("+ ZFN" ) caused only a 1.2-fold increase in gamma globulin levels compared to untreated cells ("(-)ZFN"). PB-MR-004 showed more typical untransfected levels (approximately 9%) and exhibited an approximately 2-fold increase in gamma globulin levels after 12 weeks of passage from mice.

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0025

(無)(none)

Claims (22)

一種鋅指核酸酶(ZFN)，其包含左ZFN及右ZFN，該左ZFN包含如SEQ ID NO：29中所示之胺基酸序列且該右ZFN包含如SEQ ID NO：31中所示之胺基酸序列。 A zinc finger nuclease (ZFN) comprising a left ZFN and a right ZFN, the left ZFN comprising the amino acid sequence shown in SEQ ID NO:29 and the right ZFN comprising the amino acid sequence shown in SEQ ID NO:31 amino acid sequence. 一或多種聚核苷酸，其編碼一或多種如請求項1之鋅指核酸酶。 One or more polynucleotides encoding one or more zinc finger nucleases as claimed in claim 1. 如請求項2之一或多種聚核苷酸，其中所述聚核苷酸為mRNA。 The one or more polynucleotides of claim 2, wherein the polynucleotide is mRNA. 如請求項3之一或多種聚核苷酸，其包含SEQ ID NO：28及SEQ ID NO：30。 The one or more polynucleotides of claim 3 comprising SEQ ID NO:28 and SEQ ID NO:30. 一或多種載體，其包含編碼一或多種如請求項1之鋅指核酸酶的序列。 One or more vectors comprising sequences encoding one or more zinc finger nucleases as claimed in claim 1. 一種細胞，其包含如請求項1之鋅指核酸酶、如請求項2至4中任一項之一或多種聚核苷酸或如請求項5之一或多種載體。 A cell comprising a zinc finger nuclease as claimed in claim 1, one or more polynucleotides as in any one of claims 2 to 4, or one or more vectors as in claim 5. 如請求項6之細胞，其中該細胞為幹細胞或前驅細胞。 The cell of claim 6, wherein the cell is a stem cell or a precursor cell. 如請求項7之細胞，其中該細胞為人類細胞，其中該細胞之基因組係藉由該等鋅指核酸酶來修飾。 The cell of claim 7, wherein the cell is a human cell, wherein the genome of the cell is modified by the zinc finger nucleases. 如請求項8之細胞，其中該基因組之修飾係選自由以下各項組成之群：***、缺失及其組合。 The cell of claim 8, wherein the modification of the genome is selected from the group consisting of insertions, deletions, and combinations thereof. 如請求項8或9之細胞，其中該基因組之修飾處於BCL11A增強子序列之+58區域內。 The cell of claim 8 or 9, wherein the genomic modification is within the +58 region of the BCL11A enhancer sequence. 一種經基因修飾之細胞或細胞株，其係產生自如請求項6至8中任一項之細胞、或由其傳代而來的部分或完全分化之經基因修飾之細胞或細胞株，該細胞或細胞株包含如請求項1之鋅指核酸酶。 A genetically modified cell or cell line derived from the cell of any one of claims 6 to 8, or a partially or fully differentiated genetically modified cell or cell line passaged therefrom, the cell Or the cell line comprises the zinc finger nuclease of claim 1. 如請求項11之細胞或細胞株，其中該細胞與不存在該基因組之修飾之細胞相比展現增加之γ及/或β球蛋白表現。 The cell or cell line of claim 11, wherein the cell exhibits increased gamma and/or beta globulin expression compared to a cell in the absence of the genomic modification. 一種醫藥組合物，其包含如請求項1之鋅指核酸酶、如請求項2至4中任一項之一或多種聚核苷酸或如請求項6至8中任一項之細胞或如請求項12之細胞或細胞株。 A pharmaceutical composition comprising a zinc finger nuclease as claimed in claim 1, a polynucleotide as in any one of claims 2 to 4 or a cell as in any one of claims 6 to 8 or as The cell or cell line of claim 12. 如請求項13之醫藥組合物，其包含自如請求項12中任一者之經核酸酶修飾之細胞傳代而來之繁殖與分化細胞之組合。 The pharmaceutical composition of claim 13, comprising a combination of propagated and differentiated cells passaged from the nuclease-modified cells of any one of claims 12. 一種產生在內源BCL11A增強子序列內包含基因組之修飾的經基因修飾之細胞之試管內方法，該方法包括以下步驟：(a)使細胞與如請求項2至4中任一項之一或多種聚核苷酸接觸；(b)使該細胞處於有助於自該一或多種聚核苷酸表現融合蛋白質之條件下；及(c)以該表現融合蛋白質修飾該內源BCL11A增強子序列來產生該經基因修飾之細胞，視情況其中該方法進一步包括用至少一種細胞因子(cytokine)刺激該細胞。 An in vitro method for producing a genetically modified cell comprising a modification of the genome within an endogenous BCL11A enhancer sequence, the method comprising the steps of: (a) subjecting the cell to any one of claims 2 to 4 or contacting a plurality of polynucleotides; (b) subjecting the cell to conditions conducive to expression of a fusion protein from the one or more polynucleotides; and (c) modifying the endogenous BCL11A enhancer sequence with the expression fusion protein to generate the genetically modified cell, optionally wherein the method further comprises stimulating the cell with at least one cytokine. 一種如請求項6至8中任一項之細胞或如請求項11之細胞或細胞株於製備一藥物之用途，該藥物係供用於治療血紅素病。 Use of a cell according to any one of claims 6 to 8 or a cell or cell strain according to claim 11 for the preparation of a medicament for the treatment of heme disease. 如請求項16之用途，其中該血紅素病為β型地中海型貧血或鐮形血球貧血症。 The use of claim 16, wherein the heme disease is beta thalassemia or sickle cell anemia. 一種如請求項14之醫藥組合物於製備一藥物之用途，該藥物係供用於治療血紅素病。 A use of the pharmaceutical composition as claimed in claim 14 for the preparation of a medicament for the treatment of heme disease. 如請求項18之用途，其中該血紅素病為β型地中海型貧血或鐮形血球貧血症。 The use of claim 18, wherein the heme disease is beta thalassemia or sickle cell anemia. 如請求項16之用途，其中： (i)該藥物中之細胞係在投與個體之前擴增；(ii)該藥物中之細胞係在骨髓移植中投與至個體，其中所述細胞在活體內植入、分化並且成熟；及/或(iii)在引入包含該藥物的移植物之前，個體係經輕度骨髓淨除程序(mild myeloablative procedure)或強力骨髓淨除處理方案(vigorous myeloablative conditioning regimen)治療。 As used in claim 16, where: (i) the cell line in the medicament is expanded prior to administration to the subject; (ii) the cell line in the medicament is administered to the subject in a bone marrow transplant, wherein the cells engraft, differentiate and mature in vivo; and /or (iii) the individual system was treated with a mild myeloablative procedure or a vigorous myeloablative conditioning regimen prior to introduction of the graft containing the drug. 如請求項18之用途，其中：(i)該醫藥組合物中之細胞係在投與個體之前擴增；(ii)該醫藥組合物中之細胞係在骨髓移植中投與至個體，其中所述細胞在活體內植入、分化並且成熟；及/或(iii)在引入包含該醫藥組合物的移植物之前，個體係經輕度骨髓淨除程序或強力骨髓淨除處理方案治療。 The use of claim 18, wherein: (i) the cell line in the pharmaceutical composition is expanded prior to administration to the individual; (ii) the cell line in the pharmaceutical composition is administered to the individual in bone marrow transplantation, wherein the The cells are engrafted, differentiated and matured in vivo; and/or (iii) the individual system is treated with a mild myeloablative procedure or a vigorous myeloablative treatment regimen prior to introduction into a graft comprising the pharmaceutical composition. 一種套組，其包含如請求項1之鋅指核酸酶、如請求項2至4中任一項之聚核苷酸及/或如請求項6至8中任一項之細胞或如請求項14之醫藥組合物。 A kit comprising a zinc finger nuclease as claimed in claim 1, a polynucleotide as in any one of claims 2 to 4 and/or a cell as in any one of claims 6 to 8 or as claimed in any one of The pharmaceutical composition of 14.
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Urnov, Fyodor D., et al. "Genome editing with engineered zinc finger nucleases." Nature Reviews Genetics 11.9 (2010): 636-646. *

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