TWI698526B

TWI698526B - Feeder-free derivation of human-induced pluripotent stem cells with synthetic messenger rna

Info

Publication number: TWI698526B
Application number: TW104117573A
Authority: TW
Inventors: 繼武王
Original assignee: 美商綠陽生物科技及製藥公司
Priority date: 2014-05-30
Filing date: 2015-05-29
Publication date: 2020-07-11
Also published as: KR102361849B1; PH12016502343A1; WO2015184318A1; SG11201609898WA; TW201600605A; CA2950582A1; KR20170023014A; IL249193B; CA2950582C; IL249193A0

Abstract

The present disclosure relates generally to novel methods and compositions for using engineered reprogramming factor(s) for the creation of induced pluripotent stem cells (iPSCs) through a kinetically controlled process. Specifically, this disclosure relates to establishing combinations of reprogramming factors, including fusions between conventional reprogramming factors with transactivation domains, optimized for reprogramming various types of cells. More specifically, the exemplary methods disclosed herein can be used for creating induced pluripotent stem cells from various mammalian cell types, including human fibroblasts. Exemplary methods of feeder-free derivation of human induced pluripotent stem cells using synthetic messenger RNA are also disclosed.

Description

利用合成之信使RNA進行之人類誘導型多能幹細胞之無餵養細胞衍化(DERIVATION) Feeder-free derivation of human induced pluripotent stem cells using synthetic messenger RNA (DERIVATION)

相關申請案Related applications

本申請案主張2013年5月13日申請之美國申請案序號13/893,166之權益，且為該案之部份接續申請案；其主張2012年5月13日申請之美國臨時申請案申請案序號61/646,292之優先權。各案件之內容係以其全文引用的方式併入本文中。 This application claims the rights and interests of the U.S. application serial number 13/893,166 filed on May 13, 2013, and is part of the continuation application; it claims the U.S. provisional application serial number filed on May 13, 2012 61/646,292 priority. The content of each case is incorporated into this article by way of reference in its entirety.

本發明大體上係關於使用工程化重新編程因子透過控制過程建立誘導型多能幹細胞(iPSC)之之新穎方法及組合物。具體言之，本發明係關於建立經最佳化以用於重新編程各種細胞類型之重新編程因子之組合(包括習知重新編程因子與轉活化域之間之融合)。更具體言之，本文所揭示之例示性方法可用於自各種哺乳動物細胞類型(包括非人類的靈長類動物細胞)建立誘導型多能幹細胞。亦揭示使用合成之信使RNA進行人類誘導型多能幹細胞之無餵養細胞衍化(derivation)之例示性方法。 The present invention generally relates to a novel method and composition for establishing induced pluripotent stem cells (iPSC) through a controlled process using engineered reprogramming factors. Specifically, the present invention relates to the establishment of a combination of reprogramming factors optimized for reprogramming various cell types (including the fusion between conventional reprogramming factors and transactivation domains). More specifically, the exemplary methods disclosed herein can be used to establish induced pluripotent stem cells from various mammalian cell types, including non-human primate cells. An exemplary method for feeder-free derivation of human induced pluripotent stem cells using synthetic messenger RNA is also disclosed.

下文包括可用於瞭解本發明之各種態樣及實施例之資訊。並非承認本文所提供之任何資訊為先前技術，或與當前所描述之或所主張之發明相關，或具體或含蓄引入之任何公開案或文獻為先前技術。 The following includes information that can be used to understand various aspects and embodiments of the present invention. It is not an acknowledgement that any information provided in this article is prior art, or is incompatible with what is currently described or claimed Any publications or documents related to the invention, or specifically or implicitly introduced, are prior art.

誘導型多能幹細胞(iPSC)之治療潛力促使致力於開發迴避需要基因修飾體細胞以實現重新編程至多能狀態之重新編程方法。於此點上達成成就之第一「非整合」方法(蛋白質轉導、質體轉染及使用腺病毒載體)由於所達成iPSC轉化之低效率所致而在應用上受到限制。近來，已顯示使用基因附體型DNA(episomal DNA)、仙台病毒(Sendai virus)及合成之信使RNA(mRNA)的技術以類似於或優於彼等使用整合病毒載體所達成者之效率產生「無足跡(footprint-free)」iPSC。原則上，RNA轉染因其可精確控制重新編程因子(RF)表現時程同時完全迴避「清潔」經重新編程之細胞以清除殘餘痕量之載體之任何需求而成為此等方法中最具吸引力者。然而，基於mRNA之重新編程的當前方案相當地勞動密集，因為需要持續~2週每天進行再轉染，該時間係誘導人類細胞之多能性所需。此等程序亦仰賴於餵養細胞之使用，此增加過程之複雜性及技術可變性，同時引入受非人類衍化(「異源物(xeno))之生物材料污染之可能來源。 The therapeutic potential of induced pluripotent stem cells (iPSC) has prompted efforts to develop reprogramming methods that avoid the need to genetically modify somatic cells to achieve reprogramming to a pluripotent state. The first "non-integrated" method (protein transduction, plastid transfection, and use of adenoviral vectors) to achieve this point is limited in application due to the low efficiency of the achieved iPSC transformation. Recently, techniques using episomal DNA, Sendai virus, and synthetic messenger RNA (mRNA) have been shown to produce "nothing" with efficiency similar to or better than those achieved by using integrated viral vectors. Footprint (footprint-free)" iPSC. In principle, RNA transfection is the most attractive of these methods because it can precisely control the reprogramming factor (RF) performance schedule while completely avoiding any need to "clean" the reprogrammed cells to remove residual traces of vectors. Powerhouse. However, the current protocol based on mRNA reprogramming is quite labor intensive, as it requires daily retransfection for ~2 weeks, which is required to induce pluripotency of human cells. These procedures also rely on the use of feeder cells, which increases the complexity and technical variability of the process, and at the same time introduces possible sources of biological material contamination by non-human derivatives ("xeno).

產生誘導型多能幹細胞(iPSC)之主要困難為重新編程分化細胞成多能細胞之低效率。先前已報告小鼠胚胎纖維母細胞(MEF)中有5%在連同Sox2、Klf4及c-Myc(SKM)利用包含Oct4及MyoD之轉活化域(稱作M3O)之融合基因轉導時重新編程成iPSC。此外，M3O促進大多數經轉導MEF(包括不變成iPSC之細胞)中之多能性基因之染色質重塑。此等觀察顯示細胞中超過5%已獲得在給予更有利培養條件下變成iPSC之能力之可能性。 The main difficulty in generating induced pluripotent stem cells (iPSC) is the low efficiency of reprogramming differentiated cells into pluripotent cells. It has previously been reported that 5% of mouse embryonic fibroblasts (MEF) are reprogrammed when transduced with Sox2, Klf4, and c-Myc (SKM) using a fusion gene containing Oct4 and MyoD transactivation domain (called M3O) Into iPSC. In addition, M3O promotes chromatin remodeling of pluripotency genes in most transduced MEFs (including cells that do not become iPSCs). These observations show that more than 5% of the cells have acquired the possibility of becoming iPSC under more favorable culture conditions.

使用mRNA成功重新編程非人類起源的細胞尚屬未知，由於RNA分子在其轉染至細胞(特定言之哺乳動物細胞)中時所誘導之可能細胞免疫反應的緣故。在重新編程人類纖維母細胞中，常用的操作係使用病毒蛋白質作為誘餌受體以鈍化由經轉染mRNA分子誘導之干擾素。然而，此使用誘餌受體之策略或其他類似策略在非人類物種(包括狒狒、馬及狗)之纖維母細胞之重新編程中尚未取得成功。 The use of mRNA to successfully reprogram cells of non-human origin is unknown due to the possible cellular immune response induced by RNA molecules when they are transfected into cells (in particular mammalian cells). In reprogramming human fibroblasts, the commonly used operating system uses The viral protein acts as a decoy receptor to inactivate the interferon induced by the transfected mRNA molecule. However, this strategy using decoy receptors or other similar strategies has not been successful in the reprogramming of fibroblasts of non-human species (including baboons, horses, and dogs).

因此，為解決此等不足之處，本發明提供用於產生能夠產生身體所有不同組織之幹細胞之方法及組合物。在某些態樣中，使用信使RNA分子且不需要病毒載體或餵養細胞，本文所揭示之方法可用於重新編程非人類纖維母細胞成誘導型多能幹細胞(iPSC)。使用例示性方法及組合物，達成優於先前報告之細胞重新編程方法之驚人且出人意料之改良效率，及克服先前尚未解決的抑制非人類哺乳動物細胞中之細胞免疫反應之問題。 Therefore, in order to solve these shortcomings, the present invention provides methods and compositions for generating stem cells that can produce all different tissues of the body. In some aspects, messenger RNA molecules are used and no viral vectors or feeder cells are required, the method disclosed herein can be used to reprogram non-human fibroblasts into induced pluripotent stem cells (iPSC). Using exemplary methods and compositions, it achieves amazing and unexpected improvement efficiency over previously reported cell reprogramming methods, and overcomes the previously unresolved problem of inhibiting cellular immune responses in non-human mammalian cells.

因此，提供可用於藉由改良重新編程因子(RF)混合物(cocktail)(尤其透過施用習知重新編程因子(諸如Oct4(亦稱作Oct3/4)、Sox2等)之併有已知強轉錄因子(諸如VP16及MyoD)之轉活化域之工程化變體)來促進mRNA介導之重新編程之方法、試劑及/或組合物。本文所揭示之方法及組合物達成顯著減少基於mRNA的重新編程中所涉及之時間、成本及努力之無餵養細胞方案。 Therefore, provide a cocktail that can be used to improve the reprogramming factor (RF) (especially through the administration of conventional reprogramming factors (such as Oct4 (also known as Oct3/4), Sox2, etc.)) and have known strong transcription factors (Engineered variants of the transactivation domain such as VP16 and MyoD) methods, reagents and/or compositions to promote mRNA-mediated reprogramming. The methods and compositions disclosed herein achieve a feeder cell-free solution that significantly reduces the time, cost, and effort involved in mRNA-based reprogramming.

在一個態樣中，本發明提供一種用於使體細胞去分化或重新編程之方法，該方法包括：a)利用包含有效量之選自Oct4、Sox2、Klf4、cMyc、Nanog及Lin28之合成性mRNA重新編程因子中之任何一者或多者與轉活化域之融合產物的組合物轉染經分離之體細胞，藉以使得體細胞重新編程或去分化。 In one aspect, the present invention provides a method for dedifferentiation or reprogramming of somatic cells, the method comprising: a) using an effective amount of synthetic properties selected from Oct4, Sox2, Klf4, cMyc, Nanog and Lin28 The combination of any one or more of the mRNA reprogramming factors and the fusion product of the transactivation domain is transfected into the isolated somatic cell, thereby reprogramming or dedifferentiating the somatic cell.

在一個實施例中，提供技術方案1之方法，其中該組合物包含融合至N端MyoD轉活化域之Oct4。在一個實施例中，該Oct4係以三重串接方式(in tandem triplicates)融合至N-端MyoD轉活化域。 In one embodiment, the method of technical solution 1 is provided, wherein the composition comprises Oct4 fused to the N-terminal MyoD transactivation domain. In one embodiment, the Oct4 is fused to the N-terminal MyoD transactivation domain in in tandem triplicates.

在一個態樣中，提供一種藉由使用如技術方案1之合成性mRNA重新編程因子中之一或多者重新編程哺乳動物細胞之方法，該方法包括：a)以15k至500k個細胞/標準6孔盤孔之密度使標靶細胞生長於無餵養細胞表面；b)在重新編程期間每次利用可變劑量之50ng至800ng/ml mRNA轉染細胞。 In one aspect, there is provided a method for reprogramming mammalian cells by using one or more of the synthetic mRNA reprogramming factors as in Technical Solution 1, the method includes Including: a) Growing target cells on the surface of feeder-free cells at a density of 15k to 500k cells/standard 6-well plate; b) Transfection with variable doses of 50ng to 800ng/ml mRNA each time during reprogramming cell.

在一個實施例中，該等標靶細胞係以30k、75k、100k或150k個細胞/標準6孔盤孔之密度生長於無餵養細胞表面；b)在重新編程期間每次利用可變劑量之50ng至800ng/ml mRNA轉染細胞，而較早時間點所使用之劑量低於較晚時間點；c)在未繼代培養下獲得iPSC。 In one embodiment, the target cell lines are grown on the surface of feeder-free cells at a density of 30k, 75k, 100k or 150k cells per standard 6-well plate; b) each time a variable dose is used during reprogramming. Cells were transfected with 50ng to 800ng/ml mRNA, and the dose used at the earlier time point was lower than the later time point; c) iPSC was obtained without subculture.

在一個實施例中，該等標靶細胞係以15k、75k、100k或150k個細胞/標準6孔盤孔之密度生長於無餵養細胞表面，而各孔之體積係經調整為介於0.5ml與5ml適宜培養基之間；在重新編程期間每次利用可變劑量之50ng至800ng/ml mRNA轉染細胞，而較早時間點所使用之劑量低於較晚時間點；在未繼代培養下獲得iPSC。 In one embodiment, the target cell lines are grown on the surface without feeding cells at a density of 15k, 75k, 100k or 150k cells/standard 6-well plate hole, and the volume of each well is adjusted to be between 0.5ml Between and 5ml of suitable medium; during reprogramming, cells are transfected with a variable dose of 50ng to 800ng/ml mRNA each time, and the earlier time point is lower than the later time point; under non-subculture Get iPSC.

在某些實施例中，在整個重新編程過程中使用較低劑量減小細胞免疫反應且獲得改良之iPSC成功率。在其他實施例中，使用較高純度之mRNA分子(亦即，不存在來自活體外轉錄之污染性異常性轉錄本)允許細胞以較低密度接種且在重複轉染下存活及較高iPSC產率。 In certain embodiments, lower doses are used throughout the reprogramming process to reduce cellular immune responses and obtain improved iPSC success rates. In other embodiments, the use of higher purity mRNA molecules (that is, the absence of contaminating abnormal transcripts from in vitro transcription) allows cells to be seeded at a lower density and survive repeated transfection and higher iPSC production. rate.

在一個實施例中，該等哺乳動物細胞為人類細胞。在一個實施例中，該方法係無外來物(Xeno-free)。在一個實施例中，該等哺乳動物細胞為非人類的靈長類動物細胞。在一個實施例中，該等非人類的靈長類動物細胞為馬來猴細胞。 In one embodiment, the mammalian cells are human cells. In one embodiment, the method is Xeno-free. In one embodiment, the mammalian cells are non-human primate cells. In one embodiment, the non-human primate cells are Malay monkey cells.

在一個實施例中，該一或多種因子係選自由mRNAs、調節RNA、siRNA、miRNA及其組合組成之群。 In one embodiment, the one or more factors are selected from the group consisting of mRNAs, regulatory RNA, siRNA, miRNA, and combinations thereof.

在一個實施例中，利用至少兩種不同RNA轉染該等體細胞。在一個實施例中，該等體細胞係選自係選自由單能、多潛能(multipotent)、多能及分化細胞組成之群。在一個實施例中，該一或多種RNA誘導該等體細胞去分化成單能、多潛能或多能細胞。 In one embodiment, the somatic cells are transfected with at least two different RNAs. In one embodiment, the somatic cell lines are selected from the group consisting of unipotent, multipotent, multipotent and differentiated cells. In one embodiment, the one or more RNAs induce the somatic cells to dedifferentiate into unipotent, pluripotent or pluripotent cells.

在一個實施例中，該等因子中之至少一者係選自由OCT4、SOX2、NANOG、LIN28、KLF4及MYC mRNA組成之群。在一個實施例中，該OCT4、SOX2、NANOG及LIN28 mRNA係組合投與。在一個實施例中，該OCT4、SOX2、KLF4及MYC mRNA係組合投與。 In one embodiment, at least one of the factors is selected from the group consisting of OCT4, SOX2, NANOG, LIN28, KLF4, and MYC mRNA. In one embodiment, the OCT4, SOX2, NANOG and LIN28 mRNA are administered in combination. In one embodiment, the OCT4, SOX2, KLF4 and MYC mRNA are administered in combination.

在一個實施例中，該等經轉染細胞係維持在培養物中作為誘導型多能幹(iPS)細胞。在一個實施例中，該等經轉染細胞形成誘導型多能幹細胞，進一步包括誘導iPS細胞形成分化細胞。 In one embodiment, the transfected cell lines are maintained in culture as induced pluripotent stem (iPS) cells. In one embodiment, the formation of induced pluripotent stem cells by the transfected cells further includes inducing iPS cells to form differentiated cells.

在一個態樣中，提供一種治療或抑制患者之疾病或病症之一或多種症狀之方法，該方法包括在活體外使細胞去分化及對該患者投與該等細胞。在一個實施例中，該組合物進一步包含Rarg及LrH-1交換活化域(transaction activation domains)。在一個實施例中，該組合物包含融合至VP16轉活化域之OCT4。 In one aspect, a method of treating or inhibiting one or more symptoms of a disease or condition in a patient is provided, the method comprising dedifferentiating cells in vitro and administering the cells to the patient. In one embodiment, the composition further comprises Rarg and LrH-1 transaction activation domains. In one embodiment, the composition comprises OCT4 fused to the transactivation domain of VP16.

在一個實施例中，本發明提供衍化自非人類的靈長類動物細胞之iPSC，其係用於建立疾病模型系統，因為其可產生無其他動物產物之非整合、無餵養細胞測試環境。因此，本文所述之非人類iPSC適用於臨床前測試。 In one embodiment, the present invention provides iPSC derived from non-human primate cells, which is used to establish a disease model system because it can produce a non-integrated, feeder cell-free test environment without other animal products. Therefore, the non-human iPSC described herein is suitable for preclinical testing.

本文描述並主張的發明具有許多屬性及實施例，包括(但不限於)彼等記述或描述或引用於本【發明內容】中者。無意包括全部及本文描述並主張的發明不限於或受限於本【發明內容】所述之特徵或實施例，其之包含僅基於例示之目的而非限制性。其他實施例可揭示於下文【實施方式】中。 The invention described and claimed herein has many attributes and embodiments, including (but not limited to) those described or described or cited in the [Summary of the Invention]. It is not intended to include all and the invention described and claimed herein is not limited or limited to the features or embodiments described in the [Summary of the Invention], and its inclusion is only for the purpose of illustration and not limitation. Other embodiments can be disclosed in the following [Implementation Mode].

圖1. 利用基於M3O的mRNA重新編程混合物衍化的iPSC集落。 (A)衍化自第一基於M3O的BJ重新編程試驗之兩個經擴增iPSC純系之10×明視野影像。(B)經擴增純系之用於多能性標記之免疫染色。 Figure 1. iPSC colonies derived from M3O-based mRNA reprogramming mixture. (A) 10× bright-field images derived from two amplified iPSC clones of the first M3O-based BJ reprogramming experiment. (B) Immunostaining for pluripotency markers of amplified clones.

圖2. 使用基於M3O的混合物之無餵養細胞重新編程。Figure 2. Feeder-free cell reprogramming using M3O-based mixture.

(A)顯示從50K XFF纖維母細胞上無餵養細胞衍化獲得之TRA-1-60⁺集落產率並比較基於c-Myc及L-Myc的混合物及4小時及24小時轉染方案之免疫螢光成像。對所有孔轉染9天。在實驗的第15天對4小時轉染培養物固定染色，在第11天對24小時轉染培養物固定染色。(B)相同實驗之400ng/ml Stemfect孔之10×明視野成像，其顯示在衍化的第9天超越培養物之接近長滿之似hESC集落。(C)其中使用400ng/ml Stemfect方案再次轉染100K XFF 9天之隨訪試驗中之經標記域之10×明視野時程，顯示上皮形成及於隨後出現似hESC菌落。 (A) Shows the yield of TRA-1-60 ⁺ colonies derived from feeder-free cells on 50K XFF fibroblasts and compares immunofluorescence based on the mixture of c-Myc and L-Myc and the 4-hour and 24-hour transfection protocol Light imaging. Transfect all wells for 9 days. The 4-hour transfected culture was fixed and stained on the 15th day of the experiment, and the 24-hour transfected culture was fixed and stained on the 11th day. (B) 10× bright-field imaging of 400ng/ml Stemfect wells of the same experiment, which showed that the nearly overgrown hESC-like colonies beyond the culture on the 9th day of derivation. (C) The 10× bright field time course of the labeled domain in the 9-day follow-up trial of 100K XFF retransfected with a 400ng/ml Stemfect protocol, showing epithelial formation and subsequent appearance of hESC-like colonies.

圖3. 使用4種不同mRNA混合物之重新編程效率之比較。流程圖概述四種混合物比較實驗。 Figure 3. Comparison of reprogramming efficiency using 4 different mRNA mixtures. The flow chart outlines the four mixture comparison experiments.

圖4. HDF-a無餵養細胞重新編程培養中之似hESC集落。在自經使用Stemfect轉染試劑以培養基補充劑形式遞送之400ng/ml mRNA混合物(M3O+ c-Myc⁺ Nanog⁺)處理9天之75K HDF-a成體纖維母細胞之無餵養細胞衍化第9天時的出現的似hESC集落之10×明視野影像。 Figure 4. HDF-a feeder-free reprogramming of hESC-like colonies in culture. On the 9th day of derivation of 75K HDF-a adult fibroblasts without feeder cells treated with 400ng/ml mRNA mixture (M3O+c-Myc ⁺ Nanog ⁺ ) delivered in the form of medium supplement with Stemfect transfection reagent for 9 days 10× bright-field image of hESC-like colonies appearing at the time.

圖5. 合成性mRNA混合物之產生。(A)概述製造mRNA重新編程混合物之程序之示意圖。(B)編碼SYBR E-凝膠上之若干RF及螢光報導子之合成性mRNA。每個通道中載入500ng RNA。 Figure 5. Production of synthetic mRNA mixture. (A) A schematic diagram outlining the procedure for making the mRNA reprogramming mixture. (B) Synthetic mRNA encoding several RF and fluorescent reporters on SYBR E-gel. Load 500ng RNA in each channel.

圖6. 使用用於重新編程混合物之2-硫基尿嘧啶產生合成性mRNA混合物。合成性mRNA係編碼SYBR E-凝膠上之若干RF。該等mRNA之10%尿嘧啶鹼基經2-硫基尿嘧啶置換。每個通道載入100ng RNA。 Figure 6. Using 2-thiouracil for the reprogramming mix to generate a synthetic mRNA mix. Synthetic mRNA codes for several RFs on the SYBR E-gel. 10% of the uracil bases of these mRNAs were replaced with 2-thiouracil. Load 100ng RNA per channel.

圖7. 使用具有經2-硫基尿嘧啶修飾之mRNA之mRNA混合物所建立的人類iPSC。在Pluriton(A)或Allele重新編程培養基(B)中進行不同重新編程操作期間重新編程(A)7天或(B)11天後的人類iPSC群體。 Figure 7. Human iPSC established using a mixture of mRNAs with 2-thiouracil-modified mRNA. Human iPSC populations reprogrammed (A) 7 days or (B) 11 days after different reprogramming operations in Pluriton (A) or Allele reprogramming medium (B).

圖8. 在無餵養細胞重新編程培養中使用mRNA混合物所建立的馬來猴iPSC集落。照片中所顯示為在第10天及第13天之出現的似 hESC集落之10×明視野影像。在第10天的通道中，接近中心的細胞展現自纖維母細胞至幹細胞之形態改變。在第13天的通道中，該等細胞形成完全轉形之猴iPSC之大集落。 Figure 8. Malay monkey iPSC colonies established using mRNA mixtures in feeder-free cell reprogramming culture. The photo shows the 10× bright-field images of hESC-like colonies that appeared on the 10th and 13th days. In the passage on day 10, cells near the center exhibited morphological changes from fibroblasts to stem cells. In the passage on day 13, these cells formed large colonies of fully transformed monkey iPSCs.

在描述本發明時，本文中未定義的所有術語具有其為此項技藝認可之含義。在以下描述為本發明之特定實施例或特定用途之程度上，希望僅係例示性，而非所主張發明之限制。以下描述意欲涵蓋包含於本發明之精神及範疇中之所有替代物、修改物及等效物。 When describing the present invention, all terms not defined herein have their meanings recognized by the art. To the extent that the following descriptions are specific embodiments or specific uses of the present invention, it is intended to be illustrative only and not to limit the claimed invention. The following description is intended to cover all alternatives, modifications, and equivalents included in the spirit and scope of the present invention.

分化細胞可藉由所選轉錄因子群組之表現恢復至多能狀態，開啟患者特異性細胞可用於產生任何所需類型細胞以供活體外遺傳性疾病之研究且最終供細胞替代療法之前景。該等重新編程因子之表現可透過應用整合至基因組中之病毒載體來達成，而iPSC衍化仍經常使用整合性逆轉錄病毒或慢病毒進行。基因組之隨附修飾代表iPSC之治療應用之重要障礙，而即使活體外研究仍需考量經過整合之病毒序列盒進行再活化表現之可能性。最近，已在減緩或消除基因組修飾問題之新穎表現載體之應用方面取得相當顯著進步。現可獲得一種慢病毒載體，其編碼於側接lox重組位點之單一多順反子序列盒中誘導iPSC所需之多種因子，因而容許在透過過渡表現Cre重組酶重新編程後，幾近無縫切除轉基因。亦可使用轉位子載體接著短暫表現轉位酶來***轉基因及後續之切除。已使用若干不同類型之非整合性DNA載體，其等可過渡表現重新編程因子持續一段足以誘導多能性之時間，包括腺病毒、質體及基因附體型DNA。亦已證實可藉由併有細胞穿透肽之重組RF蛋白質重複轉導細胞產生iPSC，但效率低。現可使用具有完全基於RNA的生殖週期之仙台病毒，及藉由持續轉染編碼山中因子(Yamanaka factor)之合成性mRNA轉錄本來達成相當有效之iPSC轉化。 Differentiated cells can be restored to a pluripotent state by the performance of selected transcription factor groups. Turning on patient-specific cells can be used to generate any desired types of cells for the study of in vitro genetic diseases and ultimately for the prospect of cell replacement therapy. The performance of these reprogramming factors can be achieved through the application of viral vectors integrated into the genome, and iPSC derivation is still often performed using integrated retroviruses or lentiviruses. The accompanying modification of the genome represents an important obstacle to the therapeutic application of iPSC, and even in vitro studies still need to consider the possibility of reactivation of the integrated viral sequence cassette. Recently, significant progress has been made in the application of novel expression vectors to alleviate or eliminate genome modification problems. A lentiviral vector is now available, which encodes in a single polycistronic cassette flanking the lox recombination site to induce a variety of factors required for iPSC, thus allowing the reprogramming of Cre recombinase through transitional expression, almost GMO is removed seamlessly. It is also possible to use transposon vectors followed by transient expression of the translocase for insertion of the transgene and subsequent excision. Several different types of non-integrated DNA vectors have been used, which can over-express reprogramming factors for a period of time sufficient to induce pluripotency, including adenovirus, plastid and gene appendage type DNA. It has also been confirmed that the recombinant RF protein with cell penetrating peptide can be used to repeatedly transduce cells to produce iPSC, but the efficiency is low. Now it is possible to use Sendai virus with a completely RNA-based reproductive cycle, and to achieve quite effective iPSC transformation by continuously transfecting synthetic mRNA transcripts encoding Yamanaka factor.

將mRNA轉染應用於重新編程(及有潛力應用於主導分化及轉分化)係吸引人的作法，因為此系統可以每天簡單地藉由改變添加至細胞培養基之轉錄本來調節重新編程混合物及甚至個別組分因子之表現。一旦終止特定因子之轉染，於標靶細胞內之異位性表現會因細胞質中mRNA之快速衰減而立刻停止。與非整合性DNA載體或RNA病毒對比，mRNA轉染既不需要清潔，也不會存在隨機基因組整合或持續性病毒感染之任何風險。假若設想最終可能使用多次異位性RF表現操作，從患者活檢經由iPSC中間物進行至所需類型之特化細胞時，此等優點應具有更高重要性。然而，目前實施之基於mRNA的重新編程存在缺陷。雖然RF之表現在轉染mRNA後通常穩健約24小時，但因子表現需要約兩週來誘導人類細胞之多能性，因此，採用此技術使細胞重新編程所需要的操作時間(hands-on time)相當長。並非所有細胞類型及培養基同等地利於有效之mRNA傳遞，及目前此係所述特定細胞類型(包括血液細胞)之基於mRNA的重新編程之障礙。迄今，亦已證實使用經有絲***遏制之纖維母細胞餵養層以使用mRNA方法成功使細胞重新編程為iPSC之必要性。此等餵養細胞緩衝培養物之群體密度，因為標靶細胞在iPSC誘導所需之延長時程中自低起始密度生長，甚至超出RNA及轉染試劑(兩者均具有關聯毒性)之傳遞劑量及支持標靶細胞在面對藉由重新編程過程所產生之促細胞凋亡及細胞生長抑制力之存活率。此要求增加程序之複雜性及操作時間並引入技術可變性之重要來源，尤其假若餵養細胞本身進行轉染。餵養細胞層之存在亦有礙於重新編程過程之監測及分析。最終，雖然人類餵養細胞目前係mRNA重新編程之標準，但當非人類的動物產物用於其衍化及擴增時甚至此等細胞為異源物生物污染之可能來源。 The application of mRNA transfection to reprogramming (and potentially leading differentiation and transdifferentiation) is an attractive approach, because this system can adjust the reprogramming mixture and even individually by simply changing the transcripts added to the cell culture medium every day The performance of component factors. Once the transfection of the specific factor is terminated, the heterotopic expression in the target cell will immediately stop due to the rapid attenuation of mRNA in the cytoplasm. In contrast to non-integrated DNA vectors or RNA viruses, mRNA transfection requires neither cleaning nor any risk of random genome integration or persistent viral infection. If it is envisaged that it is possible to eventually use multiple atopic RF performance procedures, from patient biopsy through iPSC intermediates to the required types of specialized cells, these advantages should be more important. However, currently implemented mRNA-based reprogramming has drawbacks. Although the expression of RF is usually robust for about 24 hours after transfection of mRNA, it takes about two weeks for factor expression to induce the pluripotency of human cells. Therefore, the hands-on time required for cell reprogramming with this technology ) Is quite long. Not all cell types and media are equally conducive to effective mRNA delivery, and this is currently an obstacle to mRNA-based reprogramming of the specific cell types (including blood cells). So far, it has also been confirmed that the use of a mitotically suppressed fibroblast feeding layer is necessary to successfully reprogram cells into iPSCs using the mRNA method. The population density of these buffered cultures of feeding cells, because the target cells grow from a low starting density during the prolonged time course required for iPSC induction, even exceeding the delivery dose of RNA and transfection reagents (both of which have associated toxicity) And to support the survival rate of target cells in the face of pro-apoptosis and cell growth inhibitory forces produced by the reprogramming process. This requirement increases the complexity of the procedure and operation time and introduces an important source of technical variability, especially if the feeder cells themselves are transfected. The presence of the feeder cell layer also hinders the monitoring and analysis of the reprogramming process. Ultimately, although human feeder cells are currently the standard for mRNA reprogramming, when non-human animal products are used for their derivation and amplification, even these cells are possible sources of foreign biological contamination.

因此，有鑑於與先前已知之程序相關聯之問題，本文中提供新穎方法、材料及及方案以產生具有改良之重新編程效率及改良之所得細胞品質之iPSC。本發明實施例成功地用於透過增強傳遞至細胞之RF混合物達成顯著驚人且意外之改良。本發明實施例亦提供新穎的方案，該等方案壓縮及精簡mRNA重新編程過程，及支持無需使用餵養細胞或任何其他潛在性地經異源物污染之試劑自人類纖維母細胞產生無足跡iPSC。本文所提供之新穎方法及組合物將延伸先前已知之mRNA方法的優點及幫助清除iPSC技術之治療應用之其餘障礙。 Therefore, in view of the problems associated with previously known procedures, this article provides novel methods, materials, and solutions to generate improved reprogramming efficiency and improved gains Cell quality iPSC. The examples of the present invention have been successfully used to achieve remarkable, surprising and unexpected improvements by enhancing the RF mixture delivered to cells. The embodiments of the present invention also provide novel solutions that compress and streamline the mRNA reprogramming process, and support the generation of footprintless iPSCs from human fibroblasts without the use of feeding cells or any other potentially contaminated reagents. The novel methods and compositions provided herein will extend the advantages of previously known mRNA methods and help clear the remaining obstacles to the therapeutic application of iPSC technology.

本發明大體上係關於使用工程化重新編程因子透過動力學控制過程建立誘導型多能幹細胞(iPSC)之方法。更具體言之，本發明係關於建立經最佳化以用於重新編程不同類型之細胞之重新編程因子之組合(包括習知重新編程因子與轉活化域之融合)；將此等因子以合成之信使RNA(mRNA)形式以較佳密度藉由導致適宜程度之轉基因表現之方法引入至培養之哺乳動物細胞中；維持細胞於所界定條件下以獲得重新編程之先前難以達成之效率。與此項技藝中已知之其他方法相比，本發明顯著縮短重新編程中涉及之時間、成本及努力，視需要完全無餵養細胞且無異源物，及無需繼代培養。本文所揭示之材料及程序可用於自不同類型之哺乳動物細胞(包括人類纖維母細胞)建立誘導型多能幹細胞。 The present invention generally relates to a method for establishing induced pluripotent stem cells (iPSC) through a dynamic control process using engineered reprogramming factors. More specifically, the present invention relates to the establishment of a combination of reprogramming factors optimized for reprogramming different types of cells (including the fusion of conventional reprogramming factors and transactivation domains); these factors are synthesized The messenger RNA (mRNA) form is introduced into cultured mammalian cells at a better density by a method that results in a suitable degree of transgene expression; the cells are maintained under defined conditions to obtain reprogramming efficiency that was previously difficult to achieve. Compared with other methods known in the art, the present invention significantly shortens the time, cost, and effort involved in reprogramming, completely free of feeder cells and foreign matter, and does not require subculture as needed. The materials and procedures disclosed herein can be used to establish induced pluripotent stem cells from different types of mammalian cells (including human fibroblasts).

本發明之態樣亦提供藉由不需要病毒載體、動物產物或餵養細胞下使用信使RNA分子產生能夠產生人體之許多不同組織之幹細胞之方法。本文所揭示之新穎方法可用於使人類纖維母細胞在最佳條件下重新編程成具有驚人且意外之效率之誘導型多能幹細胞(iPSC)。 Aspects of the present invention also provide a method for generating stem cells capable of producing many different tissues of the human body by using messenger RNA molecules without the need for viral vectors, animal products or feeder cells. The novel method disclosed herein can be used to reprogram human fibroblasts under optimal conditions into induced pluripotent stem cells (iPSC) with surprising and unexpected efficiency.

本發明提供以編碼本文所述之例示性重新編程因子之「清潔」mRNA之混合物處理非人類的哺乳動物細胞(例如纖維母細胞)之有用的方法及組合物。可使用該mRNA組合物而不引起經處理之細胞經歷與mRNA分子之引入相關聯之細胞死亡。在一個實施例中，該等非人類的哺乳動物細胞為馬來猴細胞。在另一實施例中，該等馬來猴細胞以某一梯度接種於孔中使得細胞以可變局部密度生長。 The present invention provides useful methods and compositions for treating non-human mammalian cells (e.g., fibroblasts) with a mixture of "cleaning" mRNAs encoding the exemplary reprogramming factors described herein. The mRNA composition can be used without causing the treated cells to experience cell death associated with the introduction of mRNA molecules. In one embodiment, the non-human mammalian cells are Malay monkey cells. In another embodiment, the male monkey cells Seeding in the wells with a certain gradient allows the cells to grow at variable local density.

定義definition

如本文所用，適合與該方法共同使用之細胞包括(但不限於)初級細胞及已建立之細胞系、胚胎細胞、免疫細胞、幹細胞及分化細胞(包括(但不限於)衍化自外胚層、內胚層及中胚層(包括纖維母細胞、實質細胞、造血細胞及上皮細胞)之細胞)。如本文所用，幹細胞包括單能細胞、多潛能細胞及多能細胞；胚胎幹細胞及成體幹細胞，諸如造血幹細胞、間質幹細胞、上皮幹細胞、及肌肉衛星細胞。在一個實施例中，體細胞係經去分化或重新編程。可使用任何適宜之體細胞。代表性體細胞包括纖維母細胞、角質細胞、脂肪細胞、肌細胞、器官及組織細胞及各種血液細胞(包括(但不限於)造血細胞(包括造血幹細胞及提供短期或長期造血移植之細胞))。最佳之細胞類型包括(但不限於)人類纖維母細胞、角質細胞及造血幹細胞。該等方法尤其可用於使細胞去分化或視情況再分化，而無需永久性修改細胞基因組。 As used herein, cells suitable for use with the method include (but are not limited to) primary cells and established cell lines, embryonic cells, immune cells, stem cells and differentiated cells (including but not limited to) derived from ectoderm, endoderm Germ and mesoderm (including fibroblasts, parenchymal cells, hematopoietic cells and epithelial cells). As used herein, stem cells include unipotent cells, pluripotent cells, and pluripotent cells; embryonic stem cells and adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells, and muscle satellite cells. In one embodiment, the somatic cell line is dedifferentiated or reprogrammed. Any suitable somatic cell can be used. Representative somatic cells include fibroblasts, keratinocytes, adipocytes, muscle cells, organ and tissue cells and various blood cells (including but not limited to hematopoietic cells (including hematopoietic stem cells and cells that provide short-term or long-term hematopoietic transplantation)) . The best cell types include (but are not limited to) human fibroblasts, keratinocytes and hematopoietic stem cells. These methods are particularly useful for de-differentiating or redifferentiating cells as appropriate without permanently modifying the cell genome.

可用於所揭示方法之RNA包括mRNA、調節RNA或小RNA諸如siRNA或miRNA，其中該一或多種mRNA編碼發揮使細胞去分化或重新編程之作用之多肽。轉染之效率極高。通常，經轉染細胞群體之90%將表現誘導型RNA。因此，可用一或多種不同RNA轉染細胞。例如，可用一或多種不同mRNA、一或多種不同siRNA、一或多種不同miRNA或其組合轉染細胞群體。可以單次投藥方式同時用多種RNA轉染細胞群體，或可數分鐘、數小時、數天或數週地交錯多次投藥。可交錯多種不同RNA之轉染。例如，假若期望第一RNA在一或多種其他RNA之表現之前進行表現。 The RNA that can be used in the disclosed method includes mRNA, regulatory RNA or small RNA such as siRNA or miRNA, where the one or more mRNAs encode polypeptides that function to dedifferentiate or reprogram cells. The transfection efficiency is extremely high. Generally, 90% of the transfected cell population will express inducible RNA. Therefore, cells can be transfected with one or more different RNAs. For example, a population of cells can be transfected with one or more different mRNAs, one or more different siRNAs, one or more different miRNAs, or a combination thereof. The cell population can be transfected with multiple RNAs at the same time in a single administration, or multiple administrations can be staggered in minutes, hours, days or weeks. Can stagger the transfection of multiple different RNAs. For example, if the first RNA is expected to be expressed before the expression of one or more other RNAs.

經轉染RNA之表現程度可藉由改變輸入RNA的量在寬範圍內控制，從而可個別地調節各經轉染RNA之表現程度。基於所需結果基礎上來確定輸入RNA之有效量。另外，mRNA生成之基於PCR的技術有利於具有不同結構及域組合之mRNA之設計。可用於本發明方法之RNA為此項技術中已知，及將基於標靶宿主細胞類型及意欲控制之通路或細胞活性或治療應用來選擇。可基於已知基因、mRNA或其他核苷酸或蛋白質序列構築可用於去分化細胞，例如轉化成體經分化體細胞成幹細胞之構築體。 The degree of expression of the transfected RNA can be controlled in a wide range by changing the amount of input RNA, so that the degree of expression of each transfected RNA can be individually adjusted. Determine the effective amount of input RNA based on the desired result. In addition, PCR-based techniques for mRNA generation include Facilitate the design of mRNAs with different structures and domain combinations. The RNA that can be used in the method of the present invention is known in the art and will be selected based on the target host cell type and the pathway or cell activity or therapeutic application to be controlled. Based on known genes, mRNA or other nucleotide or protein sequences, constructs that can be used to dedifferentiate cells, such as transforming into differentiated somatic cells into stem cells, can be constructed.

本文中可互換使用之術語「多核苷酸」及「核酸」係指任何長度之核苷酸之聚合形式(核糖核苷酸或脫氧核糖核苷酸)。因此，此術語包括(但不限於)單股、雙股或多股DNA或RNA、基因組DNA、cDNA、DNA-RNA混合物或包含嘌呤及嘧啶鹼基或其他天然、化學或生物修飾之非天然或衍化核苷酸鹼基之聚合物。「寡核苷酸」一般係指單股或雙股DNA之在約5與約100個核苷酸之間之多核苷酸。然而，基於本發明之目的，對寡核苷酸之長度沒有上限。寡核苷酸亦稱作寡聚物，及可自基因分離，或藉由此項技術中已知的方法化學合成。 The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to polymerized forms (ribonucleotides or deoxyribonucleotides) of nucleotides of any length. Therefore, this term includes (but is not limited to) single-stranded, double-stranded or multiple-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA mixtures, or unnatural or non-natural or non-natural or biological modifications that contain purine and pyrimidine bases or other natural, chemical or biological modifications. Polymer of derivatized nucleotide bases. "Oligonucleotides" generally refer to polynucleotides of between about 5 and about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purpose of the present invention, there is no upper limit on the length of the oligonucleotide. Oligonucleotides are also called oligomers, and can be isolated from genes or chemically synthesized by methods known in the art.

如本文所用，術語「微型RNA」係任何類型的干擾RNA，包括(但不限於)內源性微型RNA及人工微型RNA(例如，合成之miRNA)。內源性微型RNA係於基因組中自然編碼之能夠調節mRNA之生產性利用之小RNA。人工微型RNA可係除內源性微型RNA之外之能夠調節mRNA之活性之任何類型之RNA序列。微型RNA序列可係包含此等序列中之任何一者或多者之RNA分子。 As used herein, the term "microRNA" refers to any type of interfering RNA, including (but not limited to) endogenous microRNA and artificial microRNA (eg, synthetic miRNA). Endogenous microRNAs are small RNAs naturally encoded in the genome that can regulate the productive utilization of mRNA. The artificial microRNA can be any type of RNA sequence capable of regulating the activity of mRNA other than endogenous microRNA. A microRNA sequence can be an RNA molecule that includes any one or more of these sequences.

「微型RNA前驅物」(或「前miRNA」)係指其中併有微型RNA序列之具有幹環(stem-loop)結構之核酸。「成熟微型RNA」(或「成熟miRNA」)包括已自微型RNA前驅物(「前miRNA」)裂解或已合成(例如，在實驗室藉由無細胞合成法合成)，且具有約19個核苷酸至約27個核苷酸長度，例如，成熟微型RNA可具有19nt、20nt、21nt、22nt、23nt、24nt、25nt、26nt或27nt長度之微型RNA。成熟微型RNA可結合至標靶mRNA且抑制標靶mRNA之轉譯。 "MicroRNA precursor" (or "premiRNA") refers to a nucleic acid with a stem-loop structure in which a microRNA sequence is incorporated. "Mature microRNA" (or "mature miRNA") includes a microRNA precursor ("premiRNA") that has been cleaved or synthesized (for example, synthesized by a cell-free synthesis method in the laboratory) and has about 19 nuclei Glucose acid to about 27 nucleotides in length, for example, a mature microRNA may have a length of 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, or 27nt. Mature microRNA can bind to target mRNA and inhibit the translation of target mRNA.

OCT4之例示性基因組mRNA(cDNA)及蛋白質序列為此項技術中已知，參見例如(OCT4)POU5F1 POU第5類同源異型盒(homeobox)[現代人(Homo sapiens)]基因ID：5460，其提供mRNA(cDNA)序列基因庫(Genbank)寄存編號NM-001173531.1，標題為現代人POU第5類同源異型盒1(POU5F1)，轉錄本變體3，mRNA；基因庫寄存編號NM-002701.4，標題為現代人POU第5類同源異型盒1(POU5F1)轉錄本變體1，mRNA；及基因庫寄存編號NM-203289.4，標題為現代人POU第5類同源異型盒1(POU5F1)，轉錄本變體2，mRNA。SOX2之例示性基因組mRNA(cDNA)及蛋白質序列亦為此項技術中已知，參見例如SOX2 SRY(性別決定區Y)-盒2[現代人]，基因ID：6657，其提供mRNA(cDNA)序列基因庫寄存編號NM-003106.2，標題為mRNA序列現代人SRY(性別決定區Y)-盒2(SOX2)，mRNA。NANOG之例示性基因組mRNA(cDNA)及蛋白質序列亦為此項技術中已知，參見例如NANOG Nanog同源異型盒[現代人]，基因ID：79923，其提供mRNA(cDNA)序列基因庫寄存編號NM-024865.2，標題為現代人Nanog同源異型盒(NANOG)，mRNA。LIN28之例示性基因組mRNA(cDNA)及蛋白質序列亦為此項技術中已知，參見例如LIN28A同系物A(隱桿線蟲(C.elegans))[現代人]，基因ID：79727，其提供mRNA(cDNA)序列Genbank寄存編號NM-024674.4，標題為現代人lin-28同系物A(隱桿線蟲)(LIN28A)，mRNA。KLF4之例示性基因組mRNA(cDNA)及蛋白質序列為此項技術中已知，參見例如KLF4 Kruppel樣因子4(腸)[現代人]，基因ID：9314，其提供mRNA(cDNA)序列基因庫寄存編號NM-004235.4，標題為現代人Kruppel樣因子4(腸)(KLF4)，mRNA。MYC之mRNA序列亦為此項技術中已知，參見(例如)MYC v-myc髓細胞組織增生病毒致癌基因同系物(鳥類)[現代人]，基因ID：4609，其提供mRNA(cDNA)序列基因庫寄存編號NM- 002467.4，標題為現代人v-myc髓細胞組織增生病毒致癌基因同系物(鳥類)(MYC)，mRNA。 Exemplary genomic mRNA (cDNA) and protein sequences of OCT4 are known in the art, see, for example, (OCT4) POU5F1 POU Class 5 homeobox [modern human ( Homo sapiens )] Gene ID: 5460, It provides mRNA (cDNA) sequence gene bank (Genbank) accession number NM-001173531.1, titled modern human POU class 5 homeobox 1 (POU5F1), transcript variant 3, mRNA; gene bank accession number NM-002701.4 , The title is modern human POU type 5 homeobox 1 (POU5F1) transcript variant 1, mRNA; and gene bank accession number NM-203289.4, the title is modern human POU type 5 homeobox 1 (POU5F1) , Transcript variant 2, mRNA. Exemplary genomic mRNA (cDNA) and protein sequences of SOX2 are also known in the art, see, for example, SOX2 SRY (sex determining region Y)-box 2 [modern people], gene ID: 6657, which provides mRNA (cDNA) Sequence gene bank accession number NM-003106.2, titled mRNA sequence modern human SRY (sex determining region Y)-box 2 (SOX2), mRNA. The exemplary genomic mRNA (cDNA) and protein sequences of NANOG are also known in the art, see, for example, NANOG Nanog Homeobox [Modern People], Gene ID: 79923, which provides the mRNA (cDNA) sequence gene bank registration number NM-024865.2, titled Modern Human Nanog Homeobox (NANOG), mRNA. Exemplary genomic mRNA (cDNA) and protein sequences of LIN28 are also known in the art, see, for example, LIN28A homolog A ( C.elegans ) [modern people], gene ID: 79727, which provides mRNA (cDNA) sequence Genbank accession number NM-024674.4, titled modern human lin-28 homolog A (cryptobacter elegans) (LIN28A), mRNA. Exemplary genomic mRNA (cDNA) and protein sequences of KLF4 are known in the art, see for example KLF4 Kruppel-like factor 4 (intestine) [modern people], gene ID: 9314, which provides mRNA (cDNA) sequence gene library deposit No. NM-004235.4, titled modern human Kruppel-like factor 4 (gut) (KLF4), mRNA. The mRNA sequence of MYC is also known in this technology, see (for example) MYC v-myc myeloid tissue proliferation virus oncogene homolog (avian) [modern man], gene ID: 4609, which provides mRNA (cDNA) sequence The gene bank deposit number is NM-002467.4, and the title is modern human v-myc myeloid tissue proliferation virus oncogene homolog (birds) (MYC), mRNA.

「幹環結構」係指包括已知或預測形成由主要單股核苷酸(環部分)之區域連接於一側上之雙股(階梯部分)之核苷酸之區域具有次級結構之核酸。本文中亦使用術語「髮夾(hairpin)」及「折回(fold-back)」結構以指幹環結構。此等結構為此項技術中所熟知且此等術語始終以其在此項技術中已知的含義使用。幹環結構內核苷酸之實際初級序列對實踐本發明並非關鍵，只要存在次級結構即可。如此項技術中已知，次級結構不需要確切鹼基配對。因此，幹可包括一或多個鹼基錯配。或者，鹼基配對可係確切的，亦即，不包括任何錯配。 "Dry loop structure" refers to a nucleic acid with a secondary structure in a region including a region of nucleotides known or predicted to form a double strand (stepped portion) connected to one side by a region of primary single stranded nucleotides (loop portion) . The terms "hairpin" and "fold-back" structure are also used herein to refer to the stem loop structure. These structures are well known in the art and these terms are always used in their known meanings in the art. The actual primary sequence of nucleotides within the stem loop structure is not critical to the practice of the invention, as long as there is a secondary structure. As is known in the art, the secondary structure does not require exact base pairing. Therefore, stems can include one or more base mismatches. Alternatively, the base pairing may be exact, that is, not including any mismatches.

如本文所用，術語「幹細胞」係指可經誘導以增殖之未分化細胞。幹細胞能夠自穩定(self-maintenance)，意味著就每次細胞***而言，一個子細胞亦將為幹細胞。幹細胞可獲自胚胎組織、胎兒組織、出生後組織、幼年組織或成體組織。如本文所用，術語「前身細胞」係指衍化自幹細胞之未分化細胞，而自身不係幹細胞。一些前身細胞可產生能夠分化為多於一種細胞類型之子代。 As used herein, the term "stem cell" refers to undifferentiated cells that can be induced to proliferate. Stem cells can be self-maintenance, which means that for each cell division, a daughter cell will also be a stem cell. Stem cells can be obtained from embryonic tissue, fetal tissue, postnatal tissue, juvenile tissue or adult tissue. As used herein, the term "precursor cell" refers to an undifferentiated cell derived from a stem cell, but not a stem cell itself. Some precursor cells can produce offspring that can differentiate into more than one cell type.

如本文所用，術語「誘導型多能幹細胞」(或「iPS細胞」)係指自體細胞(例如，分化體細胞)誘導之且具有比該體細胞高的效力的幹細胞。iPS細胞能夠自行更新且分化為成熟細胞，例如，平滑肌細胞。iPS可亦能夠分化為平滑肌前身細胞。 As used herein, the term "induced pluripotent stem cells" (or "iPS cells") refers to stem cells that are induced by autologous cells (eg, differentiated somatic cells) and have higher potency than the somatic cells. iPS cells can renew themselves and differentiate into mature cells, for example, smooth muscle cells. iPS can also differentiate into smooth muscle precursor cells.

如本文所用，術語「經分離」在關於細胞時係指處在不同於該細胞自然產生之環境之環境中之細胞，例如，在該細胞自然產生於多細胞生物中之情況下，及自該多細胞生物移去該細胞，該細胞係「經分離」。經分離之基因修飾宿主細胞可存在於基因修飾宿主細胞之混合群體、或存在於包含基因修飾宿主細胞及未基因修飾之宿主細胞之混合群體中。例如，經分離之基因修飾宿主細胞可存在於活體外基因修飾宿主細胞之混合物群體、或存在於包含基因修飾宿主細胞及未基因修飾之宿主細胞之活體外混合群體中。 As used herein, the term "isolated" in relation to a cell refers to a cell that is in an environment different from the environment in which the cell is naturally produced, for example, when the cell is naturally produced in a multicellular organism, and from that Multicellular organisms remove the cell and the cell line is "isolated." The isolated genetically modified host cells may exist in a mixed population of genetically modified host cells, or in a mixed population including genetically modified host cells and unmodified host cells. For example, the isolated genetically modified host cell can exist in vitro. A mixed population of modified host cells, or an in vitro mixed population comprising genetically modified host cells and non-genetically modified host cells.

如本文所用，「宿主細胞」表示活體內或活體外細胞(例如，經培養作為單細胞實體之真核細胞)(該真核細胞可用作或已用作用於核酸(例如，外源核酸)之接受者)且包括該初始細胞之已經過核酸基因修飾之子代。應明瞭由於自然、意外或精密突變的緣故，單一細胞之子代在形態或基因組或總DNA互補上可不必與初始親本完全相同。 As used herein, "host cell" means a cell in vivo or in vitro (e.g., a eukaryotic cell cultured as a single cell entity) (the eukaryotic cell can be used or has been used for nucleic acid (e.g., exogenous nucleic acid) The recipient) and includes the progeny of the original cell that has been genetically modified by nucleic acid. It should be understood that due to natural, accidental or precise mutations, the progeny of a single cell need not be identical in morphology or genome or total DNA complementation to the original parent.

術語「基因修飾」係指細胞中在引入新核酸(即，細胞外源核酸)後誘導之永久性或過渡性基因改變。基因改變(「修飾」)可藉由併入新核酸至宿主細胞之基因組中，或藉由過渡性或穩定維持新核酸作為染色體外元件來達成。在細胞為真核細胞之情況下，永久性基因改變可藉由引入核酸至細胞之基因組中來達成。基因修飾之適宜方法包括病毒感染、轉染、接合作用、原生質體融合、電穿孔、粒子槍技術、磷酸鈣沉澱、直接微量注射及類似方法。 The term "genetic modification" refers to a permanent or transitional genetic change induced in a cell after the introduction of a new nucleic acid (ie, an exogenous nucleic acid from the cell). Genetic changes ("modifications") can be achieved by incorporating new nucleic acids into the genome of the host cell, or by transitional or stable maintenance of the new nucleic acids as extrachromosomal elements. In the case of a eukaryotic cell, permanent genetic changes can be achieved by introducing nucleic acid into the cell's genome. Suitable methods for gene modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection and similar methods.

如本文所用，術語「外源核酸」係指非通常或自然存在於細胞中及/或實際上非由細胞產生之核酸及/或引入至細胞中(例如，藉由電穿孔、轉染、感染、脂質體轉染或引入核酸至細胞中之任何其他手段)之核酸。 As used herein, the term "exogenous nucleic acid" refers to nucleic acid that is not normally or naturally present in a cell and/or is not actually produced by the cell and/or introduced into the cell (for example, by electroporation, transfection, infection , Liposome transfection or any other means of introducing nucleic acid into cells) nucleic acid.

如本文所用，術語「治療」及類似語係指獲得所需藥理學及/或生理學效應。該效應在完全或部分預防疾病或其症狀方面可係預防性及/或在部分或完全治癒疾病及/或可歸因於疾病之不利效應方面可係治療性。 As used herein, the term "treatment" and similar terms refers to obtaining the desired pharmacological and/or physiological effect. The effect may be preventive in terms of completely or partially preventing the disease or its symptoms and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects attributable to the disease.

如本文所用，「治療」包括針對哺乳動物(特別是人)之疾病之任何治療，且包括：(a)預防該疾病在可能易患該疾病但尚未確診為罹患該疾病之個體中發生；(b)抑制該疾病，亦即，阻止其發展；及(c)緩解該疾病，亦即，引起該疾病消退。 As used herein, "treatment" includes any treatment for a disease in mammals (especially humans), and includes: (a) preventing the disease from occurring in individuals who may be susceptible to the disease but have not yet been diagnosed with the disease; b) inhibit the disease, that is, prevent its development; and (c) alleviate the disease, that is, cause the disease to resolve.

本文中可互換使用之術語「個體(individual)」、「個體(subject)」、「宿主」及「患者」係指哺乳動物，包括(但不限於)人類、非人類的靈長類動物、嚙齒動物(例如，小鼠、大鼠等)、有蹄類動物、犬科、兔科、貓科等。在一些實施例中，所述個體為人類。在一些實施例中，個體為非人類的非人類的動物，諸如猴、嚙齒動物或兔科。 The terms "individual", "subject", "host" and "patient" used interchangeably herein refer to mammals, including (but not limited to) humans, non-human primates, rodents Animals (e.g., mice, rats, etc.), ungulates, canines, rabbits, cats, etc. In some embodiments, the individual is a human. In some embodiments, the individual is a non-human, non-human animal, such as a monkey, a rodent, or a lago.

「治療有效量」或「有效量」意指當投與個體以治療疾病時足以實現此疾病治療之化合物、核酸或許多細胞的量。「治療有效量」將視化合物或細胞、疾病及其嚴重度及待治療個體之年齡、體重等而變化。 "Therapeutically effective amount" or "effective amount" means the amount of a compound, nucleic acid, or many cells that is sufficient to achieve treatment of the disease when administered to an individual to treat the disease. The "therapeutically effective amount" will vary depending on the compound or cell, the disease and its severity, and the age and weight of the individual to be treated.

在進一步描述本發明之前，應明瞭本發明不限於所述的特定實施例，因而當然可改變。亦應明瞭用於本文中之術語僅出於描述特定實施例之目的而無意具限制性，因為本發明之範疇將僅受限於隨附申請專利範圍。 Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and therefore can of course be changed. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive, because the scope of the present invention will only be limited to the scope of the appended patents.

在提供某一範圍的值情況下，應明瞭本發明中包含介於該範圍之上限與下限之間之除非本文清楚地另作指明否則至下限之十分之一單位之各個中介值及該所述範圍中之任何其他所述或中介值。此等較小範圍之上限及下限可獨立地包含於該等較小範圍中，且亦包含於本發明中，包括所述範圍中任何明確排除之限值。在所述範圍包含限值中一者或兩者之情況下，排除彼等所包含限值中任一者或兩者之範圍亦包含於本發明中。 In the case of providing a certain range of values, it should be understood that the present invention includes each intermediate value between the upper limit and the lower limit of the range, unless otherwise clearly specified herein, to a tenth unit of the lower limit and the Any other stated or intermediate value in the stated range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, including any explicitly excluded limit in the stated range. In the case where the stated range includes one or both of the limit values, the range excluding any one or both of the included limit values is also included in the present invention.

除非另外定義，否則本文中使用的所有技術及科學術語具有本發明所屬技術之一般人員所共同瞭解的相同含義。雖然類似於或等效於彼等述於本文中者之任何方法及材料亦可用於實踐或測試本發明，然而，現仍描述較佳之方法及材料。本文所述之所有公開案以引用的方式併入本文中以揭示並描述可結合來引述公開案之方法及/或材料。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary persons of the technology to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are still described. All publications described herein are incorporated herein by reference to disclose and describe methods and/or materials that can be combined to quote publications material.

在本發明之一個態樣中，基於mRNA的重新編程可透過使用Oct4或Sox 2之併有N端MyoD轉活化域(Hirai等人，Stem Cells，2011)或C端三重重複之VP16轉活化域(Wang等人，EMBO Reports，2011；其中合成之重新編程因子係藉由使VP16轉活化域分別融合至OCT4(亦稱作Pou5f1)、NANOG及SOX2製得，此等合成因子可以增強之效率及加速之動力學重新編程小鼠及人類纖維母細胞二者)之工程化變體，或藉由以兩種其他因子Rarg及Lrh-1強化「標準」RF混合物(Wang等人，PNAS，2011)增強。各案件之內容係以引用的方式併入本文中。強轉錄激動子可在結合至DNA之特定位點時有效募集多種染色質重塑複合物。一良好的實例為MyoD，其係可切換分化細胞之命運之用於骨骼肌發生之主要轉錄因子。Hirai等人推測，由於MyoD為此一強轉錄因子，故可增加對iPS因子(假若融合在一起)之染色質可接近性。當連同Sox2及Klf4用帶有Oct-MyoD TAD融合基因之逆轉錄載體轉導小鼠或人類細胞時，與典型iPS因子相比，其等使得iPSC集落之數目增加~50倍。類似地，廣為人知之穩健轉錄激動子VP16可在融合至不同iPS因子時展現對重新編程的強刺激效應。 In one aspect of the present invention, mRNA-based reprogramming can be achieved by using Oct4 or Sox 2 with an N-terminal MyoD transactivation domain (Hirai et al., Stem Cells, 2011) or a C-terminal triple repeat VP16 transactivation domain (Wang et al., EMBO Reports, 2011; The synthetic reprogramming factor is prepared by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors can enhance the efficiency and Accelerated kinetics to reprogram engineered variants of both mouse and human fibroblasts), or by enhancing the "standard" RF mixture with two other factors Rarg and Lrh-1 (Wang et al., PNAS, 2011) Enhanced. The content of each case is incorporated into this article by reference. Strong transcriptional agonists can effectively recruit multiple chromatin remodeling complexes when they bind to specific sites in DNA. A good example is MyoD, which is the main transcription factor for skeletal myogenesis that can switch the fate of differentiated cells. Hirai et al. speculate that because MyoD is a strong transcription factor, it can increase chromatin accessibility to iPS factors (if fused together). When combined with Sox2 and Klf4, the reverse transcription vector with Oct-MyoD TAD fusion gene was used to transduce mouse or human cells, compared with typical iPS factors, they increased the number of iPSC colonies by ~50 times. Similarly, the well-known robust transcription agonist VP16 can exhibit a strong stimulating effect on reprogramming when fused to different iPS factors.

人類iPSC之例示性製法Exemplary preparation method of human iPSC

該方法亦可廣泛用於使細胞再分化或重新編程，(例如)以產生可進一步經調節以形成以下細胞之iPS細胞：造血幹細胞、間質幹細胞、上皮幹細胞及肌肉衛星細胞、或人類組織之分化細胞，包括(但不限於)紅血球、白血球(包括淋巴細胞)、血小板、基質細胞、脂肪細胞、骨骼細胞(包括破骨細胞)、上皮組織(包括皮膚細胞)、肌肉組織(包括平滑肌、骨骼肌及心肌)、血管組織(包括內皮細胞)、肝組織(包括肝細胞)及神經組織(包括神經元)。誘導iPS細胞分化成各種分化細胞類型(包括(但不限於)心肌細胞、造血幹細胞、骨骼細胞(諸如破骨細胞)、肝細胞、視網膜細胞及神經元、幹細胞(包括(但不限於)經分離之胚胎幹細胞、造血幹細胞及誘導型多能幹細胞))之方法可藉由用誘導分化之RNA過渡性轉染來誘導分化。另外或另一選擇，細胞可藉由在細胞類型特異性條件下培養該等細胞而再分化。例如，iPS細胞可維持於CF-1餵養細胞上且接著使其適應於無餵養細胞條件。iPS細胞可藉由在noggin、Dkk-1及IGF-1之存在下培養該等細胞，而誘導形成分化視網膜細胞。 This method can also be widely used to re-differentiate or reprogram cells, for example, to generate iPS cells that can be further regulated to form the following cells: hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells and muscle satellite cells, or of human tissue Differentiated cells, including (but not limited to) red blood cells, white blood cells (including lymphocytes), platelets, stromal cells, adipocytes, skeletal cells (including osteoclasts), epithelial tissues (including skin cells), muscle tissues (including smooth muscle, bone) Muscle and myocardium), vascular tissue (including endothelial cells), liver tissue (including hepatocytes) and nerve tissue (including neurons). Induce iPS cells to differentiate into various differentiated cell types (including but not limited to cardiomyocytes, hematopoietic stem cells, skeletal cells (such as osteoclasts) Cells), hepatocytes, retinal cells and neurons, stem cells (including (but not limited to) isolated embryonic stem cells, hematopoietic stem cells and induced pluripotent stem cells)) methods can be achieved by transitional transfection with induced differentiation RNA To induce differentiation. Alternatively or alternatively, the cells can be redifferentiated by culturing the cells under cell type specific conditions. For example, iPS cells can be maintained on CF-1 feeder cells and then adapted to feeder cell-free conditions. iPS cells can be induced to form differentiated retinal cells by culturing these cells in the presence of noggin, Dkk-1 and IGF-1.

先前報導的方法仰賴於整合載體，即，病毒或質體，以帶有經修飾之因子。在一個實施例中，進行重新編程試驗來比較包含用於mRNA重新編程之五種因子(Oct4、Sox2、Klf4、cMyc-T58A及Lin28)之轉錄本之6種不同mRNA組合混合物或包括Rarg及Lrh-1之7種重新編程因子RF混合物之性能，各組合係以基於野生型Oct4及MyoD-及VP16-Oct4融合構築體(分別命名為M3O及VPx3)之三種變化進行測試。在基於餵養細胞的重新編程培養物中轉染BJ纖維母細胞11天，此時，若干孔中出現進化形態。在接下來的數天，具有特徵性hESC形態之集落出現於經基於野生型Oct4及M3O的混合物轉染之孔中。雖然經VPx3轉染之培養物中之標靶細胞維持纖維母細胞形態，但同時顯示加速之生長及聚集成集落(foci)之一定傾向，且未出現集落。基於野生型Oct4及M3O的混合物之5-因子及7-因子實施例顯示相似的集落生產率，因此，於混合物中包含Rarg及Lrh-1未產生優點。然而，M3O混合物所產生集落數目為使用野生型Oct4時的若干倍。根據此結果，自M3O 5-因子孔挑選集落用於擴增及進一步分析(圖1)。藉由細胞核及細胞表面標記之免疫染色，及藉由活體外分化成三種主要胚芽層來證實M3O衍化的集落之多能性。對6種經擴增iPSC集落進行核型分析(karyotype analysis)及DNA指紋術，可在所有情況中證實該等細胞的核型正常態及BJ細胞譜系。 The previously reported methods rely on integrating vectors, ie, viruses or plastids, with modified factors. In one embodiment, a reprogramming test is performed to compare 6 different mRNA combination mixtures containing transcripts of five factors (Oct4, Sox2, Klf4, cMyc-T58A, and Lin28) used for mRNA reprogramming or including Rarg and Lrh -1 The performance of the 7 reprogramming factor RF mixtures, each combination was tested with three changes based on wild-type Oct4 and MyoD- and VP16-Oct4 fusion constructs (named M3O and VPx3, respectively). BJ fibroblasts were transfected in feeder cell-based reprogramming cultures for 11 days, at which time, evolutionary morphology appeared in several wells. In the next few days, colonies with characteristic hESC morphology appeared in wells transfected with a mixture based on wild-type Oct4 and M3O. Although the target cells in the cultures transfected with VPx3 maintained the morphology of fibroblasts, they also showed accelerated growth and a certain tendency to aggregate into foci, and no colonies appeared. The 5-factor and 7-factor examples based on a mixture of wild-type Oct4 and M3O showed similar colony productivity, therefore, including Rarg and Lrh-1 in the mixture did not produce advantages. However, the number of colonies produced by the M3O mixture was several times that of the wild-type Oct4. Based on this result, colonies were selected from the M3O 5-factor well for amplification and further analysis (Figure 1). The multipotency of colonies derived from M3O was confirmed by immunostaining of cell nucleus and cell surface markers, and by differentiation into three main germ layers in vitro. The karyotype analysis and DNA fingerprinting of the 6 amplified iPSC colonies can confirm the normal karyotype and BJ cell lineage of these cells in all cases.

該方法亦可廣泛用於使細胞再分化或重新編程(例如)以產生可進一步經調節以形成以下細胞之iPS細胞：造血幹細胞、間質幹細胞、上皮幹細胞及肌肉衛星細胞、或人類組織之經分化細胞，包括(但不限於)紅血球細胞、白血球細胞(包括淋巴細胞)、血小板、基質細胞、脂肪細胞、骨骼細胞(包括破骨細胞)、上皮組織(包括皮膚細胞)、肌肉組織(包括平滑肌、骨骼肌及心肌)、血管組織(包括內皮細胞)、肝臟組織(包括肝細胞)及神經組織(包括神經元)。誘導iPS細胞分化成各種分化細胞類型(包括(但不限於)心肌細胞、造血幹細胞、骨骼細胞(諸如破骨細胞)、肝細胞、視網膜細胞及神經元)之方法係此項技術中已知(Song等人，Cell Res.,19(11)：1233-42(2009),Lamba等人，PLoS One,5(1)：e8763(2010),Gai等人，Cell Biol Int.200933(11)：1184-93(2009).Grigoriadis等人，Blood,115(14)：2769-76(2010))。包括(但不限於)經分離之胚胎幹細胞、造血幹細胞及誘導型多能幹細胞之幹細胞可藉由用誘導分化之RNA過渡性轉染經誘導以分化。另外或另一選擇，細胞可藉由在細胞類型特異性條件下培養該等細胞再分化。例如，iPS細胞可維持於CF-1餵養細胞上且接著使其適應於無餵養細胞條件。iPS細胞可藉由在noggin、Dkk-1及IGF-1之存在下培養該等細胞誘導以形成分化視網膜細胞。在另一態樣中，mRNA混合物之效力可進一步藉由包含Nanog轉錄本增強。在此實施例中，四個含有於餵養細胞上之50K BJ纖維母細胞之孔用基於野生型Oct4或M3O的5-因子或6-因子混合物轉染6天，及接著對各培養1：6繼代培養至新鮮餵養細胞上以填滿6-孔盤(4)。各板內之轉染再持續0至5天。在第18天(其中第0天對應於第一轉染)固定該等培養並用TRA-1-60抗體染色以評估不同混合物及轉染時程對iPSC生產率的影響。結果顯示不論所使用的Oct4變體添加Nanog至混合物極度有益，同時當在M3O與Nanog一起使用時達成最大的轉化效率。 This method can also be widely used to redifferentiate or reprogram cells (for example) to generate iPS cells that can be further regulated to form the following cells: hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells and muscle satellite cells, or human tissues. Differentiated cells, including (but not limited to) red blood cells, white blood cells (including lymphocytes), platelets, stromal cells, adipocytes, skeletal cells (including osteoclasts), epithelial tissues (including skin cells), muscle tissues (including smooth muscle) , Skeletal muscle and cardiac muscle), vascular tissue (including endothelial cells), liver tissue (including hepatocytes) and nerve tissue (including neurons). Methods for inducing iPS cells to differentiate into various differentiated cell types (including but not limited to cardiomyocytes, hematopoietic stem cells, skeletal cells (such as osteoclasts), hepatocytes, retinal cells and neurons) are known in the art ( Song et al., Cell Res. , 19(11): 1233-42 (2009), Lamba et al., PLoS One , 5(1): e8763 (2010), Gai et al., Cell Biol Int. 200933(11): 1184-93 (2009). Grigoriadis et al., Blood , 115(14): 2769-76 (2010)). Stem cells including (but not limited to) isolated embryonic stem cells, hematopoietic stem cells, and induced pluripotent stem cells can be induced to differentiate by transitional transfection with differentiated RNA. Alternatively or alternatively, the cells can be re-differentiated by culturing the cells under cell type-specific conditions. For example, iPS cells can be maintained on CF-1 feeder cells and then adapted to feeder cell-free conditions. iPS cells can be induced by culturing these cells in the presence of noggin, Dkk-1 and IGF-1 to form differentiated retinal cells. In another aspect, the effectiveness of the mRNA mixture can be further enhanced by the inclusion of Nanog transcripts. In this example, four wells containing 50K BJ fibroblasts on feeder cells were transfected with 5-factor or 6-factor mixture based on wild-type Oct4 or M3O for 6 days, and then cultured for each 1:6 Subculture onto fresh feeder cells to fill the 6-well plate (4). Transfection in each plate continued for another 0 to 5 days. The cultures were fixed on day 18 (wherein day 0 corresponds to the first transfection) and stained with TRA-1-60 antibody to evaluate the effects of different mixtures and transfection schedules on iPSC productivity. The results show that it is extremely beneficial to add Nanog to the mixture regardless of the Oct4 variant used, while achieving maximum conversion efficiency when M3O is used together with Nanog.

在一個實施例中，基於M3O的5-因子或6-因子混合物之效力在使用三種其他人類纖維母細胞系(HDF-f、HDF-n及XFF)之其他基於餵養細胞的實驗中獲得證實。就所有此等三種低繼代培養細胞系而言，重新編程動力學及效率顯著改良，此展現天然群體加倍時間比正常擴增培養中之BJ快。在某些情況中，吾人從少至6天的轉染獲得似hESC集落，然而，在轉染多持續數天的實驗中產率高得多。涉及藉由添加新鮮細胞週期性強化餵養細胞層之實驗顯示雖然此策略可提供某些優點，但其等將因所得方案之複雜性而有所抵消。因此吾人決定集中於應用更有效的混合物以開發精簡無餵養細胞方案。 In one example, the efficacy of the M3O-based 5-factor or 6-factor mixture was confirmed in other feeder cell-based experiments using three other human fibroblast cell lines (HDF-f, HDF-n and XFF). For all these three low-subculture cell lines, reprogramming kinetics and efficiency are significantly improved, which shows that the natural population doubles faster than BJ in normal expansion culture. In some cases, we obtained hESC-like colonies from as little as 6 days of transfection, however, the yield was much higher in experiments where transfection lasted more than a few days. Experiments involving periodic strengthening of the feeder cell layer by adding fresh cells have shown that although this strategy can provide certain advantages, they will be offset by the complexity of the resulting solution. So we decided to focus on the application of more effective mixtures to develop a streamlined feeder-free regimen.

本發明係關於建立無餵養細胞iPSC。獨立於餵養細胞之iPSC衍化已大體上證實與使用的重新編程技術無關之一定挑戰性，而且增加持續轉染方案內容中之特定困難。對接種纖維母細胞而不折損細胞存活率及增殖活性之密度限制較小。當藉由重新編程因子之轉染及藉由重新編程因子之異位性表現對細胞施加應力時，該等細胞在稀疏培養中經歷有絲***遏制或細胞凋亡之傾向增大。除此之外，在基於餵養細胞的重新編程之高細胞密度特性下具良好耐受性之RNA劑量在分佈於較少數細胞中時產生較嚴重細胞毒性效應。同時，可藉由mRNA轉染達成之表現之外顯率在纖維母細胞達到長滿後顯著下降，可能歸因於與接觸抑制及G1遏制相關聯之內吞作用之下調。簇集培養中之此低轉染允許度似乎在重新編程期間於細胞經歷間質至上皮轉變(MET)後減緩。然而，通常，即使細胞以最低可存活初始密度接種，在使用當前mRNA混合物時人類纖維母細胞達成MET所需的~7天亦極難消除纖維母細胞過度生長之問題。細胞可藉由繼代培養稀釋以延遲此命運，但極難預測高應力之重新編程中間物之接種效率，及在任何情況中，基於繼代培養的衍化方案將不良地犧牲高通量應用之方便性及規模。在某些實施例中，細胞亦可以某一梯度接種於孔中使得不同局部細胞密度可促進重複轉染。 The present invention is about establishing feeder cell-free iPSC. The iPSC derivation independent of feeder cells has generally proven to be a certain challenge independent of the reprogramming technique used, and adds to the specific difficulties in the content of the continuous transfection protocol. The density of inoculation of fibroblasts without compromising cell survival and proliferation activity is less restricted. When stress is exerted on cells by transfection of reprogramming factors and by the ectopic performance of reprogramming factors, these cells have an increased tendency to undergo mitotic suppression or apoptosis in sparse culture. In addition, RNA doses that are well tolerated under the high cell density characteristics based on reprogramming of feeder cells produce more severe cytotoxic effects when distributed in a smaller number of cells. At the same time, the out-of-performance that can be achieved by mRNA transfection decreases significantly after fibroblasts reach fullness, which may be due to the down-regulation of endocytosis associated with contact inhibition and G1 suppression. This low transfection tolerance in cluster culture seems to slow down after cells undergo mesenchymal to epithelial transition (MET) during reprogramming. However, in general, even if the cells are seeded at the lowest viable initial density, the ~7 days required for human fibroblasts to reach MET when using the current mRNA mixture is extremely difficult to eliminate the problem of overgrowth of fibroblasts. Cells can be diluted by subculture to delay this fate, but it is extremely difficult to predict the inoculation efficiency of high-stress reprogramming intermediates, and in any case, a subculture-based derivation scheme will undesirably sacrifice high-throughput applications. Convenience and scale. In some embodiments, the cells can also be seeded in the wells with a certain gradient so that different local Cell density can promote repeated transfection.

本發明係關於MET之表型指標(纖維母細胞性過程之退化及集落及卵石形態之出現)。在一個實施例中，藉由本發明，使用經增強混合物，使用6-因子M3O混合物無需繼代培養下實現無餵養細胞重新編程，以多種低密度(每孔具有50K相對100K相對150K)接種標靶細胞，來加速MET。在另一實施例中，該等細胞係以每孔總細胞數在自約15K至500K個細胞範圍之密度梯度接種。 The present invention relates to phenotypic indicators of MET (degeneration of fibroblastic process and appearance of colony and pebble morphology). In one embodiment, with the present invention, the enhanced mixture is used, the 6-factor M3O mixture is used to achieve feeder-free cell reprogramming without subculture, and the target is inoculated at various low densities (50K vs. 100K vs. 150K per well) Cells to accelerate MET. In another embodiment, the cell lines are seeded with a density gradient ranging from about 15K to 500K cells in the total number of cells per well.

本發明之一個態樣係關於此等重新編程培養之命運，此證實對接種密度高度敏感，據推測係因為過度細胞毒性及纖維母細胞過度生長之效應在轉染方案過程中均自強化。在使用「標準」RNA給藥(1200ng/孔)之實驗中，自以100K/孔接種之HDF-n及XFF纖維母細胞獲得數十打似hESC集落，同時對應之50K及150K培養分別在發生群體劇減及纖維母細胞過度生長後僅提供少量集落。於嘗試用兩種其他纖維母細胞系BJ及HDF-a進行的衍化中，甚至最具前景(150K)培養物在達成長滿後不久變的實質上靜止，隨後僅產生具有延遲動力學之零星集落。 One aspect of the present invention relates to the fate of these reprogramming cultures, which proves to be highly sensitive to the seeding density, presumably because the effects of excessive cytotoxicity and excessive growth of fibroblasts are self-enhanced during the transfection protocol. In an experiment using "standard" RNA administration (1200ng/well), dozens of hESC-like colonies were obtained from HDF-n and XFF fibroblasts seeded at 100K/well, and the corresponding 50K and 150K cultures were taking place. Only a small number of colonies are provided after the rapid population decline and the overgrowth of fibroblasts. In attempts to derivatize with two other fibroblast cell lines, BJ and HDF-a, even the most promising (150K) culture became substantially stationary shortly after reaching full growth, and then only produced sporadic kinetics with delayed Colony.

本發明之一個態樣係關於透過尺寸排除層析純化經活體外轉錄之mRNA以移除可單獨或與mRNA一起形成dsRNA結構之異常性RNA物種，及引起細胞免疫反應。使用此等「清潔」mRNA(具有低細胞免疫原性之mRNA)，有效地使以梯度密度生長之非人類的靈長類動物細胞重新編程。 One aspect of the present invention relates to purification of in vitro transcribed mRNA by size exclusion chromatography to remove abnormal RNA species that can form a dsRNA structure alone or together with mRNA, and to induce cellular immune response. Using these "clean" mRNAs (mRNAs with low cellular immunogenicity) can effectively reprogram non-human primate cells growing at a gradient density.

在本發明之一個特定實施例中，從環境切換為5%氧培養及在轉染的前四天期間從四分之一劑量漸增至全RNA給藥以企圖減低應力誘導之細胞衰老至最低。 In a specific embodiment of the present invention, switching from environment to 5% oxygen culture and gradually increasing from a quarter dose to full RNA administration during the first four days of transfection in an attempt to minimize stress-induced cell senescence .

在本發明之一個實例中，使用此等新穎條件，本發明者測試L-Myc或c-Myc之替代是否可進一步改良重新編程混合物。 In one example of the present invention, using these novel conditions, the inventors tested whether the replacement of L-Myc or c-Myc could further improve the reprogramming mixture.

在另一實施例中，吾人評估添加RNA至細胞且每天更換培養基而非在早4小時以個別步驟遞送之簡化轉染方案。在「24-小時轉染」孔中按比例縮小RNA給藥以補償預期的細胞毒性之增加，及測試兩種不同轉染試劑(RNAiMAX及Stemfect)。以50K/孔接種XFF細胞及轉染9天。習知的「4-小時轉染」方案之每個具有基於c-Myc的混合物之孔提供約一百個TRA-1-60⁺集落，同時L-Myc混合物表現相對差(圖2)。「24-小時轉染」培養之結果仍舊更令人印象深刻。在此等孔之最具生產性者(對應於c-Myc 400ng/ml Stemfect條件)中，該培養中之似hESC細胞在第9天(於最後一次轉染後24小時)已變得幾乎過度生長。就「24-小時轉染」之優異性能而言之機制基礎可能已經具有藉由濃縮由細胞釋放之擴散因子增加稀釋培養之有效密度之效應。當此等較佳方案條件應用於另一實例實驗(在以75K個細胞/孔接種之培養中進行的使用HDF-a纖維母細胞的衍化)中時，生產率不如用高增殖性XFF細胞所達成者顯著，但許多似hESC集落早在方案的第9天再次出現(圖4)。 In another example, we evaluated a simplified transfection protocol of adding RNA to cells and changing the medium daily instead of delivering in individual steps 4 hours early. In the "24-hour transfection" wells, the RNA administration was scaled down to compensate for the expected increase in cytotoxicity, and two different transfection reagents (RNAiMAX and Stemfect) were tested. XFF cells were seeded at 50K/well and transfected for 9 days. Each well with a c-Myc-based mixture of the conventional "4-hour transfection" protocol provided approximately one hundred TRA-1-60 ⁺ colonies, while the L-Myc mixture performed relatively poorly (Figure 2). The results of the "24-hour transfection" culture are still more impressive. In the most productive of these wells (corresponding to the c-Myc 400ng/ml Stemfect condition), the hESC-like cells in this culture have become almost excessive on day 9 (24 hours after the last transfection) Grow. The mechanism basis for the excellent performance of "24-hour transfection" may already have the effect of increasing the effective density of the dilution culture by concentrating the diffusion factors released by the cells. When these optimal conditions are applied to another example experiment (derivation using HDF-a fibroblasts in a culture seeded at 75K cells/well), the productivity is not as high as that achieved with highly proliferative XFF cells The results were significant, but many hESC-like colonies reappeared as early as the 9th day of the protocol (Figure 4).

在一特定實施例中，當細胞以小於常用體積之體積例如1ml、0.75ml、0.5ml之培養基或可仍舊持續細胞培養之最小體積之培養基接種時，使用上文所揭示條件重新編程之效率明顯改良。重新編程期間之此等低體積條件因此併入本發明中。 In a specific embodiment, when cells are inoculated with a medium smaller than the usual volume, such as 1ml, 0.75ml, 0.5ml, or the smallest volume of medium that can continue cell culture, the efficiency of reprogramming using the conditions disclosed above is obvious Improved. These low volume conditions during reprogramming are therefore incorporated into the present invention.

描述於本文中之實施例絕非本發明之唯一應用。熟習此項技術者當知曉本發明亦可用於在輕度可變條件下或利用用於重新編程、定向分化或轉分化之習知或工程化因子之相似組合重新編程其他細胞。 The embodiments described herein are by no means the only application of the present invention. Those skilled in the art should know that the present invention can also be used to reprogram other cells under mildly variable conditions or using a similar combination of conventional or engineered factors for reprogramming, directed differentiation or transdifferentiation.

在其他實施例中，因子化學計量之最佳化亦可增強重新編程之步伐-mRNA方法提供界定獨立地解決ipPSC誘導之早期及晚期階段之混合物之機會。藉由使用M3O獲得之增益為最近將新穎的工程化重新編程因子應用於iPSC產生提供驗證。此等工程化重新編程因子包括至除VP16 OR MYOD、SUCH AS GAL4、GATA1、P53等外之因子之轉活化域之融合SOX2、KLF4、CMYC、LMYC、LIN28、NANOG等。用於遞送mRNA之試劑及方法亦可用於共轉染siRNA及miRNA，此早已證實其在iPSC生成中之價值。然而，本發明所揭示之無餵養細胞方案代表勝過當前方案之實質性進步，其縮短重新編程所需要的時間多達一半及勞動及材料成本減少一半或更多，從程序中省去棘手的步驟，及允許mRNA以與生長因子或細胞因子幾近相同的情況-亦即，作為培養基補充劑遞送至細胞。 In other embodiments, optimization of factor stoichiometry can also enhance the pace of reprogramming-the mRNA approach provides an opportunity to define a mixture that independently addresses the early and late stages of ipPSC induction. The gain obtained by using M3O provides verification for the recent application of novel engineered reprogramming factors to iPSC generation. These engineered reprogramming factors include To fusion SOX2, KLF4, CMYC, LMYC, LIN28, NANOG, etc. to the transactivation domain of factors other than VP16 OR MYOD, SUCH AS GAL4, GATA1, P53, etc. The reagents and methods used to deliver mRNA can also be used to co-transfect siRNA and miRNA, which has long proven its value in the generation of iPSCs. However, the feeding-cell-free solution disclosed in the present invention represents a substantial improvement over the current solution. It shortens the time required for reprogramming by as much as half and reduces labor and material costs by half or more, eliminating tricky procedures from the program. Steps, and allow mRNA to be delivered to cells in nearly the same conditions as growth factors or cytokines-that is, as a medium supplement.

在一些實施例中，細胞經重新編程以調節免疫反應。例如，淋巴細胞可經重新編程為可投與有此需要的患者以增加或轉移免疫耐受性(特定言之自耐受性)之調節T細胞。Foxp3陽性T細胞之誘導或投與可用於減小諸如移植排斥之自體免疫反應，及/或減少、抑制或減輕諸如以下之質體免疫疾病或病症之一或多種症狀：糖尿病、多發性硬化症、哮喘、發炎性腸病、甲狀腺炎、腎病、風濕性關節炎、全身性紅斑狼瘡、嚴重脫髮(alopecia greata)、僵直性脊髓炎、抗磷脂症候群、自體免疫阿迪森氏疾病(Addison's disease)、自體免疫溶血性貧血、自體免疫肝炎、自體免疫內耳病、自體免疫淋巴增生症候群(ALPS)、自體免疫血小板減少紫斑症(ATP)、貝西氏病(Behcet's disease)、大皰性類天疱瘡、心肌病、乳糜瀉-皮炎、慢性疲勞症候群免疫缺陷症候群(CFIDS)、慢性脫髓鞘多發性神經炎、瘢痕性類天皰瘡、冷凝集素病、Crest症候群、克羅恩氏病(Crohn's disease)、德高斯氏病(Dego's disease)、皮肌炎、幼年型皮肌炎、圓盤紅斑(discoid lupus)、原發性混合型冷球蛋白血症、纖維肌痛症-纖維肌炎、格蕾氏症(Grave's disease)、格-巴二氏症(Guillain-Barre)、橋本甲狀腺炎(Hashimoto's thyroiditis)、特發性肺纖維化、特發性血小板減少性紫斑(ITP)、IgA腎病、胰島素相依糖尿病(I型)、幼年型關節炎、美尼爾氏病(Meniere's disease)、混合型結締組織疾病、多發性硬化症、重症肌無力症、尋常型天疱瘡、惡性貧血、結節性多動脈炎、多軟骨炎、多腺性症候群、風濕性多肌痛、多肌炎及皮肌炎、原發性無球蛋白血症、原發性膽汁性肝硬化、銀屑病、雷諾氏現象(Raynaud's phenomenon)、雷德氏症候群(Reiter's syndrome)、風濕熱、類肉瘤病、硬皮病、休格倫氏症候群(Sjogren's syndrome)、僵人症候群(stiff-man syndrome)、高安氏關節炎(Takayasu arteritis)、顳動脈炎/巨細胞動脈炎、潰瘍性結腸炎、葡萄膜炎、血管炎、白斑病及韋格納肉芽腫(Wegener's granulomatosis)。 In some embodiments, the cells are reprogrammed to modulate the immune response. For example, lymphocytes can be reprogrammed into regulatory T cells that can be administered to patients in need to increase or transfer immune tolerance (specifically, self-tolerance). The induction or administration of Foxp3-positive T cells can be used to reduce autoimmune responses such as transplant rejection, and/or reduce, inhibit or alleviate one or more of the following plastid immune diseases or disorders: diabetes, multiple sclerosis Syndrome, asthma, inflammatory bowel disease, thyroiditis, kidney disease, rheumatoid arthritis, systemic lupus erythematosus, severe alopecia (alopecia greata), ankylosing myelitis, antiphospholipid syndrome, autoimmune Addison's disease (Addison's disease) ), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, Bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue syndrome immunodeficiency syndrome (CFIDS), chronic demyelinating polyneuritis, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, gram Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, primary mixed cryoglobulinemia, fibromyalgia Symptoms-fibromyositis, Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura ( ITP), IgA nephropathy, insulin-dependent diabetes (type I), juvenile arthritis, Meniere Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, rheumatic polymuscularis Pain, polymyositis and dermatomyositis, primary aglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever , Sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colon Inflammation, uveitis, vasculitis, leukoplakia, and Wegener's granulomatosis.

該等方法可用於產生可用於治療多種疾病及病症包括(但不限於)諸如帕金森氏症(Parkinson's)、阿茲海默氏病(Alzheimer disease)、傷口癒合及多發性硬化症之疾病之細胞。該等方法亦可用於器官再生及免疫反應之恢復或補充。例如，處在不同分化階段之細胞諸如iPS細胞、造血幹細胞、多能細胞或單能細胞(諸如前身細胞，例如上皮前身細胞)及其他細胞可經靜脈內或藉由局部手術投與。該等方法可與其他習知方法諸如處方療法、手術、激素療法、化學療法及/或放射療法組合使用。 These methods can be used to produce cells that can be used to treat a variety of diseases and disorders including (but not limited to) diseases such as Parkinson's, Alzheimer's disease, wound healing and multiple sclerosis . These methods can also be used for organ regeneration and immune response recovery or supplementation. For example, cells at different stages of differentiation such as iPS cells, hematopoietic stem cells, pluripotent cells or unipotent cells (such as precursor cells, such as epithelial precursor cells) and other cells can be administered intravenously or by local surgery. These methods can be used in combination with other conventional methods such as prescription therapy, surgery, hormone therapy, chemotherapy, and/or radiation therapy.

在一個實施例中，套組包括RNA、細胞及用於轉染RNA至細胞中之裝置。該等RNA可係凍乾或呈溶液形式。套組可視情況包括其他材料，諸如細胞培養試劑。在一替代實施例中，套組提供依所揭示方法製得之再分化、去分化或重新編程細胞及儲藏及/或冷藏或冷凍運送以供後續使用。細胞通常呈溶液儲藏，從而維持存活率。包含細胞之套組應使用與存活率一致之方法諸如在包含乾冰之冷卻器中儲藏或運送使得細胞維持低於4℃且較佳低於-20℃。 In one embodiment, the kit includes RNA, cells, and a device for transfecting RNA into cells. The RNA can be lyophilized or in solution form. The kit may optionally include other materials, such as cell culture reagents. In an alternative embodiment, the kit provides redifferentiated, dedifferentiated or reprogrammed cells prepared according to the disclosed method and storage and/or refrigerated or frozen transport for subsequent use. Cells are usually stored in a solution to maintain survival. The kit containing the cells should use a method consistent with the survival rate, such as storage or transportation in a cooler containing dry ice, so that the cells are maintained below 4°C and preferably below -20°C.

該等套組視情況包括以下各物中之一或多者：生物活化劑、培養基、賦形劑及以下各物中之一或多者：針筒、針頭、掛線、紗布、繃帶、消毒劑、抗生素、局部麻醉劑、鎮痛藥、手術線、剪刀、解剖刀、無菌流體及無菌容器。該套組之組件可個別地經包裝及可係無菌。該等套組一般提供於容器例如適用於商業規模之塑料容器、紙板容器或金屬容器中。任何該等套組可包括使用說明書。該等方法可用於產生可用於治療多種疾病或病症包括(但不限於)神經退化性疾病諸如帕金森氏症、阿茲海默氏病及多發性硬化症之細胞。本文所揭示之方法亦可用於器官再生及免疫系統之恢復或補充。例如，處在分化之不同階段之細胞諸如iPS細胞、造血幹細胞、多能細胞或單能細胞(諸如前身細胞，例如上皮前身細胞)及其他細胞可經靜脈內或藉由局部手術投與。該等方法可與其他習知方法諸如處方療法、手術、技術療法、化學療法及/或放射療法組合使用。 These kits include one or more of the following as appropriate: bioactivator, culture medium, excipient, and one or more of the following: syringe, needle, hanging thread, gauze, Bandages, disinfectants, antibiotics, local anesthetics, analgesics, surgical thread, scissors, scalpel, sterile fluid, and sterile containers. The components of the kit can be individually packaged and can be sterile. These sets are generally provided in containers such as plastic containers, cardboard containers or metal containers suitable for commercial scale. Any such kits may include instructions for use. These methods can be used to generate cells that can be used to treat a variety of diseases or disorders including but not limited to neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. The method disclosed herein can also be used for organ regeneration and immune system recovery or supplementation. For example, cells in different stages of differentiation such as iPS cells, hematopoietic stem cells, pluripotent cells or unipotent cells (such as precursor cells, such as epithelial precursor cells) and other cells can be administered intravenously or by local surgery. These methods can be used in combination with other conventional methods such as prescription therapy, surgery, technical therapy, chemotherapy, and/or radiation therapy.

本發明之態樣提供幹細胞之培養系統及用於產生用於傷口治療之皮膚組織細胞之分化方法、及用於治療關節炎、狼瘡及其他自體免疫相關疾病之幹細胞療法。 Aspects of the present invention provide a stem cell culture system and a differentiation method for producing skin tissue cells for wound treatment, and stem cell therapy for treating arthritis, lupus and other autoimmune related diseases.

實例Instance

現參考以下實例描述本發明。此等實例僅為例示說明之目的提供，且本發明並不受此等實例限制，而是包含證實為本文所提供教示之結果之所有變化。 The present invention will now be described with reference to the following examples. These examples are provided for the purpose of illustration only, and the present invention is not limited by these examples, but includes all the changes that prove to be the results of the teachings provided herein.

實例1-IVT模板之產生Example 1-Generation of IVT template

使用不依賴於連接之選殖(LIC)構築用於產生線性PCR產物活體外轉錄(IVT)模板之質體構築體。首先構築併有側有經設計以接受編碼所述蛋白質之開放閱讀框架(ORF)***物之***部位之5'及3'未轉譯區(UTR)之親本質體(pIVT)。ORF側有序列係如Warren等人，Cell Stem Cell,2010中所述，其包含低次級結構前導及強Kozak部位(5' UTR)及鼠科α-球蛋白3' UTR。PIVT載體之具有5'突出物之線性化變型係藉由再退火兩種使用定製引物自該質體擴增之PCR產物製得。藉由類似程序製造具有互補突出物之ORF PCR產物，用載體PCR產物組併在一起及藉由熱衝擊轉形至DH5α細菌中以選殖基因特異性構築體(pIVT-KLF4等)。所得質體用於模板化PCR反應以製造併有T7啟動子、側有UTR的ORF及T120尾巴以驅動添加聚A尾巴之線性IVT模板，如Warren等人，Cell Stem Cell,2010中描述。透過使用定製反向引物(T120CTTCCTACTCAGGCTTTATTCAAAGACCA)引入T120尾巴區。就M3O及VPx3融合構築體而言，轉活化域編碼序列係藉由PCR使用定製引物添加至ORF。PCR產物模板原液維持在~100ng/uL之濃度。 Use ligation-independent selection (LIC) to construct a plastid construct used to generate a linear PCR product in vitro transcription (IVT) template. First, construct a parent body (pIVT) flanked by the 5'and 3'untranslated region (UTR) of the insertion site designed to accept the open reading frame (ORF) insert encoding the protein. The ORF side has a sequence line as described in Warren et al., Cell Stem Cell, 2010, which includes a low secondary structure leader and a strong Kozak site (5' UTR) and a murine α-globulin 3'UTR. The linearized variant of the PIVT vector with 5'overhangs is prepared by re-annealing two PCR products amplified from the plastids using customized primers. By analogy Sequence production of ORF PCR products with complementary overhangs, group PCR products together with vector and transform into DH5α bacteria by thermal shock to colonize gene-specific constructs (pIVT-KLF4, etc.). The resulting plastids are used in templated PCR reactions to produce linear IVT templates with T7 promoter, UTR-flanked ORF, and T120 tails to drive poly-A tails, as described in Warren et al., Cell Stem Cell, 2010. The T120 tail region was introduced by using a customized reverse primer (T120CTTCCTACTCAGGCTTTATTCAAAGACCA). For the M3O and VPx3 fusion construct, the transactivation domain coding sequence was added to the ORF by PCR using custom primers. The stock solution of PCR product template is maintained at a concentration of ~100ng/uL.

實例2-製造mRNA混合物Example 2-Making mRNA Mix

mRNA合成方法概述於圖5中。使用ARCA帽類似物對GTP之4：1比率在IVT反應中生成合成性mRNA以產生出高比例之加帽轉錄本。在核苷三磷酸(NTP)混合物中使用CTP改由5m-CTP及UTP改由假-UTP之完全代換以降低RNA產物之免疫原性。帽類似物及經修飾NTP係購自Trilink Biotechnologies。製備2.5×NTP混合物(ARCA：ATP：5m-CTP：GTP：假-UTP為15：15：3.75：3.75：3.75mM)以更換與用於進行IVT反應之MEGAscript T7套組(Ambion)一起提供之標準NTP。各40uL IVT反應包含16uL NTP混合物、4uL 10×T7緩衝劑、16uL DNA模板及4uL T7酵素。在37℃下培養反應4至6小時且接著在37℃下用2uL TURBO Dnase再處理15分鐘，接著於MEGAclear(Ambion)離心柱上純化，該等RNA產物以100uL之體積溶出。為自未加帽轉錄本移除免疫原性5'三磷酸部分，對各製劑添加10uL南極磷酸酶(Antarctic Phosphatase)反應緩衝液及3uL南極磷酸酶(NEB)。在37℃下培養磷酸酶反應30分鐘及再純化該等IVT產物。RNA產率藉由Nanodrop(Thermo Scientific)定量化，及因此該等製劑藉由添加TE pH 7.0(Ambion)調整至100ng/uL之標準化工作濃度。RNA混合物係藉由以所需化學計量比率將代表各種RF之製劑併在一起組裝。所使用的各RF的分率考慮各別轉錄本之預測分子量，除了以3×莫耳濃度包含的Oct4及其衍生物之外，所有RF係等莫耳。添加最大10%的編碼短壽命核化單體LanYFP螢光蛋白質之mRNA至該等混合物以促進監測重新編程試驗期間之轉染效率。在其他實施例中，亦藉由使用總尿嘧啶之25%或10%的2-硫基尿嘧啶來製造mRNA(參見圖6)。 The mRNA synthesis method is summarized in Figure 5. A 4:1 ratio of ARCA cap analog to GTP was used to generate synthetic mRNA in the IVT reaction to produce a high ratio of capped transcripts. Use CTP to change from 5m-CTP and UTP to pseudo-UTP in nucleoside triphosphate (NTP) mixture to reduce the immunogenicity of RNA products. Cap analogs and modified NTP were purchased from Trilink Biotechnologies. Prepare 2.5×NTP mixture (ARCA: ATP: 5m-CTP: GTP: False-UTP 15: 15: 3.75: 3.75: 3.75 mM) to replace the MEGAscript T7 kit (Ambion) provided with the IVT reaction Standard NTP. Each 40uL IVT reaction contains 16uL NTP mix, 4uL 10×T7 buffer, 16uL DNA template and 4uL T7 enzyme. The reaction was incubated at 37°C for 4 to 6 hours and then treated with 2uL TURBO Dnase at 37°C for another 15 minutes, and then purified on a MEGAclear (Ambion) spin column. The RNA products were eluted in a volume of 100uL. To remove the immunogenic 5'triphosphate portion from the uncapped transcript, 10 uL Antarctic Phosphatase reaction buffer and 3 uL Antarctic Phosphatase (NEB) were added to each preparation. Incubate the phosphatase reaction at 37°C for 30 minutes and then purify the IVT products. The RNA yield was quantified by Nanodrop (Thermo Scientific), and therefore the preparations were adjusted to a standardized working concentration of 100 ng/uL by adding TE pH 7.0 (Ambion). The RNA mixture is assembled by combining the preparations representing various RFs in the desired stoichiometric ratio. Used The fraction of each RF takes into account the predicted molecular weight of each transcript. Except for Oct4 and its derivatives contained at a concentration of 3×mole, all RF lines are equal to mol. Add up to 10% of the mRNA encoding the short-lived nucleated monomer LanYFP fluorescent protein to these mixtures to facilitate monitoring of transfection efficiency during the reprogramming test. In other embodiments, mRNA is also produced by using 25% or 10% of the total uracil of 2-thiouracil (see Figure 6).

實例3-細胞及培養基Example 3-Cells and Media

經標靶以供重新編程之細胞包括BJ新生兒纖維母細胞(ATCC)、HDF-f胎兒纖維母細胞、HDF-n新生兒纖維母細胞及HDF-a成體纖維母細胞(ScienCell)及XFF無外源物新生兒纖維母細胞(Millipore)。就BJ、HDF及XFF細胞而言，分別在BJ培養基(DMEM+10% FBS)、ScienCell纖維母細胞培養基及FibroGRO無外源物人類纖維母細胞擴增培養基(Millipore)中進行擴增培養。所使用的餵養細胞係3001G照射之新生兒人類***纖維母細胞(GlobalStem)及FibroGRO絲裂黴素C-非活性無外源物人類新生兒纖維母細胞(Millipore)。使用TrypLE Select(Gibco)(為無動物產物細胞裂解試劑)進行與基於無外源物餵養細胞及無餵養細胞重新編程試驗相關之細胞繼代培養步驟。 Targeted cells for reprogramming include BJ neonatal fibroblasts (ATCC), HDF-f fetal fibroblasts, HDF-n neonatal fibroblasts, HDF-a adult fibroblasts (ScienCell) and XFF No foreign matter neonatal fibroblasts (Millipore). For BJ, HDF and XFF cells, they were expanded and cultured in BJ medium (DMEM+10% FBS), ScienCell fibroblast medium and FibroGRO non-exogenous human fibroblast expansion medium (Millipore). The feeding cell line used was 3001G-irradiated neonatal human foreskin fibroblasts (GlobalStem) and FibroGRO mitomycin C-inactive non-exogenous human neonatal fibroblasts (Millipore). TrypLE Select (Gibco) (an animal product-free cell lysis reagent) was used to perform cell subculture steps related to the exogenous-free feeder cell and feeder-free reprogramming test.

實例4-人類纖維母細胞之重新編程Example 4-Reprogramming of human fibroblasts

所述之所有重新編程實驗係依製造商指示在塗佈CELLstart(Gibco)無外源物受質之6孔組織培養盤中實施。在BJ重新編程實驗開始時使用包含FBS之BJ培養基以250K/孔接種GlobalStem餵養細胞。在一些稍後的基於餵養細胞之試驗中，接種密度增加及特別在培養基更換期間補充餵養細胞以企圖維持近長滿餵養細胞層以回應於使用新穎RF混合物所遭遇到的高損耗率。將無外源物餵養細胞(當使用時)接種於不含血清之基於Pluriton的重新編程培養基中。將標靶細胞接種於Pluriton無血清培養基(Stemgent)加抗生素、Pluriton補充劑及200ng/ml B18R干擾素抑制劑(eBioscience)中。在重新編程期間及之後每天更換培養基，在最終轉染後的次日停止B18R補充。在其中細胞於重新編程期間***至新鮮培養細胞上之實驗中，用於再接種之培養基中包含10uM Y27632(Stemgent)。轉染從接種標靶細胞後的次日開始，及在本文所示的持續時間以24小時間隔時間重複。在每天的培養基更換前4小時使用RNAiMAX(Invitrogen)遞送1200ng之RNA劑量至各孔，除了另外註明之外。基於RNAiMAX的轉染混合物係藉由稀釋100ng/uL RNA 5×於無鈣/鎂DPBS及稀釋5uL RNAiMAX/ug RNA 10×於相同稀釋劑中，併在一起以產生10ng/uL RNA/媒劑懸浮液及15-分鐘室溫培養後分配至培養基製得。就使用Stemfect試劑(Stemgent)之轉染而言，使RNA及Stemfect(4uL/1ug RNA)在Stemfect緩衝液中混合以提供10ng/uL之RNA濃度。培養該混合物15分鐘，接著遞送至培養基或冷藏以待後續使用。 All the reprogramming experiments described were carried out in 6-well tissue culture dishes coated with CELLstart (Gibco) substrate without foreign substances according to the manufacturer's instructions. At the beginning of the BJ reprogramming experiment, BJ medium containing FBS was used to inoculate GlobalStem feeder cells at 250K/well. In some later feeder cell-based experiments, the seeding density was increased and feeder cells were replenished during medium exchanges in an attempt to maintain a nearly overgrown feeder cell layer in response to the high wastage rate encountered with the novel RF mixture. Exogenous-free feeder cells (when used) were seeded in serum-free Pluriton-based reprogramming medium. The target cells were inoculated in Pluriton serum-free medium (Stemgent) plus antibiotics, Pluriton supplements and 200ng/ml B18R interferon inhibitor (eBioscience). During reprogramming And then change the medium every day, and stop B18R supplementation the day after the final transfection. In an experiment in which cells divide onto freshly cultured cells during reprogramming, the medium used for re-seeding contains 10 uM Y27632 (Stemgent). Transfection starts the day after inoculation of target cells, and repeats at 24 hour intervals for the duration shown here. RNAiMAX (Invitrogen) was used to deliver a dose of 1200 ng of RNA to each well 4 hours before the daily medium replacement, unless otherwise noted. The RNAiMAX-based transfection mixture is prepared by diluting 100ng/uL RNA 5× in calcium/magnesium-free DPBS and diluting 5uL RNAiMAX/ug RNA 10× in the same diluent together to produce 10ng/uL RNA/vehicle suspension It is prepared by distributing to the culture medium after 15-minute incubation at room temperature. For transfection using Stemfect reagent (Stemgent), RNA and Stemfect (4uL/1ug RNA) are mixed in Stemfect buffer to provide an RNA concentration of 10ng/uL. The mixture is incubated for 15 minutes, and then delivered to the culture medium or refrigerated for subsequent use.

在其他實施例中，人類纖維母細胞之重新編程亦在除pluriton外之培養基例如Allele重新編程培養基中進行(參見圖7)。在一些實施例中，完全不使用B18R，在其他實施例中，僅有些轉染中使用B18R。 In other embodiments, the reprogramming of human fibroblasts is also performed in a medium other than pluriton, such as Allele reprogramming medium (see Figure 7). In some embodiments, B18R is not used at all, and in other embodiments, B18R is only used in some transfections.

實例5. iPSC集落之特徵分析Example 5. Analysis of characteristics of iPSC colonies

為評估iPSC集落生產率，使用含於DPBS(具有鈣/鎂)中之4%多聚甲醛固定重新編程培養物並用100×稀釋於DPBS(具有鈣/鎂)中之StainAlive TRA-1-60 Alexa 488抗體(Stemgent)免疫染色。進行用於多能性之分子及功能性驗證之集落選取、擴增及後續免疫染色及三系分化分析。實施DNA指紋術及核型分析。進行畸胎瘤形成及在多於一組小鼠模型中得以證實。由此展現幹細胞之多能性。 To evaluate the productivity of iPSC colonies, the reprogrammed cultures were fixed with 4% paraformaldehyde in DPBS (with calcium/magnesium) and StainAlive TRA-1-60 Alexa 488 diluted with 100× in DPBS (with calcium/magnesium) Antibody (Stemgent) immunostaining. Perform colony selection, expansion and subsequent immunostaining and three-line differentiation analysis for molecular and functional verification of pluripotency. Perform DNA fingerprinting and karyotype analysis. The formation of teratomas was performed and confirmed in more than one set of mouse models. This demonstrates the pluripotency of stem cells.

揭示一種藉由使標靶細胞與工程化重新編程因子及非工程化重新編程因子之組合以使得iPSC可在約9天(有時短為6天或甚至5天)產生之方式接觸使非幹細胞高效地重新編程成多能幹細胞的新穎方法。此等iPS細胞可呈無餵養細胞、無外源物及無足跡iPSC形式產生。除藉由本發明方法顯著提高重新編程之效率外，該新穎技術亦與所有先前已知的技術的區別在於如此建立的iPSC係「清潔」，因為其從未與任何病毒或載體接觸。本發明之效用可在涉及幹細胞建立、分化、細胞及發育研究中之效用、及臨床應用之實質上所有領域中發現。相似的程序亦可用於定向分化及轉分化。 It is revealed that a combination of target cells and engineered reprogramming factors and non-engineered reprogramming factors can be contacted to make non-stem cells efficient by contacting iPSCs in about 9 days (sometimes as short as 6 days or even 5 days). A novel method of reprogramming into pluripotent stem cells. These iPS cells can be produced in the form of feeder-free cells, no foreign substances and no footprint iPSCs. except In addition to significantly improving the efficiency of reprogramming by the method of the present invention, the novel technology is also different from all previously known technologies in that the iPSC system thus established is "clean" because it has never been in contact with any virus or vector. The utility of the present invention can be found in virtually all fields related to stem cell establishment, differentiation, utility in cell and development research, and clinical applications. Similar procedures can also be used for directed differentiation and transdifferentiation.

實例6-馬來猴纖維母細胞之重新編程Example 6-Reprogramming of Malay Monkey Fibroblasts

使用馬來猴纖維母細胞之重新編程實驗係依製造商指示在塗佈CELLstart(Gibco)受質之6孔組織培養盤中實施。將標靶細胞接種於Allele Biotech的無血清培養基加抗生素中。連續12天在使用或不使用B18R下每天於重新編程期間及之後改由連同新鮮培養基一起遞送之經2-硫基修飾之mRNA/轉染試劑混合物更換培養基(圖8)。猴iPSC之集落在第9天開始出現，且達到成熟階段，在第12天達到緊緻集落階段。 The reprogramming experiment using Malay monkey fibroblasts was carried out in a 6-well tissue culture dish coated with CELLstart (Gibco) substrate according to the manufacturer's instructions. The target cells were seeded in Allele Biotech's serum-free medium plus antibiotics. The medium was replaced with a 2-thio-modified mRNA/transfection reagent mixture delivered with fresh medium every day during and after reprogramming with or without B18R for 12 consecutive days (Figure 8). The colonies of monkey iPSC began to appear on the 9th day and reached the mature stage, and reached the compact colony stage on the 12th day.

Claims

一種使哺乳動物體細胞去分化或重新編程(reprogramming)之無餵養細胞(feeder-free)、無異源物(xeno-free)、非整合(integration-free)方法，該方法包括：利用包含以下之組合物轉染經分離之非人類的靈長類動物體細胞：(i)有效量之編碼重新編程因子Oct4融合至N端MyoD轉活化域的經純化之合成mRNA；及(ii)編碼重新編程因子Sox2、Klf4、cMyc、Nanog及Lin28的經純化之合成mRNA，藉以使體細胞重新編程或去分化；其中該合成mRNA經過純化而移除在用於生產該合成mRNA的活體外轉錄反應期間產生之異常性RNA物種；且其中該等細胞係依密度梯度接種。 A feeder-free, xeno-free, and integration-free method for dedifferentiating or reprogramming mammalian somatic cells, the method comprising: using Transfection of isolated non-human primate somatic cells with the composition: (i) an effective amount of purified synthetic mRNA encoding the reprogramming factor Oct4 fused to the N-terminal MyoD transactivation domain; and (ii) encoding renewal Purified synthetic mRNA of programming factors Sox2, Klf4, cMyc, Nanog, and Lin28, thereby reprogramming or dedifferentiating somatic cells; wherein the synthetic mRNA is purified and removed during the in vitro transcription reaction used to produce the synthetic mRNA The abnormal RNA species produced; and the cell lines are inoculated according to a density gradient.

如請求項1之方法，其中該等細胞為馬來猴細胞。 The method of claim 1, wherein the cells are Malay monkey cells.

如請求項1之方法，其中該等細胞為恆河猴細胞。 The method of claim 1, wherein the cells are rhesus monkey cells.

如請求項1之方法，其中該等細胞為狒狒細胞。 The method of claim 1, wherein the cells are baboon cells.

如請求項1之方法，其中該等細胞為犬細胞。 The method of claim 1, wherein the cells are canine cells.

如請求項1之方法，其中該等細胞為馬細胞。 The method of claim 1, wherein the cells are equine cells.

如請求項1之方法，其中該等細胞為牛細胞。 The method of claim 1, wherein the cells are bovine cells.

如請求項1之方法，其中該等細胞為豬細胞。 The method of claim 1, wherein the cells are pig cells.

如請求項1之方法，其中該等細胞為羊細胞。 The method of claim 1, wherein the cells are sheep cells.

如請求項1之方法，其中Oct4係以三重串接方式(in tandem triplicates)融合至N端MyoD轉活化域。 Such as the method of claim 1, wherein Oct4 is fused to the N-terminal MyoD transactivation domain by in tandem triplicates.

一種重新編程非人類的哺乳動物細胞之無餵養細胞(feeder-free)方法，其中該方法包括：使標靶細胞以15k至500k個細胞/ml之密度生長於無餵養細胞表面上，且其中該等細胞係依密度梯度接種；及利用約50ng至約800ng/ml之劑量範圍之如請求項1之經純化之合成mRNA轉染該等細胞。 A feeder-free method for reprogramming non-human mammalian cells, wherein the method comprises: growing target cells on feeder-free cells at a density of 15k to 500k cells/ml On the surface, and wherein the cell lines are seeded according to a density gradient; and the cells are transfected with the purified synthetic mRNA as in claim 1 in a dose range of about 50ng to about 800ng/ml.

如請求項1之方法，其中該方法包括：使標靶細胞以25k個細胞/ml至300k個細胞/ml之密度生長在無餵養細胞表面上，其中該合成mRNA經過純化而移除任何來自轉錄之污染性非mRNA物種且其中該等細胞係依密度梯度接種；及利用約50ng至約800ng/ml劑量範圍之如請求項1之經純化之合成mRNA轉染該等細胞，其中較早時間點所使用之劑量低於較晚時間點；及在未繼代培養下達成iPSC；其中該合成mRNA經過純化而移除任何來自轉錄之污染性非mRNA物種。 The method of claim 1, wherein the method comprises: growing target cells at a density of 25k cells/ml to 300k cells/ml on the surface of feeder-free cells, wherein the synthetic mRNA is purified to remove any transcription from The contaminating non-mRNA species in which the cell lines are inoculated according to a density gradient; and the purified synthetic mRNA as in claim 1 in a dose range of about 50ng to about 800ng/ml is used to transfect the cells, wherein the earlier time point The dose used is lower than the later time point; and iPSC is achieved without subculture; wherein the synthetic mRNA is purified to remove any contaminating non-mRNA species from transcription.

如請求項1之方法，其中該方法包括：使標靶細胞以選自以下之任何一種密度：15k、30k、50k、75k、100k、150k、250k、500k個細胞/孔，生長於標準6孔盤之無餵養細胞表面上；其中各孔之體積係經調整介於0.5ml至5ml之適宜培養基；且其中該等細胞係依密度梯度接種；及利用約50ng至800ng/ml劑量範圍之如請求項1之經純化之合成mRNA轉染該等細胞，其中較早時間點所使用之劑量低於較晚時間點；及該等細胞在未繼代培養下達成iPSC。 The method of claim 1, wherein the method comprises: growing the target cells at a density selected from the group consisting of 15k, 30k, 50k, 75k, 100k, 150k, 250k, 500k cells/well in standard 6-wells On the surface of the plate without feeding cells; the volume of each well is adjusted between 0.5ml to 5ml of suitable medium; and the cell lines are inoculated according to a density gradient; and a dosage range of about 50ng to 800ng/ml is used as requested The purified synthetic mRNA of item 1 is transfected into these cells, where the dose used at an earlier time point is lower than that at a later time point; and these cells reach iPSC without subculture.

如請求項1之方法，其中該等體細胞包括單能、多潛能(multipotent)、多能或分化細胞。 The method of claim 1, wherein the somatic cells include unipotent, multipotent, multipotent, or differentiated cells.

如請求項1之方法，其中該合成mRNA誘導體細胞去分化為單能、多潛能或多能細胞。 The method of claim 1, wherein the synthetic mRNA induces the dedifferentiation of somatic cells into unipotent, pluripotent or pluripotent cells.

如請求項1之方法，其中該等經轉染細胞係維持在培養物中作為誘導型多能幹(iPS)細胞。 The method of claim 1, wherein the transfected cell lines are maintained in culture as induced pluripotent stem (iPS) cells.

如請求項1之方法，其中該等經轉染細胞形成誘導型多能幹細胞，進一步包括誘導該等iPS細胞形成分化細胞。 The method of claim 1, wherein the transfected cells form induced pluripotent stem cells, further comprising inducing the iPS cells to form differentiated cells.

如請求項1之方法，其中該組合物進一步包含Rarg及LrH-1轉活化域(transaction activation domains)。 The method of claim 1, wherein the composition further comprises Rarg and LrH-1 transaction activation domains.

如請求項1之方法，其中該組合物進一步包含與VP16轉活化域融合之Oct4。 The method of claim 1, wherein the composition further comprises Oct4 fused to the transactivation domain of VP16.

如請求項1之方法，其中該經純化之合成mRNA含有2-硫基尿嘧啶。 The method of claim 1, wherein the purified synthetic mRNA contains 2-thiouracil.

如請求項11之方法，其中該經純化之合成mRNA含有2-硫基尿嘧啶。 The method of claim 11, wherein the purified synthetic mRNA contains 2-thiouracil.