病毒载体技术的安全性研究及其在诱导性多能干细胞中的应用
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摘要
第一部分：逆转录病毒载体和慢病毒载体的安全性比较研究
背景：逆转录病毒载体广泛应用人类基因治疗试验中。然而载体随机插入可能会引起宿主附近基因的激活。逆转录病毒和慢病毒载体越来越多的应用于干细胞基因矫正中。由于插入诱变导致的干细胞表型改变,可能会影响其子代细胞及其分化发育性能。
目的：确定病毒载体插入部位的选择机制及对宿主基因表达的影响。
方法：分类转染的Jurkat细胞克隆,用LM-PCR方法对病毒载体插入位点进行作图。并将获得的插入位点附近序列与人类基因组草图(UCSC Human Genome Project Working Draft)进行比对。用实时荧光定量逆转录PCR技术检测病毒载体插入对宿主基因表达的影响。
结果：1)RV插入宿主基因组时偏好RIS,而LV没有类似倾向。2)RV喜好插入基因的转录起始区(TSS),而LV没有类似倾向。3)加入cHS4绝缘子后,使LV的插入模式向RV模式转变,即靠近TSS的增多。4)与LV不同,RV对宿主基因的活化频率较高,说明LV是较RV安全的载体。而且,RV可能会导致插入突变,这一特性可以用于研究基因是如何条件细胞增殖和分化的。5)宿主基因被活化与否,跟病毒载体插入位点与宿主基因启动子的远近无直接关系。
结论：RV偏好插入RIS基因附近,该类基因多是调节细胞周期和凋亡的基因。宿主基因的活化可能与所在染色质的三维空间结构有关,使得RV增强子能够激活远处的宿主基因,而对就近的基因没有影响。RV和LV在对插入位点的选择上有显著的不同。
第二部分：病毒载体技术在体细胞重编程阶段的应用
背景：虽然逆转录病毒载体插入诱变的风险高于慢病毒载体,但是存在两个明显的优势,其一体细胞重编程效率较高,其二在于细胞阶段,逆转录病毒载体的MLV启动子自发沉默,有助于后继的iPSCs向各类终末细胞分化。脊髓肌肉萎缩症(SMA)是单基因遗传病,非常适合进行发病机制和基因矫正治疗的研究。SMA疾病特异性iPSCs细胞能够提供无限量的带有疾病特征的运动神经元细胞。
目的：本实验室1)拟建立逆转录病毒载体转染正常人皮肤成纤维细胞,建立正常人iPSCs作为对照组。2)而后建立脊髓肌肉萎缩症(SMA)患者的iPSCs,探索疾病特异性iPSCs作为研究发病机制的细胞模型的可行性。3)进行重编程技术优化。
方法：来自于正常人和SMA患者的皮肤成纤维细胞被染携带有4类转录调控因子的逆转录病毒载体。4周以后,iPSCs克隆被分离培养,并进行特性鉴定,包括：1)ES细胞特异性标记基因的表达,包括TRA-1-80, hBRIX, hDNMT, NODAL, TDGF1, GDF3, NANOG, REX13, hTERT, DPPA4,内源性Oct3/4, Sox2, Klf4 and C-myc。2)胚样体形成试验,及向三胚层的诱导分化,用于证实iPSCs细胞系的多向分化潜能。3)进行重编程技术优化。
结果：(1)本实验室建立的正常iPSCs和SMA-iPSCs细胞系均具有自我更新和多向分化潜能。(2)在建立SMA-iPSCs细胞系的过程中,重编程效率类似于正常人成纤维细胞。(3)RV携带的外源基因在iPSCs细胞系中被沉默,而内源性转录因子被激活表达。(4)RV没有引起iPSCs细胞的分化阻滞。(5)重编程技术优化结果,把LV和转座子技术结合,具有可行性,但重编程效率显著降低。
结论：(1)用RV成功重编程获得正常iPSCs细胞系和疾病特异性SMA-iPSCs细胞系。(2)重编程条件优化可考虑把病毒载体技术和非病毒技术结合起来,以达到高效安全的目的。
第三部分：病毒载体技术在人诱导性多能干细胞基因矫正阶段的应用
背景：基因矫正技术和小鼠胚胎干细胞技术结合,近年来进展显著。包括利用同源基因重组技术发展而来的“基因敲出”、“基因敲入”小鼠,以及体外基因矫正技术的临床试验。但是由于人类胚胎干细胞的伦理限制、成体干细胞来源受限,体外扩增技术尚无突破,以及基因矫正的安全性问题,使人类遗传性疾病的基因矫正研究进展缓慢。
目的：1)在患者皮肤成纤维细胞中进行基因矫正的可行性；2)在疾病特异性SMA-iPSCs细胞系中进行基因矫正的可行性；3)矫正前后,细胞表型及运动神经元定向分化功能的改变。
方法：构建LV-SMN-Neo载体,1)转染SMA成纤维细胞,而后进行Neo耐药基因筛选；2)建立无滋养细胞的iPSCs培养体系,并用上述载体转染SMA-iPSCs获得成功；3)建立iPSCs向运动神经元定向分化的方案,而后用免疫荧光技术,鉴定SMN蛋白的表达情况,及Tuj-1和HB9等神经元特异性蛋白的表达情况。
结果：1)成功构建携带正常SMN基因、Neo耐药基因的慢病毒载体；2)在患者成纤维细胞水平,成功转染并筛选获得G418抗生素耐药的阳性克隆；3)转染细胞成功表达SMN蛋白,及细胞核内gem body数量的显著增加；5)在疾病特异性SMA-iPSCs水平进行转染时,成功获得SMN的特异性表达和细胞核内gem body数量的增加。6)运动神经元定向分化研究中,发现SMA-iPSCs细胞核内包含SMN蛋白的颗粒(gem body)明显减少。运动神经元定向分化过程中,正常人iPSCs可以形成正常的运动神经元,而SMA-iPSCs的运动神经元形成明显减少,神经元特异性蛋白(Tuj-1,HB9)表达减少,轴突形成减少,说明SMN蛋白缺失可能会导致运动神经元发育缺陷。7)进行基因矫正后的SMA-iPSCs向运动神经元定向分化能力明显改善,但仍低于正常对照组。
结论：慢病毒载体转染体细胞和iPSCs细胞系进行基因矫正,均具有可行性。病毒转染后的阳性克隆,其神经元特异性蛋白表达谱及运动神经元定向分化功能均得到改善。
Part I:A comparative study of retroviral and lentiviral vector-induced genotoxicity
Background:Retroviral vectors (RV) are widely used in human gene therapy trials. However, random vector integration can activate adjacent cellular genes. Study of genotoxicity has so far been limited to tumorigenesis through genetic selection. Other types of genotoxicity that permanently alter cell phenotypes receive much less attention. As RV and lentiviral vectors (LV) are increasingly used to modify stem cells, these phenotypic changes will have a long-term effect on the progeny derived from these stem cells. It is therefore important to study vector-induced genotoxicity in the absence of genetic selection.
Objectives:To determine the effect of vector integration on host gene expression.
Methods:Transduced Jurkat clones were isolated and vector integration site mapped by ligation-mediated PCR. The BLAT program was used to map sequences to the human genome (UCSC Human Genome Project Working Draft). Effect of vector integration on host gene expression was determined by Real time-PCR.
Results:(1) RV integration into the human genome prefers retrovirus integration sites (RIS) previously mapped in rodent cells whereas LV integration does not show such a preference. (2) RV preferentially integrates near a transcription start site whereas LV does not, suggesting that the mechanism for vector integration between these two systems is different. (3) Insertion of the cHS4 insulator sequence alters the integration pattern of LV toward the transcription start site. (4) In contrast to LV, RV integration leads to host gene activation at high frequencies. This profound effect suggests that LV is a safer vector to use in gene and cell-based therapy. However, RV may serve to induce insertional mutagenesis in host cells, allowing the identification of genes important for the regulation of cell proliferation and differentiation. RV demonstrated preferential integration near a group of genes termed retroviral common integration sites (CIS) whereas LV exhibited no such preference. RV activated host gene expression more frequently than LV. However, gene activation was not correlated with how far the integration site was from the host gene promoter.
Conclusions:RV prefers to integrate near CIS enriched for cell cycle and apoptosis genes. Since gene activation by RV did not depend on the distance between the promoter and the integration site, the three-dimensional architecture of the host gene might affect the RV enhancer to activate host gene transcription. These results also suggest that the process of selecting integration site or the mechanism of integration is different between RV and LV.
PartⅡ:The application of viral vector in somatic cell reprogramming
Background:Although the insertional mutagenesis in retroviral vector (RV) is more frequently than in lentiviral vector (LV), there are two advantages by using RV. First is the high efficiency of somatic cell reprogramming. Second, the MLV promoter in RV will be silenced during stem cell stage, which is helpful during the differentiation of iPSCs to lineage specific cells. Spinal muscular atrophy (SMA) is an inherited disease of single gene mutation, adapting to investigat the molecular mechanism of SMA and gene correction therapy. Development of induced pluripotent stem (iPS) cells provides an unprecedented opportunity to generate motor neurons.
Objectives:(1) To generate normal iPSCs derived from normal human skin fibroblast cells by transduced with four key transcription factors deliveried by RV. (2) To establish SMA iPS cells, and investigate the feasibility of disease-specific iPSCs as mechanism research model. (2) To modify and optimize the reprogramming process.
Methods:Fibroblasts from normal human and SMA patient were transduced with the four transcription factors (Oct3/4, Sox2, Klf4 and C-myc) by retroviral vectors respectively. iPS cell colonies were isolated four weeks after transduction and analyzed for the expression of ES cell-specific markers, including TRA-1-80, hBRIX, hDNMT, NODAL, TDGF1, GDF3, NANOG, REX13, hTERT, DPPA4, Oct3/4, Sox2, Klf4 and C-myc. Formation of embryoid bodies (EBs) and differentiation into three germ layers were carried out to confirm the pluripotent potential of these iPS cell lines. In addition, reprogramming process was optimized by combination of LV and transponse et al.
Results:(1) iPS cell lines (from normal or patient fibroblast cells) with self renewal and pluripotent potential were established. (2) There is no significant diffience of reprogramming efficiency between normal and SMA patient fibroblast cells. (3) The transgene contained by RV was silenced and the endogenous key transcription factors were reactivated. (4) RV transduced normal or SMA-iPSCs showed no differentiation block. (5) When combination of LV and transponse, the reprogramming is successful, but the efficiency is poor.
Conclusion:SMA disease specific iPSCs can be generated by the same way and same efficiency as normal iPSCs. Reprogramming comdition can be optimized by combinated both viral technique and non-viral technique, which will contribute to get safer and effective iPSCs.
Part III:The application of viral vector in gene correction of induced pluripotent stem cells
Background:Gene correction and mouse embryonic stem cells techeniques got together and developed faster during these two decades. Many gene knockout and knockin mouse models were developed by using homologous recombination (HR). Gene therapy in vitro were tested and clinic trial were designed. But due to the ethical problem and genotoxiticity issue of gene therapy, researches and application of gene correction in human monogenic disease is growing slowly.
Objectives:(1) To test the feasibility of gene correction on patient's fibroblast cells. (2) To test the feasibility of gene correction on disease specific SMA-iPSCs. (3) Compare the cell phenotype and neural lineage differentiation between SMA-iPSCs and SMA-iPSCs post correction.
Methods:(1) Successfully constructure the LV vector contained wild-type SMN gene and Neo drug resistant gene (LV-SMN-Neo). (2) Somatic cells (patient's skin fibroblast cells) were transduced with LV-SMN-Neo vector and positive clones were selected by G418 antibiotics. (3) Transduced cells expressed SMN protein and the specific "gem bodies" in nuclears were increased. (4) SMA-iPSCs was transduced with LV-SMN-Neo vector and positive clones were selected by G418 antibiotics. Positive clones expressed SMN protein and specific "gem bodies" in nuclears were increased. (5) During neural lineage differentiation study, we found motor neuron derived from normal iPSCs showed normal mortor neuron formation and neuron specific gene expression (Tuj-1, HB9). But the neuron derived from SMA-iPSCs showed sparse neuron formation and weak expression of neuron specific gene. Neurason formation was much less than normal iPSCs. (6) SMA-iPSCs corrected by LV-SMN-Neo vector were improved in their neural differentiation but still worse than normal iPSCs.
Results:It is feasible to correct gene mutation by LV vector in both somatic cell and iPSCs level. LV transduced positive clones could improve the cell phenotype and lineage specific differentiation. Confirming the presence of abnormalities not only will validate the strategy of using iPS cell to study disease pathogenesis but may also provide a platform for drug validation and screening. The availability of SMA motor neurons will also provide an opportunity to study the cellular and molecular mechanisms of SMA pathogenesis.
背景：逆转录病毒载体广泛应用人类基因治疗试验中。然而载体随机插入可能会引起宿主附近基因的激活。逆转录病毒和慢病毒载体越来越多的应用于干细胞基因矫正中。由于插入诱变导致的干细胞表型改变,可能会影响其子代细胞及其分化发育性能。
目的：确定病毒载体插入部位的选择机制及对宿主基因表达的影响。
方法：分类转染的Jurkat细胞克隆,用LM-PCR方法对病毒载体插入位点进行作图。并将获得的插入位点附近序列与人类基因组草图(UCSC Human Genome Project Working Draft)进行比对。用实时荧光定量逆转录PCR技术检测病毒载体插入对宿主基因表达的影响。
结果：1)RV插入宿主基因组时偏好RIS,而LV没有类似倾向。2)RV喜好插入基因的转录起始区(TSS),而LV没有类似倾向。3)加入cHS4绝缘子后,使LV的插入模式向RV模式转变,即靠近TSS的增多。4)与LV不同,RV对宿主基因的活化频率较高,说明LV是较RV安全的载体。而且,RV可能会导致插入突变,这一特性可以用于研究基因是如何条件细胞增殖和分化的。5)宿主基因被活化与否,跟病毒载体插入位点与宿主基因启动子的远近无直接关系。
结论：RV偏好插入RIS基因附近,该类基因多是调节细胞周期和凋亡的基因。宿主基因的活化可能与所在染色质的三维空间结构有关,使得RV增强子能够激活远处的宿主基因,而对就近的基因没有影响。RV和LV在对插入位点的选择上有显著的不同。
第二部分：病毒载体技术在体细胞重编程阶段的应用
背景：虽然逆转录病毒载体插入诱变的风险高于慢病毒载体,但是存在两个明显的优势,其一体细胞重编程效率较高,其二在于细胞阶段,逆转录病毒载体的MLV启动子自发沉默,有助于后继的iPSCs向各类终末细胞分化。脊髓肌肉萎缩症(SMA)是单基因遗传病,非常适合进行发病机制和基因矫正治疗的研究。SMA疾病特异性iPSCs细胞能够提供无限量的带有疾病特征的运动神经元细胞。
目的：本实验室1)拟建立逆转录病毒载体转染正常人皮肤成纤维细胞,建立正常人iPSCs作为对照组。2)而后建立脊髓肌肉萎缩症(SMA)患者的iPSCs,探索疾病特异性iPSCs作为研究发病机制的细胞模型的可行性。3)进行重编程技术优化。
方法：来自于正常人和SMA患者的皮肤成纤维细胞被染携带有4类转录调控因子的逆转录病毒载体。4周以后,iPSCs克隆被分离培养,并进行特性鉴定,包括：1)ES细胞特异性标记基因的表达,包括TRA-1-80, hBRIX, hDNMT, NODAL, TDGF1, GDF3, NANOG, REX13, hTERT, DPPA4,内源性Oct3/4, Sox2, Klf4 and C-myc。2)胚样体形成试验,及向三胚层的诱导分化,用于证实iPSCs细胞系的多向分化潜能。3)进行重编程技术优化。
结果：(1)本实验室建立的正常iPSCs和SMA-iPSCs细胞系均具有自我更新和多向分化潜能。(2)在建立SMA-iPSCs细胞系的过程中,重编程效率类似于正常人成纤维细胞。(3)RV携带的外源基因在iPSCs细胞系中被沉默,而内源性转录因子被激活表达。(4)RV没有引起iPSCs细胞的分化阻滞。(5)重编程技术优化结果,把LV和转座子技术结合,具有可行性,但重编程效率显著降低。
结论：(1)用RV成功重编程获得正常iPSCs细胞系和疾病特异性SMA-iPSCs细胞系。(2)重编程条件优化可考虑把病毒载体技术和非病毒技术结合起来,以达到高效安全的目的。
第三部分：病毒载体技术在人诱导性多能干细胞基因矫正阶段的应用
背景：基因矫正技术和小鼠胚胎干细胞技术结合,近年来进展显著。包括利用同源基因重组技术发展而来的“基因敲出”、“基因敲入”小鼠,以及体外基因矫正技术的临床试验。但是由于人类胚胎干细胞的伦理限制、成体干细胞来源受限,体外扩增技术尚无突破,以及基因矫正的安全性问题,使人类遗传性疾病的基因矫正研究进展缓慢。
目的：1)在患者皮肤成纤维细胞中进行基因矫正的可行性；2)在疾病特异性SMA-iPSCs细胞系中进行基因矫正的可行性；3)矫正前后,细胞表型及运动神经元定向分化功能的改变。
方法：构建LV-SMN-Neo载体,1)转染SMA成纤维细胞,而后进行Neo耐药基因筛选；2)建立无滋养细胞的iPSCs培养体系,并用上述载体转染SMA-iPSCs获得成功；3)建立iPSCs向运动神经元定向分化的方案,而后用免疫荧光技术,鉴定SMN蛋白的表达情况,及Tuj-1和HB9等神经元特异性蛋白的表达情况。
结果：1)成功构建携带正常SMN基因、Neo耐药基因的慢病毒载体；2)在患者成纤维细胞水平,成功转染并筛选获得G418抗生素耐药的阳性克隆；3)转染细胞成功表达SMN蛋白,及细胞核内gem body数量的显著增加；5)在疾病特异性SMA-iPSCs水平进行转染时,成功获得SMN的特异性表达和细胞核内gem body数量的增加。6)运动神经元定向分化研究中,发现SMA-iPSCs细胞核内包含SMN蛋白的颗粒(gem body)明显减少。运动神经元定向分化过程中,正常人iPSCs可以形成正常的运动神经元,而SMA-iPSCs的运动神经元形成明显减少,神经元特异性蛋白(Tuj-1,HB9)表达减少,轴突形成减少,说明SMN蛋白缺失可能会导致运动神经元发育缺陷。7)进行基因矫正后的SMA-iPSCs向运动神经元定向分化能力明显改善,但仍低于正常对照组。
结论：慢病毒载体转染体细胞和iPSCs细胞系进行基因矫正,均具有可行性。病毒转染后的阳性克隆,其神经元特异性蛋白表达谱及运动神经元定向分化功能均得到改善。
Part I:A comparative study of retroviral and lentiviral vector-induced genotoxicity
Background:Retroviral vectors (RV) are widely used in human gene therapy trials. However, random vector integration can activate adjacent cellular genes. Study of genotoxicity has so far been limited to tumorigenesis through genetic selection. Other types of genotoxicity that permanently alter cell phenotypes receive much less attention. As RV and lentiviral vectors (LV) are increasingly used to modify stem cells, these phenotypic changes will have a long-term effect on the progeny derived from these stem cells. It is therefore important to study vector-induced genotoxicity in the absence of genetic selection.
Objectives:To determine the effect of vector integration on host gene expression.
Methods:Transduced Jurkat clones were isolated and vector integration site mapped by ligation-mediated PCR. The BLAT program was used to map sequences to the human genome (UCSC Human Genome Project Working Draft). Effect of vector integration on host gene expression was determined by Real time-PCR.
Results:(1) RV integration into the human genome prefers retrovirus integration sites (RIS) previously mapped in rodent cells whereas LV integration does not show such a preference. (2) RV preferentially integrates near a transcription start site whereas LV does not, suggesting that the mechanism for vector integration between these two systems is different. (3) Insertion of the cHS4 insulator sequence alters the integration pattern of LV toward the transcription start site. (4) In contrast to LV, RV integration leads to host gene activation at high frequencies. This profound effect suggests that LV is a safer vector to use in gene and cell-based therapy. However, RV may serve to induce insertional mutagenesis in host cells, allowing the identification of genes important for the regulation of cell proliferation and differentiation. RV demonstrated preferential integration near a group of genes termed retroviral common integration sites (CIS) whereas LV exhibited no such preference. RV activated host gene expression more frequently than LV. However, gene activation was not correlated with how far the integration site was from the host gene promoter.
Conclusions:RV prefers to integrate near CIS enriched for cell cycle and apoptosis genes. Since gene activation by RV did not depend on the distance between the promoter and the integration site, the three-dimensional architecture of the host gene might affect the RV enhancer to activate host gene transcription. These results also suggest that the process of selecting integration site or the mechanism of integration is different between RV and LV.
PartⅡ:The application of viral vector in somatic cell reprogramming
Background:Although the insertional mutagenesis in retroviral vector (RV) is more frequently than in lentiviral vector (LV), there are two advantages by using RV. First is the high efficiency of somatic cell reprogramming. Second, the MLV promoter in RV will be silenced during stem cell stage, which is helpful during the differentiation of iPSCs to lineage specific cells. Spinal muscular atrophy (SMA) is an inherited disease of single gene mutation, adapting to investigat the molecular mechanism of SMA and gene correction therapy. Development of induced pluripotent stem (iPS) cells provides an unprecedented opportunity to generate motor neurons.
Objectives:(1) To generate normal iPSCs derived from normal human skin fibroblast cells by transduced with four key transcription factors deliveried by RV. (2) To establish SMA iPS cells, and investigate the feasibility of disease-specific iPSCs as mechanism research model. (2) To modify and optimize the reprogramming process.
Methods:Fibroblasts from normal human and SMA patient were transduced with the four transcription factors (Oct3/4, Sox2, Klf4 and C-myc) by retroviral vectors respectively. iPS cell colonies were isolated four weeks after transduction and analyzed for the expression of ES cell-specific markers, including TRA-1-80, hBRIX, hDNMT, NODAL, TDGF1, GDF3, NANOG, REX13, hTERT, DPPA4, Oct3/4, Sox2, Klf4 and C-myc. Formation of embryoid bodies (EBs) and differentiation into three germ layers were carried out to confirm the pluripotent potential of these iPS cell lines. In addition, reprogramming process was optimized by combination of LV and transponse et al.
Results:(1) iPS cell lines (from normal or patient fibroblast cells) with self renewal and pluripotent potential were established. (2) There is no significant diffience of reprogramming efficiency between normal and SMA patient fibroblast cells. (3) The transgene contained by RV was silenced and the endogenous key transcription factors were reactivated. (4) RV transduced normal or SMA-iPSCs showed no differentiation block. (5) When combination of LV and transponse, the reprogramming is successful, but the efficiency is poor.
Conclusion:SMA disease specific iPSCs can be generated by the same way and same efficiency as normal iPSCs. Reprogramming comdition can be optimized by combinated both viral technique and non-viral technique, which will contribute to get safer and effective iPSCs.
Part III:The application of viral vector in gene correction of induced pluripotent stem cells
Background:Gene correction and mouse embryonic stem cells techeniques got together and developed faster during these two decades. Many gene knockout and knockin mouse models were developed by using homologous recombination (HR). Gene therapy in vitro were tested and clinic trial were designed. But due to the ethical problem and genotoxiticity issue of gene therapy, researches and application of gene correction in human monogenic disease is growing slowly.
Objectives:(1) To test the feasibility of gene correction on patient's fibroblast cells. (2) To test the feasibility of gene correction on disease specific SMA-iPSCs. (3) Compare the cell phenotype and neural lineage differentiation between SMA-iPSCs and SMA-iPSCs post correction.
Methods:(1) Successfully constructure the LV vector contained wild-type SMN gene and Neo drug resistant gene (LV-SMN-Neo). (2) Somatic cells (patient's skin fibroblast cells) were transduced with LV-SMN-Neo vector and positive clones were selected by G418 antibiotics. (3) Transduced cells expressed SMN protein and the specific "gem bodies" in nuclears were increased. (4) SMA-iPSCs was transduced with LV-SMN-Neo vector and positive clones were selected by G418 antibiotics. Positive clones expressed SMN protein and specific "gem bodies" in nuclears were increased. (5) During neural lineage differentiation study, we found motor neuron derived from normal iPSCs showed normal mortor neuron formation and neuron specific gene expression (Tuj-1, HB9). But the neuron derived from SMA-iPSCs showed sparse neuron formation and weak expression of neuron specific gene. Neurason formation was much less than normal iPSCs. (6) SMA-iPSCs corrected by LV-SMN-Neo vector were improved in their neural differentiation but still worse than normal iPSCs.
Results:It is feasible to correct gene mutation by LV vector in both somatic cell and iPSCs level. LV transduced positive clones could improve the cell phenotype and lineage specific differentiation. Confirming the presence of abnormalities not only will validate the strategy of using iPS cell to study disease pathogenesis but may also provide a platform for drug validation and screening. The availability of SMA motor neurons will also provide an opportunity to study the cellular and molecular mechanisms of SMA pathogenesis.
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