Unlocking Regenerative Potential through in Vivo Genetic Reprogramming

描述(由申请人提供) 摘要：我们建议在体内对细胞进行基因重组，以诱导胚胎后脊椎动物的新组织发育。我们的最终目标是利用基因重新编程在成年脊椎动物身上培育出替代器官。生长替代组织的能力对于治疗和治愈糖尿病、肝病和心脏病等疾病以及修复/替代因创伤而受损或丢失的器官和肢体至关重要。我们的主要策略是测试在发育、再生和制造IPSCs(诱导多能干细胞)的过程中涉及的不同因素组合的有效性，以诱导胚胎后动物的新组织发育。我们的研究计划将专注于在前肠内胚层种植异位胰腺和肝脏组织，以及体内不同部位的异位SS细胞。目前，最流行的设计潜在治疗细胞或组织的方法主要是通过体外策略：通过胚胎干细胞(ESCs)或IPSCs的定向分化。然而，这种方法存在几个潜在的根本性障碍。培养的细胞可能会获得广泛的有害遗传和表观遗传变化(致癌或免疫原性突变)，这些变化通常会被排斥并从体内根除，但会在培养中继续茁壮成长。此外，尽管在区分培养皿中不同类型的细胞方面已经取得了很大进展，但在3D环境下培养整个器官仍然具有挑战性。此外，在培养皿中培养的组织，如心肌细胞来源的干细胞，在移植后如何整合并成为宿主心脏的功能部分仍不清楚。因此，在体外产生治疗性细胞和器官的方法出现了许多并发症。在体内产生新组织方面投入的重点要少得多，这可能会绕过这些障碍。我们建议应用我们对肝脏和胰腺前体生物学的独特见解，并将其与我们的新技术相结合，精确地诱导转基因表达，以重新编程内胚层细胞，以生长异位胰腺、肝脏和SS细胞。关于体内细胞重新编程的可行性和有效性的许多基本问题在很大程度上是未被探索的。我们的研究将使我们能够评估这些问题：是否有可能在成年动物身上培养出全新的器官？不同的体细胞(尤其是肠道内胚层的体细胞)对体内基因重新编程的可修复性如何？某些成体细胞，也许是祖细胞，更有可能进行基因重新编程？诱导器官能否有效地与邻近组织结合并发挥功能？它们是否不太可能积累突变或被免疫系统排斥？癌症风险会随着我们的基因操作而增加吗？IPSC诱导基因的表达能使体内的大多数体细胞更具可重程性吗？我们提议的工作将使我们能够开始提出这些基本问题。 公共卫生相关性：我们建议在体内对细胞进行基因重新编程，以诱导胚胎后脊椎动物的新组织发育。我们的最终目标是利用基因重新编程在成年脊椎动物身上培育出替代器官。生长替代组织的能力对于治疗和治愈糖尿病、肝脏疾病和心脏病等疾病以及修复/替代因创伤而受损或丢失的器官和肢体至关重要。

DESCRIPTION (Provided by the applicant) Abstract: We propose to genetically reprogram cells in vivo to induce new tissue development in post-embryonic vertebrate animals. Our ultimate goal is to grow replacement organs in an adult vertebrate animal using genetic reprogramming. The ability to grow replacement tissues will be critical for treating and curing diseases such as diabetes, liver disease, and heart diseas and for repairing/replacing damaged or lost organs and limbs sustained from traumatic injury. Our primary strategy is test for the efficacy of different combinations of factors implicated in th process of development, regeneration, and of making iPSCs (induced pluripotent stem cells) to induce new tissue development in post-embryonic animals. Our research proposal will focus on growing ectopic pancreas and liver tissues in the foregut endoderm, and ectopic ss-cells in different various places in the body. Currently, the most popular approach to engineering potential therapeutic cells or tissues is primarily via an in vitro strategy: through directed differentiation of embryonic stem cells (ESCs) or from iPSCs. However, there are several potential fundamental obstacles with this approach. Cultured cells may acquire extensive deleterious genetic and epigenetic changes (oncogenic or immunogenic mutations) that would normally be rejected and eradicated from the body, but would continue to thrive in culture. Also, although much progress has been made in differentiating various cell types in the dish, it remains challenging to grow whole organs in 3D. Further, it is still unclear how tissues cultured in a dish such as cardiomyocytes derived stem cells would integrate and become a functional part of the host heart following transplantation. Therefore, many complications have arisen with in vitro approaches to generating therapeutic cells and organs. Much less emphasis has been invested in generating new tissues in vivo, which may circumvent these obstacles. We propose to apply our unique insight on liver and pancreas progenitor biology and combine it with our new technology to precisely induce transgene expression to reprogram endoderm cells to grow ectopic pancreas, liver, and ss-cells. Many fundamental questions regarding the feasibility and effectiveness of in vivo cell reprogramming is largely unexplored. Our studies will allow us to evaluate these issues: Is it possible to grow whole new organs in an adult animal? How amendable are different somatic cells (particularly in the gut endoderm for this proposal) to in vivo genetic reprogramming? Are certain adult cells, maybe progenitors, more poised for genetic reprogramming? Will induced organs integrate effectively to adjacent tissues and be functional? Are they less likely to accumulate mutations or be rejected by the immune system? Will cancer risks increase with our genetic manipulations? Can expression of iPSC induction genes make most somatic cells in vivo more reprogrammable? Our proposed work will allow us to begin to ask these fundamental questions. Public Health Relevance: We propose to genetically reprogram cells in vivo to induce new tissue development in post-embryonic vertebrate animals. Our ultimate goal is to grow replacement organs in an adult vertebrate animal using genetic reprogramming. The ability to grow replacement tissues will be critical for treating and curing diseases such as diabetes, liver disease, and heart disease and for repairing/replacing damaged or lost organs and limbs sustained from traumatic injury.