The rhesus macaque as a preclinical model for induced pluripotent stem cells

恒河猴作为诱导多能干细胞的临床前模型

• 批准号: 8344862
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 35.04万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344862
• 关键词: Ablation; Achievement; Adult; Alkaline Phosphatase; Allogenic; Antibodies; Autologous; Binding; Biological Assay; CD34 gene; Cell Therapy; Cells; Characteristics; Clinical; Clinical Trials; Codon Nucleotides; Collaborations; Derivation procedure; Development; Ectoderm Cell; Ectopic Expression; Embryo; Embryonic Development; Endoderm Cell; Excision; FK506; Fibroblasts; Ganciclovir; Gene Expression; Generations; Genes; Genomics; Goals; Health; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Homing; Human; Immunodeficient Mouse; Implant; In Vitro; Inbreeding; Insertional Activations; Insertional Mutagenesis; Laboratory Procedures; Laboratory mice; Lentivirus Vector; Location; Longevity; Macaca; Macaca mulatta; Marrow; Mediating; Mesenchymal; Mesoderm Cell; Methods; Modeling; Morphology; Mouse Strains; Mus; National Institute of Dental and Craniofacial Research; Oral Administration; Organoids; Paper; Pharmaceutical Preparations; Physiological; Play; Pre-Clinical Model; Preparation; Prodrugs; Proliferating; Property; Proteins; Proto-Oncogenes; Protocols documentation; Publishing; Reagent; Regenerative Medicine; Relative (related person); Residual state; Resources; Retrieval; Retroviral Vector; Risk; Rodent Model; Role; Safety; Site; Skin; Somatic Cell; Specific qualifier value; Staging; Stem cells; Stromal Cells; Structure of beta Cell of islet; Suicide; System; T-Lymphocyte; TK Gene; Tacrolimus Binding Proteins; Teratoma; Testing; Thymidine Kinase; Tissues; Transcription factor genes; Transplantation; United States National Institutes of Health; Xenograft Model; Xenograft procedure; base; blastocyst; bone; caspase-9; cell killing; cell type; cross reactivity; cytokine; design; embryonic stem cell; gene therapy; graft vs host disease; human embryonic stem cell; immunogenic; immunogenicity; improved; in vivo; induced pluripotent stem cell; model development; neutrophil; nonhuman primate; novel; osteogenic; pluripotency; prevent; programs; scale up; small molecule; suicide gene; telomere; tissue regeneration; tool; transcription factor; tumor; vector

The re-programming of post-natal somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of stem cell specifying transcription factors has many exciting potential applications for improving human health. iPSCs were initially developed in the murine model, and shown to have the potential to contribute to all tissues via blastocyst complementation assays. Just a few years later, human iPS cells were created using a similar panel of transcription factors, and demonstrated to form teratomas in immunodeficient mice and share functional and gene expression characteristics with human embryonic stem cells. However, there are numerous hurdles to moving iPSC forward into clinical regenerative medicine applications. First and most important are safety concerns. Both murine and human iPSCs were initially derived by introducing the required transcription factor genes into target cells using integrating vectors, associated with risks due to ongoing or reactivated ectopic expression of the transcription factors, or insertional activation of genomic proto-oncogenes. Novel non-integrating vectors or protein transfer systems have begun to surmount this problem. However, much more serious concerns relate to the consequences of administering primitive pluripotent cells that may have the potential to form tumors, if differentiation is incomplete or inefficient. Second, there are significant challenges to the efficient differentiation of iPSCs into functional adult tissues. Protocols for differentiation of iPSCs towards even well-characterized hematopoietic stem cells are inefficient, inconsistent and result in aberrant or embryonic hematopoiesis. Design of methods for direct delivery or facilitation of homing of iPSCs or their progeny to appropriate locations in the body will also be a major challenge. While murine models are invaluable tools, it will be critical to develop more relevant models for clinical development of iPSCs. Murine and human embryonic stem cells behave quite differently in culture, require different cytokines and handling, and may be derived from different stage of embryonic development. Generation of murine iPSCs appears to be at least an order of magnitude more efficient that generation of human iPSCs. Telomeres in inbred laboratory mice are significantly longer than human telomeres, and may impact on the relative ease of immortalization of murine versus human cells and thus oncogenicity. Human iPSCs can be implanted in immunodeficient mouse strains and form teratomas, but the next steps in development, requiring functional differentiation and appropriate delivery or homing, may be impossible to model in xenografts. Scale-up of laboratory procedures developed in mice to human therapies would also be very difficult to develop solely using murine-murine or human-murine xenograft models. The rhesus macaque non-human primate (NHP) model will be a valuable resource to clear hurdles preventing clinical development. The close physiologic and genomic relationship between humans and NHPs results in cross-reactivity for most cytokines, antibodies and other reagents. Teratoma formation and other safety issues can be directly assessed utilizing autologous rhesus iPSCs. Differentiation, homing and other parameters critical for efficacy can be modeled. Tissue damage models such as pancreatic beta cell or hematopoietic stem cell ablation are well established in macaques. Rhesus embryonic stem cells were isolated prior to human ESCs, and their properties are well-characterized. Development of rhesus iPSCs at the NIH takes advantage of our unique expertise in NHP transplantation and in the development of novel cell and gene therapies in this valuable model. Our plans also focuses on the development of a suicide gene strategy to increase safety of utilization of iPSCs for tissue regeneration. There is a significant risk that residual pluripotent cells remaining following direct differentiation of iPSCs could form tumors in vivo. If integrating vectors are used to generate iPSCs, tumors could result from vector-related insertional mutagenesis or re-activation of reprogramming factors. Even if differentiation is complete and successful, iPSC-derived progeny might localize or proliferate inappropriately. In all these scenarios, the ability to ablate iPSCs in vivo would be desirable. Several promising suicide gene strategies have been developed over the past decade, allowing efficient killing of cells carrying the suicide gene vector via administration of a non-toxic drug. Several clinical trials utilizing allogeneic T cells carrying the herpes thymidine kinase (tk) suicide gene have been performed. Ganciclovir, a pro-drug only toxic to cells expressing herpes tk, was shown to ablate alloreactive T cells and successfully treat graft-versus-host disease. To avoid the immunogenicity of herpes tk, another promising suicide system utilizes human caspase 9 fused to the FK506 binding domain, allowing inducible dimerizer and caspase 9 activation following administration of an oral small molecule dimerizer. These suicide gene strategies hold great promise for iPSC safety, but need further clinical development in a relevant model such as the rhesus macaque. Thsi project began this year, and we have already published a paper taking advantage of our expertise in vector integration site retrieval and analysis to demonstrate that in human iPSCs, vector integration sites do not appear to play a role in promoting successful reprogramming of iPSCs. This is reassuring for at least preclinical and model development, allowing continued use of lentiviral vectors for reprogramming, given their much greater efficiency compared to non-integrating vectors. We have now optimized conditions to derive rhesus iPSCs, and have successfully shown that rhesus iPSCs can be created from rhesus marrow mesenchymal cells or from skin fibroblasts, utilizing either retroviral or lentiviral vectors. These clones are pluripotent as assayed in a murine teratoma assay, express all pluripotency markers, and can be differentiated to endodermal, mesodermal and ectodermal cell types. The conditions utilized for murine and human ESCs and iPSCs were not successful using rhesus cells, and we have developed new conditons, based on the optimal conditions for growing rhesus ESCs. Our rhesus iPSCs express alkaline phosphatase, shut off the reprogramming vectors and morphologically resemble rhesus ESCs. For in vivo studies in autologous rhesus, excision of the potentially-immunogenic reprogramming factors is likely required, so we have now created rhesus iPSCs with an exicisable reprogramming cassette, and shown all retained functions following cre-mediated excision. These cells are about to be tested in an autologous rhesus teratoma model. We have developed an in vivo bone organoid model in the rhesus, in collaboration with Pam Robey's group in NIDCR. We are currently testing the model with rhesus MSCs, and plan to move into rhesus iPSCs in the next several months. We have also begun to differentiate rhesus iPSCs to mature neutrophils, and will test their lifespan and function in vivo. Finally, we have introduced suicide genes into rhesus iPSCs in preparation for beginning in vivo suicide ablation studies.

通过干细胞特异转录因子的异位表达将出生后体细胞重新编程为诱导多能干细胞（iPSC），对于改善人类健康具有许多令人兴奋的潜在应用。 iPSC 最初是在小鼠模型中开发的，并通过囊胚互补测定显示出具有对所有组织做出贡献的潜力。仅仅几年后，使用一组类似的转录因子创建了人类 iPS 细胞，并证明可以在免疫缺陷小鼠中形成畸胎瘤，并与人类胚胎干细胞共享功能和基因表达特征。 然而，将 iPSC 推进临床再生医学应用存在许多障碍。 首先也是最重要的是安全问题。小鼠和人类 iPSC 最初都是通过使用整合载体将所需的转录因子基因引入靶细胞而衍生的，这与转录因子持续或重新激活的异位表达或基因组原癌基因的插入激活带来的风险相关。新型非整合载体或蛋白质转移系统已开始克服这个问题。然而，更严重的问题涉及如果分化不完全或效率低下，使用可能形成肿瘤的原始多能细胞的后果。其次，iPSC 有效分化为功能性成体组织面临重大挑战。 将 iPSC 分化为特征明确的造血干细胞的方案效率低下、不一致，并会导致异常或胚胎造血。设计直接递送或促进 iPSC 或其后代归巢到体内适当位置的方法也将是一个重大挑战。 虽然小鼠模型是非常宝贵的工具，但开发更相关的 iPSC 临床开发模型至关重要。小鼠和人类胚胎干细胞在培养中的表现截然不同，需要不同的细胞因子和处理，并且可能源自胚胎发育的不同阶段。鼠 iPSC 的生成似乎比人类 iPSC 的生成效率至少高一个数量级。 近交实验室小鼠的端粒明显长于人类端粒，可能会影响小鼠细胞与人类细胞永生化的相对容易程度，从而影响致癌性。人类 iPSC 可以植入免疫缺陷小鼠品系并形成畸胎瘤，但下一步的开发，需要功能分化和适当的递送或归巢，可能无法在异种移植物中建模。仅使用鼠-鼠或人-鼠异种移植模型，也很难将在小鼠中开发的实验室程序扩大到人类疗法。 恒河猴非人灵长类动物 (NHP) 模型将成为清除临床开发障碍的宝贵资源。 人类和 NHP 之间密切的生理学和基因组关系导致大多数细胞因子、抗体和其他试剂发生交叉反应。畸胎瘤形成和其他安全问题可以利用自体恒河猴 iPSC 直接评估。 可以对分化、归巢和其他对功效至关重要的参数进行建模。胰腺β细胞或造血干细胞消融等组织损伤模型在猕猴中已得到很好的建立。 恒河猴胚胎干细胞在人类 ESC 之前就已被分离出来，并且其特性已得到充分表征。 NIH 恒河猴 iPSC 的开发利用了我们在 NHP 移植以及在这一有价值的模型中开发新型细胞和基因疗法方面的独特专业知识。 我们的计划还侧重于开发自杀基因策略，以提高 iPSC 用于组织再生的安全性。 iPSC 直接分化后残留的多能细胞存在在体内形成肿瘤的重大风险。 如果使用整合载体生成 iPSC，则载体相关的插入突变或重编程因子的重新激活可能会导致肿瘤。 即使分化完全且成功，iPSC 衍生的后代也可能不适当地定位或增殖。在所有这些情况下，体内消除 iPSC 的能力将是令人期望的。在过去的十年中，已经开发了几种有前途的自杀基因策略，可以通过施用无毒药物来有效杀死携带自杀基因载体的细胞。已经进行了几项利用携带疱疹胸苷激酶 (tk) 自杀基因的同种异体 T 细胞的临床试验。更昔洛韦是一种前药，仅对表达疱疹 tk 的细胞有毒，它被证明可以消除同种反应性 T 细胞并成功治疗移植物抗宿主病。为了避免疱疹 tk 的免疫原性，另一种有前途的自杀系统利用与 FK506 结合结构域融合的人 caspase 9，在给予口服小分子二聚体后允许诱导二聚体和 caspase 9 激活。这些自杀基因策略对于 iPSC 的安全性有着巨大的希望，但需要在恒河猴等相关模型中进行进一步的临床开发。 该项目于今年开始，我们已经发表了一篇论文，利用我们在向量整合位点检索和分析方面的专业知识来证明，在人类 iPSC 中，向量整合位点似乎并未在促进 iPSC 成功重编程方面发挥作用。这至少对于临床前和模型开发来说是令人放心的，允许继续使用慢病毒载体进行重编程，因为与非整合载体相比，慢病毒载体的效率要高得多。我们现在已经优化了衍生恒河猴 iPSC 的条件，并成功证明可以利用逆转录病毒或慢病毒载体从恒河猴骨髓间充质细胞或皮肤成纤维细胞中产生恒河猴 iPSC。 这些克隆在鼠畸胎瘤测定中具有多能性，表达所有多能性标记物，并且可以分化为内胚层、中胚层和外胚层细胞类型。用于鼠类和人类 ESC 和 iPSC 的条件在恒河猴细胞中并不成功，我们根据恒河猴 ESC 生长的最佳条件开发了新的条件。我们的恒河猴 iPSC 表达碱性磷酸酶，关闭重编程载体，并且在形态上类似于恒河猴 ESC。对于自体恒河猴的体内研究，可能需要切除潜在的免疫原性重编程因子，因此我们现在创建了具有可切除重编程盒的恒河猴 iPSC，并显示了 cre 介导的切除后所有保留的功能。这些细胞即将在自体恒河猴畸胎瘤模型中进行测试。我们与 NIDCR 的 Pam Robey 团队合作，开发了恒河猴体内骨类器官模型。我们目前正在用恒河猴 MSC 测试该模型，并计划在未来几个月内转向恒河猴 iPSC。我们还开始将恒河猴 iPSC 分化为成熟的中性粒细胞，并将测试它们的寿命和体内功能。 最后，我们将自杀基因引入恒河猴 iPSC 中，为开始体内自杀消融研究做好准备。