Primate iPS lines using Retrovirus and TAT Protein Transduction

描述（由申请人提供）：本申请的最重要假设是，非人灵长类动物（NHP）诱导的多能干细胞系（iPS）的产生和表征将加速人类替代组织和器官的发育。猕猴与人类有着极其密切的系统发育关系，并发展出自然或实验诱导的疾病状态，准确地模仿人类的状况。NHPs可以提供一个独特的临床前模型来测试iPS细胞和再生医学方法在治疗和治愈疾病方面的有效性，而其他模型可能无法做到这一点。通过相对简单的重编程步骤，从皮肤成纤维细胞中产生自体多能干细胞的能力是一项量子科学进步，将在基础研究和应用临床医学的许多领域产生重大影响。然而，在重编程方案中使用病毒载体（就像现在存在的那样）在患者中使用是极不可能的。因此，研究无病毒重编程的替代方法是至关重要的。TAT是一种短的11个氨基酸蛋白转导结构域（PTD），由HIV反激活子衍生而来，已被用于在体外和体内将大的（~110kD）生物活性蛋白直接传递到细胞中。TAT融合蛋白和肽也被用于治疗癌症、炎症和其他疾病的小鼠模型。这是我们的假设，重组TAT和能够重编程细胞进入多能状态。一旦很好地表征，通过病毒或蛋白质转导方法衍生的细胞系可用于许多不同疾病的NHP临床前模型。因此，我们提出：(1)从非人灵长类动物——麻瓜猕猴（cynomolgus macaque）中产生诱导多能干细胞（iPS）；(2)利用HIV衍生的TAT细胞穿透基序修饰的重组蛋白来确定是否可以产生非病毒的麻瓜猕猴iPS细胞，以帮助蛋白质在质膜上易位；(3)表征和比较病毒和蛋白质转导产生的iPS细胞。

DESCRIPTION (provided by applicant): The overriding hypothesis of this application is that the generation and characterization of non Human primate (NHP) induced pluripotent stem cell lines (iPS) will hasten the development of replacement tissues and organs in Humans. Macaque monkeys share an extremely close phylogenetic relationship with Man, and develop natural or experimentally induced disease states which accurately mimic the Human condition. NHPs can provide a unique preclinical model to test the usefulness of iPS cells and regenerative medicine approaches to the treatment and cure of disease, where other models may fail. The ability to generate autologous, pluripotent stem cells from skin fibroblasts, by relatively simple reprogramming steps, is a quantum scientific advance which will have major ramifications in many areas of basic research and applied clinical medicine. It is highly unlikely, however, that the use of viral vectors in the reprogramming protocol (as it exists now) will be acceptable for use in patients. It is vital, therefore, that alternative ways of reprogramming, without virus, be investigated. The short 11 amino acid protein transduction domain (PTD) derived from the HIV transactivator, TAT has been used to deliver large (~110kD), biologically active proteins directly into cells in vitro and in vivo. TAT fusion proteins and peptides have also been used to treat mouse models of cancer, inflammation and other diseases. It is our hypothesis that recombinant TAT and be able to reprogram cells into a pluripotent state. Once well characterized, lines derived by viral or protein transduction methods can be used in NHP preclinical models for a number of different diseases. We therefore propose to: (1) generate induced pluripotent stem cell lines (iPS) from the non-human primate, Macaca fascicularis (cynomolgus macaque) (2) To determine if M. fascicularis iPS cells can be produced non-virally using recombinant proteins modified with an HIV derived, TAT cell penetrating motif to aid in protein translocation across the plasma membrane and (3) characterize and compare iPS lines generated by viral and protein transduction.