Testing the safety and efficacy of inhibitory RNA mediated purine analog resistan

描述（由申请人提供）：基因疗法长期以来一直被吹捧为有望治愈各种先天性疾病。然而，由于一些问题阻碍了当前技术的广泛应用，基因治疗试验在数量和规模上仍然很小。体外操作细胞的低转导、低长期表达和植入失败延缓了基因治疗的实际进展。因此，从单基因异常患者中选择或扩增基因校正细胞群的能力可能会增强基因治疗的适用性。先前研究的体内细胞选择策略有效性有限和/或需要使用高毒性药物。我们发现，提供干扰RNA介导的嘌呤类似物抗性（iPAR）允许使用嘌呤类似物6-硫鸟嘌呤（6TG）高效地在体外和体内选择慢病毒转导的小鼠造血祖细胞。我们假设iPAR和嘌呤类似物（iPAR/PA）可以作为一种安全有效的手段，在体内选择自体的、基因校正的造血细胞来治疗人类疾病。虽然我们相信我们的初步数据是令人信服的，但大量的临床前研究对于确定iPAR在人类患者中的适用性至关重要。通过本文提出的研究，我们计划为iPAR/PA在造血细胞选择中的有效性提供进一步的实验证据。该项目的具体目标是：1)确定iPAR/PA在人类造血祖细胞中的有效性；2)确定慢病毒递送iPAR和6TG治疗的毒性，因为它与病毒整合到基因组有关。这些目标将主要通过以下方法实现：1)人类CD34+脐带血细胞（UCBC）在免疫功能低下的NOD/SCID小鼠中的异种移植；2)通过LAM-PCR和测序鉴定慢病毒整合位点，以及功能转化分析，包括改良的髓系永生化实验和肿瘤易感小鼠模型。这些研究的积极结果将证明进一步在大型动物或非人类灵长类动物以及最终在人类患者中研究iPAR/PA。多种人类血液病将受益于这种体内细胞选择策略，包括但不限于先天性骨髓衰竭综合征（Fanconi和Diamond-Blackfan贫血）、慢性肉芽肿病、wiscottt - aldrich综合征、血红蛋白病（镰状细胞病、地中海贫血），甚至血友病。公共卫生相关性：这些实验的目标是改善目前治疗血液系统遗传疾病的努力。该策略包括为基因校正后的细胞提供耐药性，然后在体内选择这些细胞，使用一种可能比之前研究过的其他药物更安全的药物。这一策略可应用于多种人类疾病，如骨髓衰竭综合征、血红蛋白病和血友病。

DESCRIPTION (provided by applicant): Gene therapy has long been touted to hold the promise of cure for a variety of congenital diseases. However, gene therapy trials are still small in number and scale due to several problems that have prevented wider application of current technology. Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy. Therefore, the ability to select for or amplify a genetically corrected population of cells from patients with single gene abnormalities might enhance the applicability of gene therapy. Previously investigated strategies for in vivo cell selection have had limited effectiveness and/or require the use of highly toxic medications. We have found that the provision of interfering RNA mediated purine analog resistance (iPAR) allows for efficient in vitro and in vivo selection of lentivirus transduced murine hematopoietic progenitor cells using the purine analog, 6-thioguanine (6TG). We hypothesize that iPAR and purine analogs (iPAR/PA) can be used as a safe and effective means for in vivo selection of autologous, genetically corrected hematopoietic cells to cure human diseases. While we believe that our preliminary data are compelling, substantial pre-clinical studies are essential to determine the applicability of iPAR for use in human patients. With the studies proposed herein, we plan to provide further experimental evidence of the effectiveness of iPAR/PA in hematopoietic cell selection. The specific aims of this project are to 1) determine the effectiveness of iPAR/PA in human hematopoietic progenitor cells and 2) determine the toxicity of lentivirus delivered iPAR and treatment with 6TG as it relates to virus integration into the genome. These aims will be accomplished primarily by using 1) xenografts of human CD34+ umbilical cord blood cells (UCBC) in immunocompromised NOD/SCID mice and 2) identification of lentiviral integration sites by LAM-PCR and sequencing, as well as functional transformation assays including a modified myeloid immortalization assay and a tumor prone mouse model. Positive findings from these studies will justify further investigation of iPAR/PA in larger animals or non-human primates and eventually human patients. A variety of human hematologic diseases would benefit from this strategy of in vivo cell selection, including, but not limited to congenital bone marrow failure syndromes (Fanconi and Diamond-Blackfan anemias), chronic granulomatous disease, Wiscott-Aldrich syndrome, hemoglobinopathies (sickle cell disease, thalassemias), and even hemophilias. PUBLIC HEALTH RELEVANCE: The goal of these experiments is to improve current efforts to cure genetic diseases of the blood system. The strategy involves providing drug resistance to the cells that have been genetically corrected, followed by selection of these cells within the body using a medication that may be safer than others that have been previously studied. This strategy could be applied to a variety of human diseases such as bone marrow failure syndromes, hemoglobinopathies, and hemophilias.