AAV TRANSDUCTION OF QUIESCENT HEMATOPOIETIC STEM CELLS

静态造血干细胞的 AAV 转导

• 批准号: 6189141
• 负责人: Saswati Chatterjee
• 金额: $ 34.71万
• 依托单位: CITY OF HOPE NATIONAL MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6189141
• 关键词: CD34 molecule; SCID mouse; adeno associated virus group; bone marrow transplantation; cell transplantation; clinical research; gene therapy; hematopoietic stem cells; human subject; laboratory mouse; polymerase chain reaction; technology /technique development; transfection /expression vector; xenotransplantation

The transfer of therapeutic genes into hematopoietic stem cells (HSC) offers the potential of effective and permanent treatment for a wide spectrum of diseases including metabolic diseases, infections and cancer. However, despite promising results in vitro and in murine models, the results of human gene therapy trials to date have revealed disappointingly low retrovirus-mediated gene transfer frequencies into pluripotential long-term repopulating cells. While the lentivirus vectors may offer one alternative, their inability to integrate in quiescent cells and safety issues make the continued pursuit of alternative approaches for gene delivery to HSC imperative for continued progress. Adeno-associated irus (AAV) vectors are rapidly emerging as gene transfer vectors with unique properties including those of non-pathogenicity and low immunogenicity. We and others have shown the ability of AAV vectors to efficiently transduce a variety of non- dividing cells both in vivo and in vitro. We have also recently demonstrated the capacity of AAV vectors to transduce primitive clonogenic CD34+ and CD34+/CD38- human hematopoietic progenitor cells and to display chromosomal integration. These properties make AAV vectors attractive for further evaluation to for gene delivery to HSC. Here we propose to test the proficiency of AAV vectors to transduce non-dividing and metabolically quiescent CD34+38- hematopoietic progenitor cells. AAV transduction will be tested in vitro in populations defined as being in G0 based upon their 2n DNA and low RNA content and multi-potential clonogenic properties. The full range of hematopoietic potentials of AAV transduced CD34+38- and G0 cells will be tested in a chimeric xenograft model utilizing transplantation of AAV transduced hematopoietic cells into immunodeficient NOD/SCID mice. AAV transduction of a newly defined lin-CD34-38- primitive hematopoietic progenitor cell will also be tested. Experiments directly comparing retroviral and AAV transduction will test the transduction potentials of the two vector systems in vitro and in NOD/SCID mice. Transduction of self-renewing pluripotential stem cells will be tested in a murine serial transplant model. In all three systems to be utilized, the ability of a single transduced progenitor cell to give rise to multi-lineage progeny will be tested using sensitive PCR assays developed for our vectors. Lastly, information obtained from these experiments will be used to design a therapeutic marrow transplant strategy utilizing AAV transduction of the ApoAI Milano gene for the treatment of atherosclerosis. It is expected that knowledge gained from these experiments will provide an accurate assessment of the transduction potential of AAV vectors for hematopoietic stem cell gene therapy.

将治疗性基因转移到造血干细胞（HSC）中，为包括代谢性疾病、感染和癌症在内的广泛疾病提供了有效和永久治疗的潜力。然而，尽管在体外和小鼠模型中取得了令人鼓舞的结果，但迄今为止人类基因治疗试验的结果显示，逆转录病毒介导的基因转移到多能长期再生细胞的频率很低，令人失望。虽然慢病毒载体可能提供了一种替代方法，但它们无法整合到静止细胞中，并且存在安全性问题，因此继续寻求将基因传递到HSC的替代方法是继续取得进展的必要条件。腺相关病毒（Adeno-associated virus， AAV）作为一种基因转移载体正迅速兴起，它具有非致病性和低免疫原性等独特特性。我们和其他人已经证明了AAV载体在体内和体外有效转导多种非分裂细胞的能力。我们最近也证明了AAV载体转导原始克隆性CD34+和CD34+/CD38-人造血祖细胞并显示染色体整合的能力。这些特性使得AAV载体对进一步评估基因传递到HSC具有吸引力。在这里，我们提出测试AAV载体转导非分裂和代谢静止的CD34+38-造血祖细胞的能力。AAV的转导将在体外测试，根据其2n DNA和低RNA含量以及多潜在克隆特性，定义为G0的人群。AAV转导的CD34+38-和G0细胞的造血潜能将在嵌合异种移植模型中进行测试，该模型利用AAV转导的造血细胞移植到免疫缺陷的NOD/SCID小鼠中。新定义的lin-CD34-38原始造血祖细胞的AAV转导也将被测试。直接比较逆转录病毒和AAV转导的实验将测试两种载体系统在体外和NOD/SCID小鼠中的转导潜力。自我更新的多能干细胞的转导将在小鼠系列移植模型中进行测试。在这三种系统中，单个转导祖细胞产生多系后代的能力将使用为我们的载体开发的敏感PCR检测方法进行测试。最后，从这些实验中获得的信息将用于设计一种利用AAV转导ApoAI米兰基因治疗动脉粥样硬化的治疗性骨髓移植策略。预计从这些实验中获得的知识将为AAV载体在造血干细胞基因治疗中的转导潜力提供准确的评估。