STEM CELL GENE THERAPY USING LONG TERM BONE MARROW CULTURE TRANSDUCTION PROTOCOL

使用长期骨髓培养转导方案的干细胞基因治疗

• 批准号: 6453734
• 负责人: M ROSENZWEIG
• 金额: $ 11.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6453734
• 关键词: Mammalia; animal tissue; gene therapy; hematology; immunology; inflammation

Attempts to introduce genes into hematopoietic stem cells (HSCs) have been disappointing In general, using murine retroviral based vectors to transduce CD34+ cells in vitro have resulted in a low level (0 1-1%) of gene marking for a limited period of time Studies in canines demonstrated that LTBMC transduced with retroviral vectors gave higher levels of gene marking in vivo The advantage of this technique is that by using unfractionated marrow there is no preselection; stem cells and retrovirus are able to colocalize on the stroma; the system selects for LTCIC; transduction occurs 3 times over 21 days following feeding so that cells are in cycle, improving transduction efficiency We examined the utility of this technique in rhesus macaques In 2 animals that underwent this autologous transplantation procedure, no conditioning regime was implemented Unfractionated marrow was transduced for 15 - 18 days with a PA317LN based vector Animals were monitored f or "neo" the marker gene in peripheral blood and bone marrow We observed between 1% and 3% gene marking in peripheral blood for approximately 3 months The frequency of neo in bone marrow was generally lower than that in peripheral blood This technique appears encouraging as our data are similar to what has been observed by others, but our protocol is in the absence of any conditioning regimen Recent efforts have focused on attempts to use LTBMC to transduce HSC using a retroviral vector encoding GFP Initial experiments using a retroviral vector provided by R Howley resulted in a failure to maintain LTBMC, which appears to be due to soluble factors provided by the retroviral producer cell line These results suggest that the LTBMC technique may be difficult to generalize to other retroviral vectors Results of these studies may prove useful in efforts to optimize genetic modification of human hematopoietic stem cells

试图将基因引入造血干细胞（HSC） 通常，使用鼠逆转录病毒感到失望 体外转导CD34+细胞的向量导致低水平 （0 1-1％）基因标记在有限的时间研究中 犬类证明了用逆转录病毒向量转导的LTBMC 给出了更高水平的基因标记体内的优势 技术是通过使用未分离的骨髓没有 预选；干细胞和逆转录病毒能够在 基质；系统选择LTCIC；转移发生3次 进食后21天，以使细胞处于周期状态，改善 转导效率我们检查了此技术的实用性 经历了这种自体的两只动物的恒河猕猴 移植程序，未实施任何条件制度 用PA317LN转导未分离的骨髓15-18天 监测基于基于的载体动物或“ neo”标记基因 外周血和骨髓我们观察到1％至3％的基因 在外周血中标记大约3个月的频率 骨髓中的NEO通常低于外周 血液这种技术似乎令人鼓舞，因为我们的数据与 其他人观察到的是什么，但是我们的协议是在不存在的情况下 在任何条件方案中，最近的努力都集中在尝试 使用LTBMC使用编码GFP的逆转录病毒矢量转导HSC 使用R Howley提供的逆转录病毒载体的初始实验 导致无法维护LTBMC，这似乎是由于 逆转录病毒生产商细胞系提供的可溶性因子 结果表明LTBMC技术可能很难 将这些研究的其他逆转录病毒载体结果推广 证明在优化人类的遗传修饰方面有用 造血干细胞