Stem Cell Cycling and Transduction

描述（由申请人提供）：近期和长期的职业目标。将抗HIV组分导入造血干细胞（HSC）为治疗艾滋病和开发治疗性艾滋病疫苗提供了巨大的希望。目前通过使用重组细胞因子的干细胞的离体操作和扩增通常导致体内再增殖的减少或HSC多能性的丧失。这一领域的进展需要详细说明确定的干细胞周期条件和艾滋病毒复制的知识。该奖项的候选人计划扩大她在干细胞研究方面的科学背景，并研究细胞周期检查点蛋白p21 ciplNvAF 1/sd_l（以下简称p21）在HSC循环中的生理作用以及在HIV衍生载体的转导效率中的作用。一旦K18奖项完成，学员计划与赞助商合作开发基因治疗策略，以产生HSC，从而产生对HIV-1致病作用具有抵抗力的CD 4 + T细胞。 研究计划大纲。艾滋病的基因治疗，即将抗HIV基因转移到HSC中的策略，是近年来尚未实现的重要前提之一，因为转导效率中等（用HIV衍生载体为30 - 65%），并且体内携带转染基因的细胞极低，例如动物和人中为0.1-1%（4，21，33）。HSC的周期状态在转导效率和造血细胞再增殖中起重要作用。单个分子p21能够在体内决定干细胞动力学（15），并且在其不存在的情况下，干细胞离开静止期并进入细胞周期（15）。我们推测，细胞周期检查点分子p21的修饰可以将HSC从静止状态释放到循环状态，这可能会增加转导效率而不丧失多能性。为了验证这一假设，我们计划：1）采用RNAi技术沉默p21基因在HSC中的表达;和2）使用不同的体外和体内模型，以确定转导效率和HSC多潜能性后，与常用的载体在基因治疗策略。 本申请的目的是使初级研究员能够拓宽她的科学背景，特别是在她的研究中使用干细胞。这个K18奖将使她有机会将干细胞循环的知识应用于艾滋病毒/艾滋病研究，并找到更好的预防或治疗艾滋病毒/艾滋病的方法。

DESCRIPTION (provided by applicant): Immediate and long-term career goals. Transduction of anti-HIV components into hematopoietic stem cell (HSC) provides great promise for the treatment of AIDS and the development of a therapeutic AIDS vaccine. The current ex-vivo manipulation and expansion of stem cells by using recombinant cytokines generally results in a decrease of in vivo repopulation or a loss of HSC multipotentiality. Progress in this area needs elaboration of defined stem cell cycle conditions and knowledge of HIV replication. The candidate of this award plans to broaden her scientific background in stem cell research, and to study the physiologic role of a cell cycle checkpoint protein p21 ciplNvAF1/sd_l (hereafter p21) in HSC cycling as well as in transduction efficiency with HIV derived vectors. Once the K18 award is completed, the trainee plans to collaborate with the sponsor to develop gene therapy strategies to generate HSC and therefore CD4+ T cells which are resistant to the pathogenic effects of HIV-1. Outline of the research plan. Gene therapy of AIDS, the strategy of transferring anti-HIV genes into HSC, has been one of the great yet unfulfilled premises of recent years, since the efficiency of transduction is moderate (30 - 65% with HIV derived vectors) and the cells which remain to carry transfected genes are extremely low in vivo, such as 0.1-1% in animals and in human (4,21,33). The cycle status of HSC plays a cardinal role in efficiency of transduction and in hematopoietic cell re-population. A single molecule, p21, is able to dictate stem cell kinetics in vivo (15), and in its absence, stem cells leave a quiescent stage and enter the cell cycle (15). We hypothesize that modification of the cell cycle checkpoint molecule, p21, can release HSC from quiescence into cycling, and this may increase transduction efficiency without loss of multipotentiality. To test this hypothesis we plan to: 1) employ the RNAi technique to silence p21 gene expression in HSC; and 2) use different in vitro and in vivo models to determine the transduction efficiency and HSC multipotentiality after transduction with vectors commonly used in gene therapy strategies. The objective of this application is to enable a junior investigator to broaden her scientific background, in particular, to use stem cells in her research. This K18 award will give her an opportunity to apply the knowledge of stem cell cycling to HIV/AIDS research and to find the better ways of prevention or cure of HIV/AIDS.