Modeling RNA-based HIV gene therapeutics in SCID-hu mice

描述(由申请人提供)：动物模型对 基因治疗策略的临床前评估。SCID-HU小鼠， 拥有正常功能的人胸腺，维持AS的胸腺生成 长达一岁，容易感染艾滋病毒。我们和其他人有 进一步发展了该系统用于外源干细胞转移。纯化的CD34 逆转录病毒载体转导基因后的造血祖细胞 被引入胸腺移植物，发育成正常的人T淋巴细胞。我们有 最近还证明了载体递送抗HIV基因治疗 核酶等构建物在这些细胞中保留和表达 成熟。病毒诱导的细胞耗竭与基因转导的结合 CD34细胞重建在相对较短的时间内发生，为我们提供了 具有独特的实验系统，在其中解决许多关键问题 与基因治疗方法的成功相关。另外，目前， SCID-HU系统是唯一一个在体内准确评估 载体转导的造血细胞的胸腺生成潜能和对HIV的抗性 祖细胞。最近发生了几个令人兴奋的新发展 干细胞生物学、慢病毒基因转移载体、核酶 靶向和基于RNA的疗法，因此，目前阶段是 准备取得成功。在本提案中，我们希望利用这些 新技术，并在我们最新进展的基础上再接再厉。这里概述的实验 (项目2)是互动的，与随行的目标是互补的 互动R01提案(项目1)，由J.Rossi提出，标题为“组合使用 抗艾滋病毒核糖核酸疗法。“我们提议的具体目标是： L)确定逆转录病毒转导的PolIII启动子驱动的效果 核仁、核仁和细胞质靶向的抗HIV核酶TAR和RBE诱饵， 无论是单独的还是组合的，都是关于谱系的具体区分 CD34细胞体外进入巨噬细胞和体内进入胸腺细胞 SCID-Hu THY/Liv移植及基于RNA的作用机制研究 分化细胞中的治疗学。 2)确定不同抗HIV-1 RNA的体内保护作用， HIV-1后SCID-Hu小鼠THY/LIV移植的单独和联合研究 挑战。 3)确定新一代基于SIV的慢病毒载体的能力 转导各种造血祖细胞，包括CD34细胞和 新发现的原始造血细胞，即CD34和KDR 前体细胞以及侧群(SP)细胞。 4)测定慢病毒载体的植入和胸腺生成能力。 转导的原始造血祖细胞，即a)CD34和KDR 细胞，b)SCID-Hu小鼠胸腺微环境中的SP细胞。

DESCRIPTION (provided by applicant): Animal models are critical for the preclinical evaluation of gene therapeutic strategies. The SCID-hu mouse, harboring a normally functioning human thymus, sustains thymopoiesis for as long as one year and is susceptible to HIV infection. We and others have developed this system further for exogenous stem cell transfer. Purified CD34+ hematopoletic progenitor cells when gene transduced with retroviral vectors and introduced into thymic grafts, develop into normal human T lymphocytes. We have also recently demonstrated that vector delivered anti-HIV gene therapeutic constructs such as ribozymes were retained and expressed in these cells as they mature. The combination of virus-induced cell depletion and gene transduced CD34+ cell reconstitution taking place in a relatively short time, provides us with a unique experimental system in which to address many critical issues relevant for the success of gene therapy approaches. Furthermore, at present, the SCID-hu system is the only in vivo system to accurately evaluate the thymopoietic potential and HIV resistance of vector transduced hematopoietic progenitor cells. Several new exciting developments occurred recently in the areas of stem cell biology, lentiviral gene transfer vectors, ribozyme targeting, and RNA-based therapeutics and, therefore, the stage is currently set to achieve success. In the present proposal, we would like to exploit these new technologies and build upon our recent progress. Experiments outlined here (Project 2) are interactive and complimentary to the objectives of accompanying interactive R01 proposal (Project 1) by J. Rossi entitled "Combinatorial use of anti-HIV RNA-based therapeutics." The specific objectives of our proposal are: l) Determine the effect of retrovirally transduced pol III promoter driven nucleolar, nuclear and cytoplasmtargeted anti-HIV nbozymes TAR and RBE decoys, either individually or in combination, on the lineage specific differentiation of CD34+ cells into macrophages in vitro and into thymocytes in vivo in the SCID-hu thy/liv grafts and investigate the mechanism of action of RNA-based therapeutics in differentiated cells. 2) Determine the in vivo protective effects of different anti-HIV-1 RNAs, individually and in combination, in SCID-hu mice thy/liv grafts after HIV-1 challenge. 3) Determine the ability of new generation SIV-based lentiviral vectors to transduce various hematopoietic precursor cells that include CD34+ cells and the newly described primitive hematopoietic cells, namely CD34+ and KDR+ precursor cells, as well as side population (SP) cells. 4) Determine the engraftment and thymopoietic potential of lentivirally transduced primitive hematopoietic precursor cells i.e., a) CD34+ and KDR+ cells, b) SP cells in the SCID-hu mouse thymic microenvironment.