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

描述（由申请方提供）：动物模型对于 基因治疗策略的临床前评估。SCID-hu小鼠， 拥有正常功能的人类胸腺，维持胸腺生成， 长达一年，易感染艾滋病病毒。我们和其他人已经 进一步开发了用于外源性干细胞转移的该系统。纯化的cd 34 + 当用逆转录病毒载体进行基因转导时， 引入胸腺移植物，发育成正常的人类T淋巴细胞。我们有 最近也证明了载体传递的抗HIV基因治疗剂 构建体如核酶被保留并在这些细胞中表达， 成熟病毒诱导的细胞耗竭和基因转导的组合 CD 34+细胞在相对较短的时间内发生重建，为我们提供了 通过独特的实验系统来解决许多关键问题 与基因治疗方法的成功有关。此外，目前， SCID-hu系统是唯一能准确评估 载体转导的造血干细胞的胸腺生成潜能和HIV抗性 祖细胞最近，一些新的令人兴奋的发展发生在 干细胞生物学领域，慢病毒基因转移载体，核酶 靶向和基于RNA的治疗，因此，目前的阶段是 准备取得成功。在本提案中，我们希望利用这些 新技术，并建立在我们最近的进展。这里概述的实验 （项目2）是互动和互补的目标，陪同 J. Rossi的交互式R 01提案（项目1），题为“ 基于抗HIV RNA的疗法。“我们建议的具体目标是： l）确定逆转录病毒转导的pol III启动子驱动的细胞因子的作用。 核仁、核和细胞质靶向的抗HIV核酶TAR和RBE诱饵， 单独或组合地，对谱系特异性分化 CD 34+细胞在体外转化为巨噬细胞，在体内转化为胸腺细胞。 SCID-hu thy/liv移植物，并研究基于RNA的 分化细胞中的治疗剂。 2)确定不同抗HIV-1 RNA的体内保护作用， 在HIV-1感染后，在SCID-hu小鼠中单独和联合进行thy/liv移植， 挑战. 3)确定新一代基于SIV的慢病毒载体 包括CD 34+细胞在内的多种造血前体细胞， 新描述的原始造血细胞，即CD 34+和KDR+ 前体细胞以及侧群（SP）细胞。 4)确定慢病毒的植入和胸腺生成潜力 转导的原始造血前体细胞即，a）CD 34+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.