Humanized Rag2-/-gammac-/-mice for HIV infection and gene therapy

描述(申请人提供)：通过使用合适的动物模型来模拟正常运作的人类造血系统所提供的生理条件，将极大地促进艾滋病毒发病机制的研究和艾滋病毒-1基因治疗结构的临床前评估。由于以前使用的异种移植SCID-HU小鼠模型缺乏产生初级免疫反应的能力，因此只能在没有主动免疫反应的情况下评估病毒对人类细胞的致病作用。因此，在这些系统中缺少一个重要的生理维度。这一缺陷现在有可能通过使用新出现的具有免疫能力的人源化小鼠模型来纠正。将人CD34造血干细胞注射到免疫缺陷的新生Rag2-/-YC-/-小鼠体内，导致多血统的人类造血和功能免疫系统的产生。最近的证据表明，这些人源化小鼠对播散性艾滋病毒感染是允许的。由于具有多系人类细胞植入能力和对HIV感染的易感性，该模型显示出在基于干细胞的环境中评估抗HIV基因治疗结构的巨大潜力。基因修饰的细胞现在可以测试它们的免疫重建能力。我们最近在构建和评估几个有前景的抗HIV RNA抑制分子方面取得了进展，如siRNAs，并建议利用这个新的小鼠模型来回答与基因治疗方法成功相关的几个重要问题。我们的具体目标是：1.进一步建立人源化的Rag2-/-YC-/-小鼠HIV感染模型，以评价抗HIV基因的治疗构建；2.评价抗HIV基因转导的CD34造血祖细胞在人源化Rag2-/-YC-/-小鼠体内的植入潜能和多系分化；3.检测抗HIV基因在终末分化的T细胞和巨噬细胞中的表达，并评价其对HIV-1攻击的体内外抗性。4.评价转基因造血细胞亚群的功能能力和人源化Rag2-/-YC-/-小鼠产生正常免疫反应的能力：这些研究将有助于开发新的HIV/AIDS研究动物模型，并评估控制HIV感染的新的基因治疗方法。

DESCRIPTION (provided by applicant): Studies on HIV pathogenesis and preclinical evaluation of HIV-1 gene therapeutic constructs would be greatly facilitated by the use of a suitable animal model that can mimic the physiological conditions afforded by a normally functioning human hematopoietic system. As the previously used xenograft SCID-hu mouse models lacked the capacity for generating primary immune responses, viral pathogenic effects on the human cells could only be evaluated in the context of the absence of an active immune response. Thus an important physiological dimension was missing in these systems. This deficiency can now be potentially rectified by the use of a newly emerged immunocompetent humanized mouse model. Injection of human CD34 hematopoietic stem cells into immunodeficient neonatal Rag2-/-yc-/- mice results in multi-lineage human hematopoiesis and the generation of a functional immune system. Recent evidence showed that these humanized mice are permissive for disseminated HIV infection. With the capacity for multi-lineage human cell engraftment and susceptibility for HIV infection, this model shows great potential to evaluate anti-HIV gene therapeutic constructs in a stem cell-based setting. Gene modified cells can now be tested for their capacity for immune reconstitution. We recently made progress in the construction and evaluation of several promising anti-HIV RNA inhibitory molecules such as siRNAs and propose to utilize this new mouse model to answer several important questions relevant for the success of gene therapy approaches. Our specific aims are to: 1. Further develop the humanized Rag2 common gamma chain double knock out (Rag2-/-yc-/-) mouse model for HIV infection to evaluate anti-HIV gene therapeutic constructs, 2. Evaluate the engraftment potential and multi-lineage differentiation of anti-HIV gene transduced CD34 hematopoietic progenitor cells in humanized Rag2-/-yc-/- mice, 3. Determine the expression of anti-HIV genes in terminally differentiated T cells and macrophages and evaluate their resistance to HIV-1 challenge in vitro and in vivo. 4. Evaluate the functional competence of transgenic hematopoietic cell subsets and the capacity of humanized Rag2-/-yc-/- mice bearing transgenic cells to give rise to normal immune responses: These studies will be helpful in developing new animal models for HIV/AIDS research, and in evaluating novel gene therapy approaches to control HIV infection.