Efficacy and Safety of Foamy Retroviral Vectors for AIDS Gene Therapy

描述(申请人提供)：治愈艾滋病的一种有希望的方法是使用含有抑制艾滋病毒复制的转基因的造血干细胞(HSCs)进行基因治疗。造血干细胞可以产生参与HIV致病的所有细胞：T淋巴细胞、巨噬细胞、树突状细胞和小胶质细胞，因此有望成为艾滋病基因治疗的靶点。然而，无法有效地向造血干细胞输送抗艾滋病毒转基因一直是临床试验的障碍。我们建议开发抑制HIV复制的泡沫逆转录病毒载体，以实现针对艾滋病的HSC基因治疗。泡沫载体能高效、稳定地转导HSCs，可能非常适合艾滋病的基因治疗。将评估表达靶向TAT/REV的短发夹状RNA(ShRNAs)、靶向CCR5的核酶、TAR诱饵和抑制HIV进入的多肽的泡沫载体是否能够稳定和特异性地抑制CD34来源的巨噬细胞的HIV复制。将进行实验以证实在转导的HIV耐药细胞中HIV vRNAs特异性减少，并将评估shRNA表达的潜在毒性。我们将确定转导的造血祖细胞的集落形成能力是否发生改变，或者shRNA表达是否影响抗HIV巨噬细胞中宿主基因的表达。非肥胖糖尿病/重度NOD/SCID联合免疫缺陷异种移植模型将用于评估体内分化的人巨噬细胞和T淋巴细胞是否保持对HIV的耐药性，并确定是否可以通过体内选择来增加功能性抗HIV再繁殖细胞的数量。最后，在这些NOD/SCID实验中，我们还将确定泡沫载体在人类再生细胞中的整合位置，这是目前面临逆转录病毒基因治疗的关键问题。拟议的实验旨在系统地开发安全有效的泡沫shRNA载体，以便我们在未来的研究中评估其在灵长类动物模型中的有效性，并最终在临床试验中使用。

DESCRIPTION (provided by applicant): One promising approach to cure AIDS is by gene therapy using hematopoietic stem cells (HSCs) containing transgenes that inhibit HIV replication. HSCs are promising targets for gene therapy of AIDS as they produce all of the cells involved in HIV pathogenesis; T lymphocytes, macrophages, dendritic cells and microglial cells. However, the inability to efficiently deliver anti-HIV transgenes to HSCs has been a roadblock in clinical trials. We propose to develop foamy retroviral vectors that inhibit HIV replication towards a HSC gene therapy for AIDS. Foamy vectors may be well suited for gene therapy of AIDS as they can efficiently and stably transduce HSCs. Foamy vectors expressing short hairpin RNAs (shRNAs) targeted to tat/rev, a ribozyme targeted to CCR5, a TAR decoy, and a peptide that inhibits HIV entry will be evaluated for their ability to deliver stable and specific inhibition of HIV replication to CD34-derived macrophages. Experiments will be performed to confirm that HIV vRNAs are specifically reduced in transduced HIV-resistant cells and potential toxicity from shRNA expression will be evaluated. We will determine if the colony-forming ability of transduced hematopoietic progenitors is altered or if shRNA expression affects host gene expression in HIV-resistant macrophages. The non-obese diabetic/severe combined immunodeficient NOD/SCID xenotransplantation model will be used to evaluate whether HIV resistance is maintained in human macrophages and T lymphocytes differentiated in vivo and determine if we can increase the numbers of functional HIV-resistant repopulating cells by in vivo selection. Finally in these NOD/SCID experiments we will also determine foamy vector integration sites in human repopulating cells which is a critical issue currently facing retroviral gene therapy. The proposed experiments are designed to systematically develop safe and effective foamy shRNA vectors for future studies where we assess their efficacy in primate models and eventually in clinical trials.