Efficacy and Safety of Foamy Retroviral Vectors for AIDS Gene Therapy

描述（由申请人提供）：一种有希望治愈艾滋病的方法是使用含有抑制HIV复制的转基因的造血干细胞（HSC）进行基因治疗。造血干细胞是艾滋病基因治疗的有希望的靶点，因为它们产生参与HIV发病机制的所有细胞; T淋巴细胞、巨噬细胞、树突状细胞和小胶质细胞。然而，无法有效地将抗HIV转基因递送到HSC一直是临床试验中的障碍。我们建议开发泡沫状逆转录病毒载体，抑制艾滋病病毒的复制对HSC基因治疗艾滋病。泡沫载体可以高效稳定地转染造血干细胞，非常适合于艾滋病的基因治疗。将评价表达靶向达特/rev的短发夹RNA（shRNA）、靶向CCR 5的核酶、TAR诱饵和抑制HIV进入的肽的泡沫载体向CD 34衍生巨噬细胞提供稳定和特异性HIV复制抑制的能力。将进行实验以确认HIV vRNA在转导的HIV抗性细胞中特异性减少，并将评价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改变，或者shRNA表达是否影响HIV抗性巨噬细胞中宿主基因的表达。非肥胖糖尿病/严重联合免疫缺陷NOD/SCID异种移植模型将用于评估HIV抗性是否在体内分化的人巨噬细胞和T淋巴细胞中维持，并确定我们是否可以通过体内选择增加功能性HIV抗性再增殖细胞的数量。最后，在这些NOD/SCID实验中，我们还将确定人再生细胞中的泡沫载体整合位点，这是目前逆转录病毒基因治疗面临的关键问题。拟议的实验旨在系统地开发安全有效的泡沫状shRNA载体，用于未来的研究，我们在灵长类动物模型中评估其疗效，并最终在临床试验中。

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.