SV40-BASED COMBINATION GENETIC THERAPIES FOR HIV/SIV

本IP/CP申请拟使用重组SV 40衍生载体（rSV 40）提供抗HIV/SIV的联合基因治疗。这种方法基于三个原则：1。使用攻击慢病毒复制周期的不同阶段的抗慢病毒转基因的组合可以实现对HIV/SIV的更好抑制; 3.骨髓祖细胞的转导可以提供能够产生抗慢病毒感染的分化后代的干细胞库。我们将应用这些原理，使用rSV 40载体连续转染相同的细胞，选择3个转基因来抑制逆转录病毒复制的互补阶段：（a）MIP-1 β-KDEL，在内质网中隔离HIV/SIV的CCR 5共受体，并阻止HIV/SIV进入细胞;（B）RT#3，单链抗体对HIV-1逆转录酶（RT），在感染早期阻断RT;和（c）RevM 10，核输出蛋白Rev上的显性负突变体，以防止未剪接的慢病毒转录物离开核。一个多机构的合作者小组将在T淋巴细胞和CD 34+细胞中体外分析HIV/SIV的转导、毒性和抑制，并在两种不同的艾滋病动物模型中体内分析。我们提出了重要的初步数据来支持我们的论点：这些转基因-和指定的替代物-将在这种情况下抑制HIV/SIV; rSV 40载体将它们有效地递送到CD 34+细胞、T细胞系和原代人外周血单核细胞（pbmc）; rSV 40载体将稳定地用多个转基因转染这些细胞，效率>98%;并且所提出的rSV 40转导将保护易感细胞免受HIV-1的攻击。项目#1由大卫斯特雷耶指导，与罗杰Pomerantz合作，将设计和优化策略，抑制HIV-1在培养的细胞系和pbmc，使用rSV 40载体的组合;以最大限度地减少毒性，最大限度地提高表达，并提供高水平的保护，从HIV-1。项目#2和#3将研究离体向人和猿CD 34+细胞的基因递送组合，以及随后在体外和体内再植入后对这些细胞的分化后代的保护。由Harris Goldstein指导的项目#2将评估人CD 34+细胞的组合rSV 40转导，测试这种组合转导对SCID-hu小鼠中这些细胞成熟的影响，并测量这些动物免受HIV-1攻击的保护。由Paul约翰逊指导的项目#3将测试恒河猴CD 34+细胞的离体rSV 40转导，评估分化后代在体外和体内免受SIV或SHIV（RT）攻击的保护，并确定治疗的任何副作用。项目#3还将测试rSV 40载体的高转导效率是否可以用于将基因治疗直接递送到恒河猴的骨髓中。这些项目得到了载体中心和管理中心的支持，载体中心将生产和表征用于这些项目的rSV 40载体，并检测经处理的动物血清中的抗SV 40抗体，管理中心将确保各个赠款组成单位的顺利运作。因此，我们提出了一个财团IP/CP研究，以评估rSV 40衍生的组合抗慢病毒基因治疗在相关的实验系统，在体外和体内，并将这些发现转化为临床研究。

This IP/CP application proposes to use recombinant SV40-derived vectors (rSV40) to deliver combination genetic therapy against HIV/SIV. This approach is based on three principles: 1. better inhibition of HIV/SIV can be achieved using combinations of anti-lentiviral transgenes that attack different stages of the lentiviral replicative cycle; 3. transduction of bone marrow progenitor cells may provide a reservoir of stem cells capable of producing differentiated progeny that are resistant to lentivirus infection. We will apply these principles, using rSV40 vectors to transduce the same cells consecutively with 3 transgenes chosen to inhibit complementary stages of retrovirus replication: (a) MIP-1beta-KDEL, to sequester CCR5 co-receptor for HIV/SIV in the endoplasmic reticulum and impede HIV/SIV entry into the cell; (b) RT#3, single chain antibody vs. HIV-1 reverse transcriptase (RT), to block RT early in infection; and (c) RevM10, dominant negative mutant on the nuclear export protein, Rev, to prevent unspliced lentivirus transcripts from exiting the nucleus. A multi- institutional group of collaborators will assay transduction, toxicity, and inhibition of HIV/SIV in vitro in T lymphocytes and CD34+ cells, and in vivo in two different animal models of AIDS. We present significant preliminary data to support our contention that: these transgenes- and designated alternates-will inhibit HIV/SIV in this setting; rSV40 vectors deliver them effectively to CD34+ cells, T cell lines and primary human peripheral blood mononuclear cells (pbmc); rSV40 vectors will stably transduce these cells with multiple transgenes, with >98% efficiency; and the proposed rSV40 transduction will protect susceptible cells from challenge with HIV-1. Project #1, directed by David Strayer, in collaboration with Roger Pomerantz, will devise and optimize strategies for inhibiting HIV-1 in cultured cell lines and pbmc, using combinations of rSV40 vectors; to minimize toxicity, maximize expression, and provide high levels of protection from HIV-1. Projects #2 and #3 will study will combination gene delivery to human and simian CD34+ cells ex vivo, and consequent protection of differentiated progeny of these cells in vitro and following reimplantation in vivo. Project #2, directed by Harris Goldstein, will evaluate combination rSV40 transduction of human CD34+ cells, test effects of such combination transduction on maturation of these cells in SCID-hu mice, and measure protection of these animals from challenge with HIV-1. Project #3, directed by Paul Johnson, will test ex vivo rSV40 transduction of rhesus CD34+ cells, assess protection of differentiated progeny from challenge with SIV or SHIV(RT) challenge in vitro and in vivo, and identify any side effects of the treatment. Project #3 will also test whether the high transduction efficiency of rSV40 vectors can be exploited to deliver genetic therapy directly into the bone marrow in rhesus macaque monkeys. These projects are supported by a Vector Cor, which will produce and characterize rSV40 vectors for use in these projects, and test for anti- SV40 antibody in treated animal sera, and by an Administrative Core, which will assure smooth functioning of the individual grant component units. Thus, we propose an consortium IP/CP study to evaluate rSV40-derived combination anti-lentiviral gene therapy in relevant experimental systems, in vitro and in vivo, and to translate these findings into clinical studies.