SV40-BASED COMBINATION GENETIC THERAPIES FOR HIV/SIV

该IP/CP申请建议使用重组sv40衍生载体（rSV40）来提供针对HIV/SIV的联合基因治疗。这种方法基于三个原则：1。利用攻击慢病毒复制周期不同阶段的抗慢病毒转基因组合，可以更好地抑制HIV/SIV；3. 骨髓祖细胞的转导可能提供了一个干细胞库，能够产生能够抵抗慢病毒感染的分化后代。我们将运用这些原则，使用rSV40载体连续转导相同的细胞，选择3种转基因来抑制逆转录病毒复制的互补阶段：(a) mip -1 β - kdel，将HIV/SIV的CCR5共受体隔离在内质网中，阻止HIV/SIV进入细胞；(b) RT#3，单链抗体对抗HIV-1逆转录酶（RT），在感染早期阻断RT；(c) RevM10，核输出蛋白Rev上的显性负突变体，用于阻止未剪接的慢病毒转录本离开细胞核。一个多机构合作小组将在体外T淋巴细胞和CD34+细胞以及体内两种不同的艾滋病动物模型中检测HIV/SIV的转导、毒性和抑制作用。我们提供了重要的初步数据来支持我们的论点：这些转基因-和指定的替代品-将在这种情况下抑制HIV/SIV；rSV40载体将其有效地传递到CD34+细胞、T细胞系和原代人外周血单个核细胞（pbmc）；rSV40载体可以稳定地用多个转基因转导这些细胞，转导效率约为98%；提出的rSV40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（David Strayer）与罗杰·波莫兰兹（Roger Pomerantz）合作指导，将设计并优化在培养细胞系和pbmc中抑制HIV-1的策略，使用rSV40载体的组合；最小化毒性，最大化表达，并提供高水平的HIV-1保护。项目#2和#3将研究将基因结合到人类和类人猿CD34+细胞的体外传递，以及随后在体外和体内重新植入这些细胞的分化后代的保护。由Harris Goldstein指导的项目#2将评估人类CD34+细胞的rSV40组合转导，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 Johnson领导的项目#3将测试恒河猴CD34+细胞的体外rSV40转导，评估体外和体内SIV或SHIV（RT）攻击对分化后代的保护作用，并确定治疗的任何副作用。项目#3还将测试是否可以利用rSV40载体的高转导效率将基因治疗直接传递到恒河猴的骨髓中。这些项目由一个Vector Cor和一个Administrative Core支持，后者将生产和表征用于这些项目的rSV40载体，并在处理过的动物血清中测试抗SV40抗体，后者将确保单个资助组成单位的顺利运作。因此，我们提出了一项联合IP/CP研究，以评估rsv40衍生的联合抗慢病毒基因治疗在相关实验系统中的体外和体内应用，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.