ANIMAL MODELS OF AIDS--POLYTAR INHIBITION OF SIV

我们提出了一个合作项目，以评估小说的能力， 递送TAR诱饵构建体高效基因递送系统 （polyTAR）抑制人类和猿猴免疫缺陷病毒（HIV-1和 SIV）体外复制和SIV在恒河猴体内的复制。 基因疗法在HIV-1预防和治疗中的有效应用 感染仍然是一个难以实现的目标。 本项目将实现这一目标 使用一系列独特的工具。 这些包括体外CD 34+细胞 由Paul约翰逊博士开发的分化培养系统，其中 在胸腺基质上培养的祖细胞向CD 3 +4+8-T分化 淋巴细胞，易受SIV感染。 该系统将测试 祖细胞基因对SIV诱导分化细胞的保护作用 转移 逆转录病毒载体向选定祖细胞递送PolyTAR 细胞抑制SIV和HIV-1在衍生T细胞中的复制， 巨噬细胞 我们还将应用一种非常有效的基因传递载体， SV40 我们已经证明，SV 40可以使正常人和猴子 骨髓祖细胞在体外具有非常高的效率， 选择. 由于SV 40衍生病毒的滴度大于 10/10 ml，并且由于它们没有免疫原性，因此SV 40递送polyTAR可以 允许SIV和HIV-1的体内基因治疗。 我们假设SV 40可以有效地将聚TAR递送至T细胞， 骨髓祖细胞，有效抑制HIV-1和SIV 在体外和体内。 为了验证这一假设，我们将生成SV 40 含有聚TAR（SV（聚TAR）），并将测试其交付， 在易感细胞系中的聚TAR和SIV和HIV-1的抑制。 将测量PolyTAR表达和细胞对SIV的易感性 在源自SV的巨噬细胞和T淋巴细胞（polyTAR）中， 转导的祖细胞。将检测SV（polyTAR）的抑制作用 SIV在恒河猴中的祖细胞转导 离体和体内再输注，以及在体内直接转导后， vivo. 基于这些数据，将修改向量设计以增强 SIV-和HIV-1-的polyTAR表达和慢病毒抗性 易感细胞系和正常猿猴CD 34+细胞，与 逆转录病毒转导的polyTAR。 这些修饰的SV（polyTAR）载体将 设计用于更长和更大的polyTAR表达。 的疗效 这些优化的SV（polyTAR）载体在抑制SIV和HIV-1中的作用 将类似地评估体外感染和体内SIV感染。 SV 40递送、polyTAR活性和干细胞的组合方法 转导为HIV-1感染的治疗提供了巨大的希望。 在非人类灵长类动物中进行的研究将有助于定义 这种方法对艾滋病治疗的效用。

We propose a collaborative project to assess the ability of a novel, highly effective gene delivery system to deliver TAR decoy constructs (polyTAR) to inhibit human and simian immunodeficiency virus (HIV-1 and SIV) replication in vitro and SIV in vivo in rhesus macaque monkeys. Effective use of genetic therapy for prophylaxis and treatment of HIV-1 infection remains an elusive goal. This project will address that goal using a series of unique tools. These include an in vitro CD34+ cell differentiation culture system developed by Dr. Paul Johnson, in which progenitor cells cultured on thymic stroma differentiate to CD3+4+8-T lymphocytes, susceptible to SIV infection. This system will test protection of differentiated cells from SIV by progenitor cell gene transfer. PolyTAR delivered by retroviral vector to selected progenitor cells inhibits SIV and HIV-1 replication in derivative T cells and macrophages. We will also apply a very effective gene delivery vehicle, SV40. We have shown that SV40 can transduce normal human and monkey bone marrow progenitor cells in vitro with very high efficiency without selection. As SV40-derived viruses are made in titers greater than 10/10ml and as they are not immunogenic, SV40 delivery of polyTAR may allow in vivo gene therapy of SIV and -HIV-1. We hypothesize that SV40 can deliver polyTAR efficiently to T cells and to bone marrow progenitor cells, to effectively inhibit HIV-1 and SIV in vitro and in vivo. To test this hypothesis we will generate SV40 containing polyTAR (SV(polyTAR)) and will test both its delivery of polyTAR and inhibition of SIV and HIV-1 in susceptible cell lines. PolyTAR expression and cellular susceptibility to SIV will be measured in both macrophages and T lymphocytes derived from SV(polyTAR)- transduced progenitor cells. SV(polyTAR) will be tested for inhibition of SIV in rhesus mecaque monkeys following progenitor cell transduction ex vivo and reinfusion in vivo, and following direct transduction in vivo. Based on these data, vector design will be modified to enhance polyTAR expression and lentivirus resistance of SIV- and HIV-1- susceptible cell lines and normal simian CD34+ cells, compared to retrovirus-transduced polyTAR. These modified SV(polyTAR) vectors will be designed for longer and greater polyTAR expression. The efficacy of these optimized SV(polyTAR) vectors in inhibiting SIV and HIV-1 infection in vitro and SIV infection in vivo will be assessed similarly. The combined approach of SV40 delivery, polyTAR activity, and stem cell transduction offers great promise for the therapy of HIV-1 infection. Studies proposed here in nonhuman primates will help to define the utility of this approach to AIDS therapy.