ANIMAL MODELS OF AIDS--POLYTAR INHIBITION OF SIV

我们提出了一个合作项目来评估一部小说的能力， 高效基因递送系统递送TAR诱饵构建物 (Polytar)抑制人类和猿类免疫缺陷病毒(HIV-1和 SIV)在恒河猴体内和体外复制。 有效利用基因疗法预防和治疗HIV-1 感染仍然是一个难以捉摸的目标。该项目将实现这一目标 使用一系列独特的工具。其中包括体外培养的CD34+细胞 由Paul Johnson博士开发的分化培养系统，其中 胸腺基质上培养的祖细胞向CD3+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 调职。通过逆转录病毒载体向选定的祖细胞递送聚焦油 细胞抑制衍生T细胞中SIV和HIV-1的复制 巨噬细胞。我们还将应用一种非常有效的基因输送工具， SV40。我们已经证明SV40可以转导正常人和猴子 体外非常高效的骨髓祖细胞 选择。因为SV40衍生病毒的滴度大于 10/10ml，由于它们不是免疫原性，SV40递送的Polytar可以 允许SIV和-HIV-1的体内基因治疗。 我们假设SV40可以有效地将Polytar运送到T细胞和 对骨髓祖细胞，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SV40 包含Polytar(SV(Polytar))，并将测试其交付的 聚焦油与SIV和HIV-1在敏感细胞系中的抑制作用。 将测量Polytar的表达和细胞对SIV的敏感性 在巨噬细胞和来自SV(Polytar)的T淋巴细胞中- 转导的祖细胞。将对SV(Polytar)进行抑制测试 SIV在恒河猴祖细胞转导后的表达 体外和体内回输，以及在体内直接转导 活着。基于这些数据，矢量设计将被修改以增强 SIV-和HIV-1-多聚体的表达及其对慢病毒的抗性 易感细胞系和正常猿猴CD34+细胞 逆转录病毒转导的Polytar。这些修改后的SV(Polytar)向量将 为更长更大的Polytar表达而设计。的功效。 这些优化的SV(Polytar)载体抑制SIV和HIV-1 体外感染和体内SIV感染将进行类似的评估。 SV40传递、Polytar活性和干细胞的联合方法 转导为HIV-1感染的治疗提供了巨大的希望。 这里提出的在非人类灵长类动物中的研究将有助于定义 这一方法在艾滋病治疗中的实用性。

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.