SIMIAN RETROVIRUSES--NOVEL GENE DELIVERY VECTORS

说明（申请摘要） 逆转录病毒载体作为基因传递载体， 在单基因疾病的人类治疗中的潜在用途的载体。 重组鼠逆转录病毒已被开发用于基因转移 将功能性基因产物递送到细胞中的载体， 无功能或有缺陷的产品。 最近，我的实验室分离出一种 一种血清组2猿猴逆转录病毒（SRV; D2/RHE/OR/V1）。 这种逆转录病毒表现出广泛的 非人类灵长类动物的向性。 该分子的序列分析 克隆使我们能够确定包膜的遗传结构（env） 糖蛋白基因，并建立一个结构模型， 逆转录病毒-细胞相互作用。 具体目标1的总体目标是 把猿猴逆转录病毒作为基因传递载体。 该载体将 代表了第一个专门设计的灵长类定向逆转录病毒载体 作为潜在的基因传递载体。 我们将评估SRV的能力 基因转移载体感染并表达转基因 非人灵长类动物和人细胞系，然后利用这种载体作为 用于在非人中表达报告基因的基因递送载体 灵长类动物 具体来说，我们将a）识别猿类逆转录病毒psi 包装信号，和B）构建SRV包装细胞系和基因 转移载体并在已建立的猴中检测逆转录病毒重组体 和人类细胞系，最终在恒河猴体内测试。 大分子的核质转运，包括 RNA是由携带明确输出信号的特定蛋白质介导的。 SRV基因组包含一个顺式作用元件（组成性运输 与细胞蛋白质结合，促进细胞核内 输出未剪接的SRV RNA。 在猿类中发现的基因间区 逆转录病毒Mason-Pfizer猴病毒（MPMV）能够使不依赖于Rev的人 免疫缺陷病毒-1（HIV-1）复制，并可促进核 在不存在rev和rev应答的情况下输出未剪接的HIV mRNA 元素（RRE）。 因此，我们假设基因间间隔区 env基因与3 '-long末端重复序列（LTR）之间存在一个潜在的 CTE，其促进SRV RNA的核输出。 中描述的研究 具体目标2将帮助我们确定重要的决定因素， 逆转录病毒运输可能提供载体的进一步完善 发展 具体来说，我们将a）定义 SRV CTE，使用RNA迁移率变化和RNA酶消化/足迹分析， B）证明CTE序列在以下中的功能重要性： 利用定点突变，细胞核质输出SRV RNA 分级分离和原位杂交程序，以及c）鉴定 编码参与核质 利用酵母三杂交选择系统，输出SRV RNA。

DESCRIPTION (Abstract of the application) Retrovirus vectors have received considerable attention as gene delivery vehicles for potential use in human therapy of single gene disorders. Recombinant murine retroviruses have been developed as gene transfer vehicles to deliver functional gene products into cells that contain nonfunctional or deficient product. Recently, my laboratory isolated an infectious molecular clone of a serogroup 2 simian retrovirus (SRV; D2/RHE/OR/V1) found within Asian macaques. This retrovirus exhibits broad tropism within the nonhuman primate. Sequence analysis of this molecular clone has allowed us to define the genetic structure of the envelope (env) glycoprotein gene and to develop a structural model of simian retrovirus-cell interaction. The overall goal of Specific Aim 1 is to develop the simian retrovirus as a gene delivery vehicle. This vector will represent the first primate-directed retrovirus vector specifically designed as a potential gene delivery vehicle. We will assess the ability of the SRV gene transfer vehicle to infect and express transgenes in established nonhuman primate and human cell lines, and then utilize this vector as a gene delivery vehicle for the expression of reporter genes in nonhuman primates. Specifically, we will a) identify the simian retrovirus psi packaging signal, and b) construct SRV packaging cell lines and gene transfer vehicles and test the retroviral recombinants in established monkey and human cell lines, with ultimate in vivo testing in rhesus macaques. Nucleocytoplasmic transport of macromolecules, including nuclear export of RNA, is mediated by specific proteins that harbor explicit export signals. The SRV genome contains a cis-acting element (constitutive transport element; CTE) that binds to cellular protein(s), facilitating the nuclear export of unspliced SRV RNA. The intergenic region identified in the simian retrovirus, Mason-Pfizer monkey virus (MPMV), enables rev-independent human immunodeficiency virus-1 (HIV-1) replication, and can promote the nuclear export of unspliced HIV mRNA in the absence of rev and rev-responsive element (RRE). We therefore hypothesize that the intergenic spacer region between the env gene and 3'-long terminal repeat (LTR) contains a potential CTE that facilitates nuclear export of SRV RNA. The research described in Specific Aim 2 will help us identify important determinants of simian retrovinus trafficking that may provide further refinement in vector development. Specifically, we will a) define the precise sequence of the SRV CTE, using RNA mobility shift and RNase digestion/footprinting analyses, b) demonstrate the functional importance of the CTE sequence in nucleocytoplasmic export of SRV RNA, using site-directed mutagenesis, cell fractionation, and in situ hybridization procedures, and c) identify the gene(s) encoding cellular protein factor(s) involved in nucleocytoplasmic export of SRV RNA, using the yeast three-hybrid selection system.