LENTIVIRAL VECTORS FOR HIV GENE THERAPY

这一临床前IPCP项目的主要目标是开发慢病毒载体用于HIV载体的基因治疗，这些载体以造血细胞为靶点，包括用于抗病毒和基于免疫的治疗的干细胞和树突状细胞。我们的具体目标是：1.建立优化的HIV-2基因转移载体和稳定的包装体系-在优化载体滴度的同时，努力将产生PCR的机会降至最低。2.为了证明慢病毒载体转导对原代T细胞、巨噬细胞和CD34+细胞后代的保护作用，本研究将检测表达抗HIV-1和抗CCR5核酶的HIV-2载体。我们还将确定这些载体的转导是否会对这些靶细胞的细胞增殖、分化和/或功能产生任何有害影响。3.探讨慢病毒载体转导对树突状细胞功能及与T细胞相互作用的影响--树突状细胞(DC)是HIV感染的促进者和防御者，因为它们既能向T细胞递呈病毒抗原，又能向T细胞递呈感染性病毒。最近的数据表明，虽然只有未成熟的DC才能感染HIV，但成熟的DC需要表达功能性趋化因子受体CCR5和CXC4，以分别将M嗜性、T嗜性和T嗜性病毒传递到CD4+T细胞。我们将构建表达Gag-Poll抗原以及抗CCR-5和/或抗CXCR4核酶的HIV-1载体，并确定这些载体的转导对树突状细胞呈递CD4和CD8细胞抗原或将HIV感染传递给CD4细胞的能力有何影响。除了这些目标外，该项目还将与项目2和项目3密切合作，在体外和体内直接比较基于HIV和FIV的慢病毒载体和基于MLV的逆转录病毒载体的安全性和有效性。

The major goal of this preclinical IPCP project is the development of lentiviral vectors for gene therapy of HIV-based vectors that target hematopoietic cells, including stem cells and dendritic cells for antiviral and immune-based therapies. Our specific aims are: 1. To establish optimal HIV-2 gene transfer vector and stable packaging lines-Efforts will be made to minimize the chance of PCR generation, while optimizing vector titers. 2. To demonstrate protection of primary T cells, macrophages and progeny of CD34+ cells by lentiviral vector transduction against HIV challenge-HIV-2 vectors expressing anti-HIV-1 and anti-CCR5 ribozymes will be tested in these studies. We will also ascertain whether transduction by these vectors will have any deleterious effect on cell proliferation, differentiation and/or function of these target cells. 3. To examine the effect of lentiviral vector transduction on dendritic cell function and interaction with T cells-Dendritic cells (DC) are both facilitators and defenders of HIV infection since they can present both viral antigens and infectious virus to T cells. Recent data suggest that while only immature DC's are infectiable by HIV, mature DC's need to express functional chemokine receptors CCR5 and CXC4 to transmit M-tropic and T-tropic and T-tropic viruses to CD4+ T cells respectively. We will construct HIV- 1 vectors expressing gag-pol antigens as well as anti-CCR-5 and/or anti CXCR4 ribozymes, and determine how transduction by these vectors will impact on the ability of dendritic cells to be present antigen to CD4 and CD8 cells or to transmit HIV infection to CD4 cells. In addition to these aims, this project will collaborate closely with Projects 2 and 3 to directly compare the safety and efficacy of HIV-based and FIV-based lentiviral vectors and MLV-based retroviral vectors in vitro and in vivo.