Blocking of HIV Chemokine Receptors By Intrabodies

趋化因子受体在HIV进入和发病机制中的作用的阐明使人们能够深入了解这种疾病和可能的治疗干预要点。对CCR5基因自然发生的多态性的鉴定表明，基因破坏提供了保护，使其免受CCR5病毒的侵袭。由于这些多态存在于其他看起来健康的个体中，防止或限制CCR5表达的基因干预策略可能提供对初始感染的保护，并可能限制病毒传播。我们已经证明，含有内质网滞留信号的CCR5特异性单链抗体(即体内)的细胞内表达有效地干扰了CCR5的表面表达。体内表达的细胞株对游离HIV-1或HIV感染细胞的攻击具有很高的抵抗力。我们认为，HIV载体将CCR5和CXCR4体内基因传递到选定的造血细胞群体将限制HIV进入。我们的研究将探索这种方法，以限制艾滋病毒对髓系细胞和T细胞的感染。在特定的目标1中，我们将研究通过HIV载体分别在体内传递CCR5和CXCR4基因来干扰CD4T细胞、巨噬细胞和树突状细胞表面CCR5和CXCR4的细胞表面表达的病毒学和生物学后果。在特定的目的2中，我们评估了HIV载体将CCR5体内基因传递到CD34+干细胞是否促进和维持了CCR5在人-NOD/SCID小鼠的髓系分化过程中的干扰。在具体目标3中，我们将使用SCID-Hu小鼠来研究转导CCR5体内基因的HIV载体转导的CD34+细胞产生的胸腺细胞是否保持体内表达。在具体目标4中，我们将使用人PBL-NOD/SCID小鼠来确定通过使用HIV载体在体内传递CCR5基因来破坏成熟T细胞中CCR5的生物学和病毒学后果。最后，我们提出的研究将有助于对使用HIV载体在造血细胞中传递和维持异位基因表达的可行性提供基础和临床前的见解。

The elucidation of the roles of chemokine receptors in entry of HIV and pathogenesis has allowed insights into the disease and possible points of therapeutic intervention. The identification of naturally occurring polymorphisms of the CCR5 gene has shown that gene disruption provides protection from CCR5 using viruses. Since these polymorphisms are present in otherwise healthy appearing individuals, genetic intervention strategies that prevent or limit expression of CCR5 might provide protection from initial infection and possibly limit viral spread. We have demonstrated that intracellular expression of a CCR5- specific single-chain antibody (i.e., intrabody), containing endoplasmic reticulum retention signals efficiently disrupted surface expression of CCR5. Intrabody-expressing cell lines were highly refractory to challenge with free HIV-1 or HIV-infected cells. We propose that HIV vector delivery of CCR5 and CXCR4 intrabody genes to selected hematopoietic cell populations will limit HIV entry. Our studies will investigate this approach to limit HIV infection of myeloid cells and T cells. In Specific Aim 1 we will investigate the in vitro virological and biological consequences of disrupting cell surface expression of CCR5 and CXCR4 in CD4 T cells, macrophages and dendritic cells through HIV vector delivery of CCR5 and CXCR4 intrabody genes, respectively. In Specific Aim 2 we evaluate whether HIV vector delivery of CCR5 intrabody genes to CD34+ stem cells promotes and maintains disruption of CCR5 throughout myeloid differentiation in human-NOD/SCID mice. In Specific Aim 3 we will use SCID-hu mice to investigate whether thymocytes arising from CD34+ cells transduced with HIV vectors delivering CCR5 intrabody genes maintain intrabody expression. In Specific Aim 4 we will use human PBL-NOD/SCID mice to determine the biological and virological consequences of disrupting CCR5 in mature T cells by using an HIV vector to deliver CCR5 intrabody genes. Lastly, our proposed studies will contribute basic and pre-clinical insights into the feasibility of using HIV vectors to deliver and maintain ectopic gene expression in hematopoietic cells.