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疾病和可能的治疗干预点。自然发生的CCR5基因多态性的鉴定表明，基因破坏提供了对CCR5使用病毒的保护。由于这些多态性存在于看似健康的个体中，阻止或限制CCR5表达的遗传干预策略可能提供保护，防止初始感染，并可能限制病毒传播。我们已经证明，含有内质网保留信号的CCR5特异性单链抗体的细胞内表达（即体内）有效地破坏了CCR5的表面表达。体内表达细胞系对游离HIV-1或hiv感染细胞的攻击具有高度的难治性。我们提出，HIV载体将CCR5和CXCR4体内基因递送到选定的造血细胞群将限制HIV的进入。我们的研究将探讨这种方法来限制骨髓细胞和T细胞的HIV感染。在Specific Aim 1中，我们将分别通过HIV载体递送CCR5和CXCR4体内基因，研究破坏CD4 T细胞、巨噬细胞和树突状细胞中CCR5和CXCR4细胞表面表达的体外病毒学和生物学后果。在Specific Aim 2中，我们评估了HIV载体将CCR5体内基因递送到CD34+干细胞中是否促进并维持了人类nod /SCID小鼠骨髓分化过程中CCR5的破坏。在Specific Aim 3中，我们将使用SCID-hu小鼠来研究由携带CCR5体内基因的HIV载体转导的CD34+细胞产生的胸腺细胞是否维持体内表达。在Specific Aim 4中，我们将使用人类PBL-NOD/SCID小鼠，通过使用HIV载体递送CCR5体内基因，来确定破坏成熟T细胞中CCR5的生物学和病毒学后果。最后，我们提出的研究将为利用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.