973. Targeting of CCR5 through Intrabody Mediated HIV-1 Vector Gene Delivery Is Sufficient for Blocking HIV-1-Infection and Confers a T-Cell Survival and Growth Advantage

Abstract

Top of pageAbstract With the emergence of HAART resistant HIV-1s, there is a need for additional therapies to preserve T-effector cells and reduce viral burden. Moreover, since it may be impossible to protect all susceptible cells in an individual through gene delivery, it is therefore necessary to develop and verify that genetic means of protection against HIV-1 provides a cellular survival and growth advantage. To this purpose we have developed single chain antibodies to CCR5 which targets protein. Efficacy of HIV-1 protection and enrichment was assessed by free viral and HIV-1 loaded dendritic cell challenge. HIV-1 vector delivery of CCR5-intrabody (termed the CAD-R5 vector), coupled to a KDEL endoplasmic retention sequence, to primary CD4 T-cells efficiently disrupted CCR5 cell surface expression 4-fold as determined by mean fluorescence intensity (MFI) as compared to a control vector without the intrabody, 8 and 34 MFI, respectfully. CAD-R5 transduced primary CD4 T-cells were resistant to robust, free, R5-tropic SF162 challenges as shown by 50 to 60-fold reductions in HIV-1 p24 concentrations. Moreover, CCR5-intrabody gene expressing CD4 T-cells, as compared to cells with a control vector, demonstrated a selective advantage and enriched from 2-10-fold in dendritic cell cultures. Similar levels of HIV-1 protection were seen in CD34 cell derived macrophages; 3-weeks after challenge with SF162, macrophages expressing CCR5-intrabody generated 42-, 57-, and 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral loads in CD4 T-cells and macrophages. To assess hematopoietic developmental alterations from CCR5-intrabody expression, CD34 cells were transduced and colony forming (CFU) and macrophage assays were performed. No differences were seen in numbers/types of macrophages or CFUs produced when comparing parental, control vector, and intrabody expressing cells (n=3). To assess in vivo correlates of hematopoietic function and intrabody-mediated protection, CAD-R5 and control vector transduced, fetal liver derived CD34 cells were engrafted in human thymi implants in SCID-hu thy/liv mice (n = 2, 2 groups), 6-weeks later the thymi displayed an average of 25% reporter gene expressing T-cells, similar to controls. Development was not altered as compared to controls as determined by CD4/CD8 profile. Furthermore, CCR5-intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge. These results validate the efficacy of HIV-1 delivered CCR5-intrabody mediated protection from HIV-1 challenges in primary T-cells and macrophages and, importantly, demonstrates a survival and growth advantage of protected CD4 T-cells. The findings underscore the potential advantage of intrabody gene delivery to CD34 cells, which allows differentiation of protected T-cells, macrophages, and dendritic cells.