Potent Inhibition of CXCR4- and CCR5- tropic HIV-1 infection by lentivirally co-expressed shRNAs targeting CCR5 and HIV-1

Downregulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. However, CCR5 knockdown will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published short hairpin RNAs (shRNAs) targeting highly conserved regions of HIV-1 and identified an shRNA targeting the R-region of the HIV-1 long terminal repeat. Here, we report that co-expression of two shRNAs targeting 1. CCR5 and 2. the R region of HIV-1 within a single lentiviral vector stably down-regulates CCR5 and inhibits CXCR4- and CCR5- tropic HIV-1 replication. Transduction with vectors bearing both shRNAs inhibited CXCR4- and CCR5- tropic viral infection in cell lines and peripheral blood lymphocytes. No obvious cytotoxicity or interferon response was observed. Transplantation of vector-tra nsduced hematopoietic stem/progenitor cells (HSPC) into humanized bone marrow/liver/thymus (hu-BLT) mice resulted in stable gene marking and CCR5 downregulation in human CD4+ T cells in peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT-derived human splenocytes ex vivo. These results demonstrate that simultaneous delivery of shRNAs targeting both CCR5 and HIV-1 into HSPC may be a potential therapeutic reagent against HIV disease.