Engineering HIV-1-Resistant T-Cells from Short-Hairpin RNA-Expressing Hematopoietic Stem/Progenitor Cells in Humanized BLT Mice

• DOI: 10.1371/journal.pone.0053492
• Publication Date: 2012-12-31T22:14:11Z
• Authors: Gene-Errol E. Rin...; Saki Shimizu; Hubert Arokium; Joanna Camba-Colón; Maria V. Carroll; Ruth Cortado; Yiming Xie; Patrick Y. Kim; Anna Sahakyan; Emily L. Lowe; Munetoshi Narukawa; Fadi N. Kandarian; Bryan P. Burke; Geoff P. Symonds; Dong Sung An; Irvin S. Y. Chen; Masakazu Kamata
• Keywords: 0301 basic medicine; Receptors, CCR5; General Science & Technology; Science; T-Lymphocytes; Immunology; 610; Down-Regulation; HIV Infections; Regenerative Medicine; Small Interfering; Mice; 03 medical and health sciences; Stem Cell Research - Nonembryonic - Human; 616; Receptors; Genetics; Animals; RNA, Small Interfering; Transplantation; 0303 health sciences; Biomedical and Clinical Sciences; 5.2 Cellular and gene therapies; Q; R; Hematopoietic Stem Cell Transplantation; Gene Therapy; Stem Cell Research; Hematopoietic Stem Cells; Human Fetal Tissue; 3. Good health; Infectious Diseases; Medical Microbiology; HIV-1; Sexually Transmitted Infections; HIV/AIDS; RNA; Medicine; Stem Cell Research - Nonembryonic - Non-Human; Infection; CCR5; Biotechnology; Research Article

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation 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 shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4(+) T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+) T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+) T-cells ex vivo. Furthermore, levels of gene-marked CD4(+) T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.