title: Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.
abstract: 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.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Gene-Errol E
authors.initials: GE
authors.lastname: Ringpis
authors.forename: Saki
authors.initials: S
authors.lastname: Shimizu
authors.forename: Hubert
authors.initials: H
authors.lastname: Arokium
authors.forename: Joanna
authors.initials: J
authors.lastname: Camba-Colón
authors.forename: Maria V
authors.initials: MV
authors.lastname: Carroll
authors.forename: Ruth
authors.initials: R
authors.lastname: Cortado
authors.forename: Yiming
authors.initials: Y
authors.lastname: Xie
authors.forename: Patrick Y
authors.initials: PY
authors.lastname: Kim
authors.forename: Anna
authors.initials: A
authors.lastname: Sahakyan
authors.forename: Emily L
authors.initials: EL
authors.lastname: Lowe
authors.forename: Munetoshi
authors.initials: M
authors.lastname: Narukawa
authors.forename: Fadi N
authors.initials: FN
authors.lastname: Kandarian
authors.forename: Bryan P
authors.initials: BP
authors.lastname: Burke
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Symonds
authors.forename: Dong Sung
authors.initials: DS
authors.lastname: An
authors.forename: Irvin S Y
authors.initials: IS
authors.lastname: Chen
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kamata
mesh_terms: D000818:Animals; D015536:Down-Regulation; D015658:HIV Infections / Q000276:immunology / Q000628:therapy* / Q000821:virology; D015497:HIV-1 / Q000276:immunology*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000276:immunology* / Q000821:virology; D051379:Mice; D034741:RNA, Small Interfering; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000821:virology
chemical_list: D034741:RNA, Small Interfering; D019713:Receptors, CCR5