Journal Article

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

Symonds, Geoff P., Lowe, Emily L., Camba-Colón, Joanna, Cortado, Ruth, Burke, Bryan P., Sahakyan, Anna, Kandarian, Fadi N., Carroll, Maria V., An, Dong Sung, Shimizu, Saki, Chen, Irvin S. Y., Kamata, Masakazu, Arokium, Hubert, Xie, Yiming, Ringpis, Gene-Errol E., Kim, Patrick Y., Narukawa, Munetoshi

2012

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Acquired immune deficiency syndrome / AIDS / Analysis / Animal tissues / Animals / Biocompatibility / Biological response modifiers / Biology / Blood / Bone marrow / CCR5 protein / CD4 antigen / Cell lines / Cells (biology) / CXCR4 protein / Cytotoxicity / Disseminated infection / Down-Regulation / Gene expression / Gene therapy / Genes / Genetic modification / Genetically modified organisms / Genetics / Gut-associated lymphoid tissues / Health aspects / Hematology / Hematopoietic Stem Cell Transplantation / Hematopoietic stem cells / Hematopoietic Stem Cells - immunology / Hematopoietic Stem Cells - virology / HIV / HIV Infections - immunology / HIV Infections - therapy / HIV Infections - virology / HIV-1 - immunology / Human immunodeficiency virus / Immune system / Immunology / Infection / Infections / Interferon / Leukocytes (mononuclear) / Life cycle analysis / Liver / Long terminal repeat / Lymphocytes T / Lymphoid tissue / Medicine / Mice / Nursing schools / Oncology / Peripheral blood mononuclear cells / Progenitor cells / Receptors, CCR5 - genetics / Receptors, CCR5 - metabolism / Replication / Ribonucleic acid / RNA / RNA polymerase / RNA, Small Interfering / Rodents / Spleen / T cells / T-Lymphocytes - immunology / T-Lymphocytes - virology / Thymus / Toxicity / Transplantation / Vectors (Biology) / Virus replication / Viruses