RESUMO
Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.
Assuntos
Vacinas contra a AIDS , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Proteínas Recombinantes de Fusão/genética , Animais , Sequência de Bases , Linhagem Celular , Bases de Dados Genéticas , Engenharia Genética , Humanos , Memória Imunológica , Imunofenotipagem , Macaca mulatta , Modelos Animais , Dados de Sequência Molecular , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Transdução Genética/métodos , Integração ViralRESUMO
The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Terapia Genética/métodos , Inibidores da Fusão de HIV/uso terapêutico , Infecções por HIV/prevenção & controle , HIV-1 , Células-Tronco Hematopoéticas/metabolismo , Vacinas contra a AIDS/genética , Animais , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Infecções por HIV/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , O(6)-Metilguanina-DNA Metiltransferase/análise , Retroviridae/genética , Transdução Genética/métodosRESUMO
Introduction of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) into the 3' untranslated region of retroviral and lentiviral gene transfer vectors enhances both titer and transgene expression. Optimal use of the PRE is often necessary to obtain vectors with sufficient performance for therapeutic applications. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced. However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that the PRE might potentially contribute to tumorigenesis, especially if encoding a truncated version of the WHV X protein. Oncogenic side effects of lentiviral vectors containing the PRE have reinforced these safety concerns, although a causal role of the PRE remained unproven. Here, we demonstrate that PRE mutants can be generated that are devoid of X protein open reading frames (ORFs) as well as other ORFs exceeding 25 amino acids, without significant loss of RNA enhancement activity. Furthermore, the X protein promoter could be deleted without compromising the enhancement of vector titers and transgene expression. Such a modified PRE sequence appears useful for future vector design.