title: Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection.
abstract: Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.
authors.affiliation: New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772, USA.
authors.forename: M
authors.initials: M
authors.lastname: Rosenzweig
authors.forename: D F
authors.initials: DF
authors.lastname: Marks
authors.forename: D
authors.initials: D
authors.lastname: Hempel
authors.forename: M
authors.initials: M
authors.lastname: Heusch
authors.forename: G
authors.initials: G
authors.lastname: Kraus
authors.forename: F
authors.initials: F
authors.lastname: Wong-Staal
authors.forename: R P
authors.initials: RP
authors.lastname: Johnson
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000628:therapy*; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007114:Immunization; D008253:Macaca mulatta; D008264:Macrophages / Q000821:virology*; D016337:RNA, Catalytic / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D015302:Simian Immunodeficiency Virus / Q000502:physiology*; D013601:T-Lymphocytes / Q000821:virology*; D014779:Virus Replication
chemical_list: D018952:Antigens, CD34; D016337:RNA, Catalytic