title: RNA-based anti-HIV-1 gene therapeutic constructs in SCID-hu mouse model.
abstract: Effective suppression of HIV-1 replication requires inhibition of critical viral target molecules. Tat and Rev are indispensable regulatory factors for HIV-1 replication, whereas Env mediates virus entry by direct interaction with surface receptor CD4 and coreceptor CCR5 or CXCR4. Anti-HIV-1 tat-rev and env ribozymes and Rev aptamers were previously demonstrated to provide relatively long-term protection against HIV-1 infection in vitro. However, further improvements in these constructs for clinical application in a stem-cell-based gene therapy setting requires in vivo characterization. Toward this end, we introduced these constructs into CD34(+) hematopoietic progenitor cells by retrovirus-mediated gene transduction. Ribozyme- and aptamer-transduced CD34(+) cells differentiated normally into multiple lineages of erythroid and myeloid progenies in a colony-forming unit assay. Macrophages that differentiated from the transduced CD34(+) cells expressed anti-tat-rev and -env ribozymes and Rev aptamers and displayed their normal characteristic surface markers CD14, CD4, and CCR5. Using the SCID-hu mouse in vivo human thymopoiesis model, we demonstrated that ribozyme- and aptamer-transduced CD34(+) cells retained their normal capacity to reconstitute human fetal thymus and liver tissue (thy/liv) grafts. Reconstitution by ribozyme- and aptamer-transduced CD34(+) cells reached levels of up to 87% based on HLA surface marker staining. Differentiated thymocytes derived from reconstituted grafts expressed anti-tat-rev and -env ribozymes and Rev aptamers and showed significant resistance to HIV-1 infection upon challenge. Analysis of reconstituted thymocytes by hybridization revealed an average of 0.4 to 2 copies of vector sequences per cell. Southern analysis of proviral integration junctions in progeny thymocytes demonstrated that the human thy/liv grafts were reconstituted by a few primitive hematopoietic stem cells. These results highlight the utility of RNA-based anti-HIV-1 gene therapeutic approaches and their preclinical testing in a surrogate animal model harboring human tissue.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523, USA.
authors.forename: Jirong
authors.initials: J
authors.lastname: Bai
authors.forename: Nirmal
authors.initials: N
authors.lastname: Banda
authors.forename: Nan Sook
authors.initials: NS
authors.lastname: Lee
authors.forename: John
authors.initials: J
authors.lastname: Rossi
authors.forename: Ramesh
authors.initials: R
authors.lastname: Akkina
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D016207:Cytokines / Q000494:pharmacology; D004195:Disease Models, Animal; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D015658:HIV Infections / Q000235:genetics* / Q000628:therapy*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000821:virology; D006801:Humans; D016031:Liver Transplantation; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008934:Mitogens / Q000494:pharmacology; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use*; D019718:Receptors, CXCR4 / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000196:embryology / Q000276:immunology / Q000821:virology; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; D015694:Gene Products, rev; D015696:Gene Products, tat; D008934:Mitogens; D016337:RNA, Catalytic; D019718:Receptors, CXCR4; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus