title: Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene.
abstract: Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMT(P140K) marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.
authors.affiliation: Department of Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Chung
authors.affiliation: Department of Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Scherer
authors.affiliation: Laboratory for Cellular Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Angel
authors.initials: A
authors.lastname: Gu
authors.affiliation: Laboratory for Cellular Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Agnes M
authors.initials: AM
authors.lastname: Gardner
authors.affiliation: Laboratory for Cellular Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Torres-Coronado
authors.affiliation: Laboratory for Cellular Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Elizabeth W
authors.initials: EW
authors.lastname: Epps
authors.affiliation: 1] Laboratory for Cellular Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA [2] Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Digiusto
authors.affiliation: 1] Department of Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, California, USA [2] Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Rossi
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000235:genetics / Q000473:pathology / Q000628:therapy* / Q000821:virology; D000818:Animals; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000627:therapeutic use; D006678:HIV / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D064168:Minichromosome Maintenance Complex Component 7 / Q000235:genetics; D020871:RNA Stability / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7; D045643:DNA Repair Enzymes