title: In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene.
abstract: Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.
authors.affiliation: National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. larochea@nhlbi.nih.gov
authors.forename: Andre
authors.initials: A
authors.lastname: Larochelle
authors.forename: Uimook
authors.initials: U
authors.lastname: Choi
authors.forename: Yan
authors.initials: Y
authors.lastname: Shou
authors.forename: Nora
authors.initials: N
authors.lastname: Naumann
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Loktionova
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Clevenger
authors.forename: Allen
authors.initials: A
authors.lastname: Krouse
authors.forename: Mark
authors.initials: M
authors.lastname: Metzger
authors.forename: Robert E
authors.initials: RE
authors.lastname: Donahue
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kang
authors.forename: Clinton
authors.initials: C
authors.lastname: Stewart
authors.forename: Derek
authors.initials: D
authors.lastname: Persons
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
authors.forename: Brian P
authors.initials: BP
authors.lastname: Sorrentino
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002330:Carmustine / Q000494:pharmacology; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015316:Genetic Therapy; D005822:Genetic Vectors; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D018398:Homeodomain Proteins / Q000502:physiology; D016086:Lentivirus / Q000235:genetics*; D008253:Macaca mulatta; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D000077204:Temozolomide
chemical_list: D018906:Antineoplastic Agents, Alkylating; D018398:Homeodomain Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine; D000077204:Temozolomide