Efficiency and safety of O⁶-methylguanine DNA methyltransferase (MGMT(P140K))-mediated in vivo selection in a humanized mouse model.
Efficient O⁶-methylguanine DNA methyltransferase (MGMT(P140K))-mediated myeloprotection and in vivo selection have been demonstrated in numerous animal models and most recently in a phase I clinical study in glioblastoma patients. However, this strategy may augment the genotoxic risk of integrating vectors because of chemotherapy-induced DNA damage and the proliferative stress exerted during the in vivo selection. Thus, to improve the safety of the procedure, we evaluated a self-inactivating lentiviral MGMT(P140K) vector for transduction of human cord blood-derived CD34⁺ cells followed by transplantation of the cells into NOD/LtSz-scid/Il2rγ⁻/⁻ mice. These experiments demonstrated significant and stable enrichment of MGMT(P140K) transgenic human cells in the murine peripheral blood and bone marrow. Clonal inventory analysis utilizing linear amplification-mediated polymerase chain reaction and high-throughput sequencing revealed a characteristic lentiviral integration profile. Among the bone marrow insertions retrieved, we observed considerable overlap to previous MGMT(P140K) preclinical models or the clinical study. However, no significant differences between our chemotherapy-treated and nontreated cohorts were observed. This also hold true when specific cancer gene databases and a functional annotation of hit genes by the Panther Database with respect to molecular function, biological process, or cellular component were assessed. Thus, in summary, our data demonstrate efficient and long-term in vivo selection without overt hematological abnormalities using the lentiviral MGMT(P140K) vector. Furthermore, the study introduces humanized mouse models as a novel tool for the pre-clinical assessment of human gene therapy related toxicity.
1 REBIRTH Research Group Reprogramming and Gene Therapy, Hannover Medical School , 30625 Hannover, Germany .
Ruhi
R
Phaltane
Reinhard
R
Haemmerle
Michael
M
Rothe
Ute
U
Modlich
Thomas
T
Moritz
D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D015870:Gene Expression; D023061:Gene Order; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D006085:Graft Survival / Q000187:drug effects; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D064593:Heterografts; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes; D016662:Virus Integration
D000970:Antineoplastic Agents; D019853:O(6)-Methylguanine-DNA Methyltransferase