Drug selection of mutant methylguanine methyltransferase from different oncoretroviral backbones results in multilineage hematopoietic transgene expression in primary and secondary recipients.
Optimized hematopoietic gene therapy requires vectors with strong expression in the desired target cell population and the ability to select for the expressing transduced cells. In the context of drug resistance selection of repopulating hematopoietic stem cells in the mouse, we examined tissue expression after transduced marrow transplantation of the drug selection gene, G156A mutant O6-methylguanine-DNA methyltransferase (G156A MGMT). To gain more experience with the rigor of the impact of selection on tissue-specific gene expression, we also asked whether there are expression differences between three different onco-retroviral backbones--MPSV, SF, and MFG. MGMT expression was compared after O6-benzylguanine (BG) and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) drug selection in vivo. After mice were transplanted with cells transduced with MPSV, MFG, or SF retroviral vectors expressing G156A MGMT and drug treated, nearly complete replacement by transduced progenitors was observed in the marrow. Each backbone supported MGMT expression in all four hematopoietic lineages in vivo indicating that MGMT-mediated selection is indeed robust. Expression in marrow, spleen, and thymus was very similar between the vectors and differences were most likely due to differences in gene copy number per selected cell. In primary and secondary recipients, the highest expression was observed in MFG and this was the vector that transduced at the greatest proviral copy number per cell. These data indicate that strong selection pressure using the MGMT gene to protect primary and secondary repopulating murine stem cells from the toxicity of BCNU. Regardless of the vector backbone used, multiorgan expression was observed without evidence of gene silencing. These data help establish mutant, BG-resistant MGMT as a potent selection gene for stem cell selection in vivo.
Davis
Brian M
BM
Division of Hematology/Oncology and the Comprehensive Cancer Center at Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106-4937, USA.
Reese
Jane S
JS
Lingas
Karen
K
Gerson
Stanton L
SL
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D019070:Cell Lineage; D002469:Cell Separation; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D020868:Gene Silencing; D005822:Genetic Vectors; D016678:Genome; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008809:Mice, Inbred C3H; D008957:Models, Genetic; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors; D019076:Transgenes
D000970:Antineoplastic Agents; D015682:Retroviridae Proteins, Oncogenic; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase