In vivo selection of retrovirally transduced hematopoietic stem cells

Abstract

One of the main impediments to effective gene therapy of blood disorders is the resistance of human hematopoietic stem cells to stable genetic modification. We show here that a small minority of retrovirally transduced stem cells can be selectively enriched in vivo, which might be a way to circumvent this obstacle. We constructed two retroviral vectors containing an antifolate-resistant dihydrofolate reductase cDNA transcriptionally linked to a reporter gene. Mice were transplanted with transduced bone marrow cells and then treated with an antifolate-based regimen that kills unmodified stem cells. Drug treatment significantly increased the percentage of vector-expressing peripheral blood erythrocytes, platelets, granulocytes, and T and B lymphocytes. Secondary transplant experiments demonstrated that selection occurred at the level of hematopoietic stem cells. This system for in vivo stem-cell selection provides a means to increase the number of genetically modified cells after transplant, and may circumvent an substantial obstacle to successful gene therapy for human blood diseases.

Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice

“…49 DHFR-transduced hematopoietic cells can be enriched in vivo following administration of antifolates. 50,51 Although taxol selection has been shown to result in enrichment of MDR1-transduced bone marrow cells, 19 …”

Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene

“…Cells were plated in thymidine-phosphorylase-treated methylcellulose 19 culture medium (MethoCult GF M3434; Stem Cell Technologies Vancouver, BC, Canada) without and with pemetrexed at or above the concentration (100 nM) previously determined to be 100% lethal for mocktransduced mouse bone marrow-derived colonies (100 nM). The plates were stored at 371C in a 5% CO 2 incubator, and colonies of more than 50 cells were counted after 12 days.…”

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

“…13 Several alternative drug resistance genes have been studied in vitro, including O 6 -alkylguanine-DNA-alkyltransferase, which confers protection to some alkylating agents 14,15 and mutant dihydrofolate reductase. 16,17 Although the strategy using the mutant dihydrofolate reductase has proven feasible in vivo in mice, 17 confirmatory studies in nonhuman primates or humans have not yet been reported.…”