660. The Effect of the B2 Mutation on Hematopoietic Reconstitution in the French X- SCID Gene Therapy Trial

Abstract

Top of pageAbstract The B2 mutation arose in Murine Leukemia Virus (MLV) vectors selected for expression in embryonal carcinoma cells, and is postulated to prevent silencing resulting from the binding of undetermined factors to the integrated proviral DNA (Virology 193:690|[ndash]|699, 1993). This B2 mutation is also one of the few differences between the otherwise strikingly similar common |[gamma]|-chain retrovirus vectors that were used in the two well-known French and British X-SCID clinical trials. To date, three leukemias have occurred in the French trial, and none have occurred in the British trial. Although leukemias may still occur in the British trial, their absence to date impels us to investigate methodological differences between the two trials. An unusual property of the B2 mutation is derived from the fact that it resides embedded within the tRNA primer binding site of the retroviral genome, which causes it to revert at high frequency. This reversion occurs mechanistically when the tRNA primer binding site is reverse transcribed both from the full length genomic RNA during minus strand synthesis, and subsequently when the tRNA primer (covalently linked to the 5' end of minus strand cDNA) is copied into plus sense DNA. These DNAs (one copied from the B2 mutated RNA genome and one copied from the tRNA primer) later form a duplex prior to elongation of the 3' ends and synthesis of the remainder of the proviral genome. As a result, the proviral genome from B2 mutant retroviral vectors contains a single base pair mismatch at the site of the mutation. Progeny cells from such integrations show roughly equal distribution of wild-type and mutant proviral genomes, as would be predicted by equal probabilities of mismatch repair and/or segregation of the two strands into daughter cells. Although the B2 mutation has been shown to prevent silencing in murine systems, the evidence for an effect on gene expression in human cells is considerably weaker. If the level of gene expression in human cells is increased by the B2 mutation, and if the resulting increase in common |[gamma]|-chain gene expression increases proliferation and the efficiency of hematopoietic reconstitution after retrovirally transduced autologous X-SCID bone marrow transplantation, then we would expect that reconstituted blood samples from the French X-SCID gene therapy trial would show a bias in the fraction of cells that contained the B2 mutation relative to those that had reverted to wild type. With this in mind, we analyzed the proportion of B2 mutated genomes in peripheral blood mononuclear cells from 2 successfully treated X-SCID gene therapy patients which have not contracted leukemia, and compared them with control infections of HeLa cells using similar MFG-B2-|[gamma]|-chain vectors. Although we did observe frequent reversion of the B2 mutation, as expected, the data do not support the hypothesis that cells with B2 mutated vector genomes improve reconstitution significantly. In fact, B2 mutant genomes constituted approximately 30|[ndash]|45% of the total, suggesting that the B2 mutated genomes may have been occasionally selected against during hematopoietic reconstitution.