Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy

Abstract

Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (43/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.

Human cytochrome P450 2E1 and sulfotransferase 1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis

“…Chromosomal instability was found to occur at the site of integration of the Cyp2b1 expression vector. In another report, V79 cells were treated with an attenuated retrovirus [Themis et al, 2003], wherein an increased mutation rate at the Environmental and Molecular Mutagenesis. DOI 10.1002/em Fig.…”

Evidence for endogenous retroviruses in human chemokine receptor gene introns: possible evolutionary inferences and biological roles

“…A BLAST search demonstrates that the virus-derived sequences present in the CKR introns are in fact present at many other sites in the human genome. For instance, a BLAST search (without filter for low complexity regions) for the HERV-H/env60 sequence, of which a homolog is present in the CCR10 intron (290 nt; 85% conserved; 4.E-88; Table 1), yielded 34,192 Blast Hits with E values equal to or lower than the virus/CKR alignment. Thus, it may be concluded that the question, whether CKRs were primarily infected or other genes were first infected and then a SINE duplicated itself in some of CKR gene introns, cannot at present be answered.…”

“…(31,32) Potential genotoxicity of retroviral integration is also discussed in. (33)(34)(35)(36)(37)(38)(39)(40) In still other instances, sequences of infecting retroviruses, after reverse transcription, became permanently integrated into the intronic genome of germ-line cells and are thus transmitted in a Mendelian manner: these sequences are generally referred to as "endogenous retroviruses" (ERVs) or more specifically "human ERVs" (HERVs) if present in the human genome. (41)(42)(43)(44)(45) Such sequences are widespread in many animal species, including humans, in which they are estimated to account for 1-5% of the genome.…”

Effect of endothelial cell-based iNOS gene transfer on cavernosal eNOS expression and mouse erectile responses

“…7 The reversal of eNOS downregulation by L-NIL suggests a NOS dependent mechanism of eNOS downregulation, and not a non-specific effect of retroviral gene transduction such as insertional mutation. 21,22 The elevated nitrite levels measured in the iNOStransduced EC, but not L-NIL treated cells, support a mechanism related to NO or one of its downstream mediators. However, iNOS overexpression could also downregulate eNOS via a superoxide-mediated mechanism, independent of NO.…”