No abstract

The limited efficiency of in vivo gene transfer by replication-deficient retroviral vectors remains an obstacle to achieving effective gene therapy for solid tumors. One approach to circumvent this problem is the use of replication-competent retroviral vectors. However, the application of such vectors is at a comparatively early stage and the effects which virus strain, transgene cassette position, and target cell can exert on vector spread kinetics, genomic stability, and transgene expression levels remain to be fully elucidated. Thus, in this study a panel of vectors allowing the investigation of different design features on an otherwise genetically identical background were analyzed with respect to these readout parameters in cultures of both murine and human cells and in preformed tumors in nude mice. The obtained data revealed that (i) Moloney murine leukemia virus (Mo-MLV)-based vectors spread with faster kinetics, drive higher levels of transgene expression, and are more stable than equivalent Akv-MLV-based vectors; (ii) vectors containing the transgene cassette directly downstream of the envelope gene are genomically more stable than those containing it within the 3-long terminal repeat U3 region; and (iii) the genomic stability of both strains seems to be cell line dependent.

Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human ␣1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver-and ␤-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.Because simple retroviruses can transduce only actively dividing cells (3,26,36,44), their use in cancer gene therapy has been extensively investigated, and over the last decade, numerous preclinical in vivo studies and clinical trials have been carried out using replication-defective retroviral (RDR) vectors (13). Although promising results have been obtained with animal models, therapeutic benefit in clinical settings has remained elusive, especially for cancer gene therapy, since the infection efficiency of solid tumors is too low (34). Of late, therefore, the use of replication-competent retroviral (RCR) vectors has been advocated, and it has been demonstrated by various groups that these are much more efficacious than their RDR counterparts (15, 23, 26-29, 40-42, 45). Mitotic cells, of course, are not unique to tumors, and although it may be expected that RCR vectors would not replicate efficiently outside of the immune-privileged environment of a solid tumor in a healthy individual, the possibility of spread occurring in dividing cells outside of the tumor mass must nevertheless be considered (7,33,35). Moreover, not least due to recent events demonstrating that retroviral vectors are capable, albeit in rare circumstances, of inducing oncogenesis in humans (19,30), safety is a primary concern in retroviral vect...

Transcriptionally targeted MLV-based ProCon vectors allow expression of the transduced gene in a promoter-specific manner by replacement of the viral U3 region with a heterologous promoter. In order to evaluate the effects of sequence elements present in ProCon vectors on transgene expression (enhanced green fluorescence protein, EGFP), a series of deletion constructs mimicking the situation in proviral DNA following promoter conversion, where expression of the EGFP gene is driven by three different constitutive promoters (MLV U3, mCMV, and hCMV) in the context of a 5'LTR, respectively, were generated and tested in transient transfection experiments. We discovered that modifications in the 3'LTR have only marginal effects on the EGFP expression and the sequence between the promoter and the transgene did not influence EGFP expression at all. On the other hand, EGFP expression was reduced by up to 17-fold in cells transfected with constructs containing SV40neo and/or pBR322ori sequences. To study this effect in transduced cells, we generated a series of retroviral vectors in which these elements were deleted in various combinations and found that an increase in EGFP expression and viral titer was also consistently obtained using vectors lacking these elements, although this was much smaller than that observed using the expression constructs. A vector containing the gene for puromycin resistance (pac) in place of the neomycin resistance gene (neo) was also tested, and found to result in improved vector titers and transgene expression. We conclude that, where possible, the inclusion of neo and ori sequences in retroviral vectors should be avoided, and that, if selection of infected cells is necessary, the pac, rather than neo gene should be used.