Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors.

BACKGROUND: Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. METHODS: The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. RESULTS: In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. CONCLUSIONS: This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.

In situ gene transfer into animal tendons by injection of naked DNA and electrotransfer.

BACKGROUND: Degenerative or traumatic tendon injuries are extremely common but often heal poorly, not restoring the normal function of the injured tissues. Gene transfer could improve the repair process, by permitting local production of therapeutic substances, e.g. growth factors. METHODS AND RESULTS: Injection of a plasmid carrying the lacZ marker gene was performed into the Achilles tendons of rat and mouse, and the patellar tendons of rabbit. At 48 h, transduced cells were found in the injected zones of the tendons but represented a minority of the tendon cells. A kinetics study in rats permitted observation of a gradual decrease with time in the beta-gal-expressing cell number; at day 42 no more gene expression was detected. Noteworthy, no inflammatory reaction was observed. We then investigated whether electrotransfer could improve gene transfer efficacy in rat tendon by delivering in situ electric pulses after DNA injection. Gene transfer was improved at best by approximately 50% under certain electrical conditions (200 V for 10 ms or 1200 V for 100 micro s). Finally, multiple injections of plasmid permitted an increase in the number of transduced cells by approximately 400%. CONCLUSIONS: In situ injection of naked DNA into tendons is a very simple technique that permits delivery of genes with a duration of expression sufficient for clinical application aimed at modulating healing or restoration of a degenerative tendon. Despite a low transfer efficiency, this method should be compatible with clinical applications aimed at delivering therapeutic substances acting at low concentration.

Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene.

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4(+) cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from the CD4 gene--a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10(7) infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2-deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4 gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4(+) T cells in reconstituted mice and in ex vivo-transduced human T cells. Non-T CD4(+) dendritic and macrophage cells derived from human CD34(+) cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4(+) T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4(+) cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.

Replicative retroviral vectors for cancer gene therapy.

Poor efficiency of gene transfer into cancer cells constitutes the major bottleneck of current cancer gene therapy. We reasoned that because tumors are masses of rapidly dividing cells, they would be most efficiently transduced with vector systems allowing transgene propagation. We thus designed two replicative retrovirus-derived vector systems: one inherently replicative vector, and one defective vector propagated by a helper retrovirus. In vitro, both systems achieved very efficient transgene propagation. In immunocompetent mice, replicative vectors transduced >85% tumor cells, whereas defective vectors transduced <1% under similar conditions. It is noteworthy that viral propagation could be efficiently blocked by azido-thymidine, in vitro and in vivo. In a model of established brain tumors treated with suicide genes, replicative retroviral vectors (RRVs) were approximately 1000 times more efficient than defective adenoviral vectors. These results demonstrate the advantage and potential of RRVs and strongly support their development for cancer gene therapy.