Targeting human glioma cells using HSV-1 amplicon peptide display vector.

Targeting cell infection using herpes simplex virus type 1 (HSV-1) vectors is a complicated issue as the process involves multiple interactions of viral envelope glycoproteins and cellular host surface proteins. In this study, we have inserted a human glioma-specific peptide sequence (denoted as MG11) into a peptide display HSV-1 amplicon vector replacing the heparan sulfate-binding domain of glycoprotein C (gC). The modified MG11:gC envelope recombinant vectors were subsequently packaged into virions in the presence of helper virus deleted for gC. Our results showed that the tropism of these HSV-1 recombinant virions was increased for human glioma cells in culture as compared with wild-type virions. The binding of these recombinant virions could also be blocked effectively by pre-incubating the cells with the glioma-specific peptide, indicating that MG11 peptide and the recombinant virions competed for the same or similar receptor-binding sites on the cell surface of human glioma cells. Furthermore, preferential homing of these virions was shown in xenograft glioma mouse model following intravascular delivery. Taken together, these results validated the hypothesis that HSV-1 binding to cells can be redirected to human gliomas through the incorporation of MG11 peptide sequence to the virions.

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (BM-hMSCs) are nonhematopoietic stem cells that have the potential to differentiate into adipocytes, osteocytes and chondrocytes. Because of its propensity to migrate to the sites of injury and the ability to expand them rapidly, BM-hMSCs have been exploited as potential gene transfer vehicles to deliver therapeutic genes. Herein, we evaluated the feasibility of employing herpes simplex virus type I (HSV-1) amplicon viral vector as a gene delivery vector to BM-hMSCs. High transduction efficiencies were consistently observed in different isolates of BM-hMSCs following infection with HSV-1 amplicon viral vectors. Furthermore, we demonstrated that transduction with HSV-1 amplicon viral vector did not alter the intrinsic properties of the BM-hMSCs. The morphology and cellular proliferation of the transduced BM-hMSCs were not altered. Chromosomal stability, as confirmed by karyotyping and soft agar colony assays, of the transduced BM-hMSCs was not affected. Similarly, transduction with HSV-1 amplicon viral vectors has no effect on the pluripotent differentiation potential and the tumor tropism of BM-hMSCs. Taken together, these results demonstrated that BM-hMSCs could be transduced efficiently by HSV-1 amplicon viral vector in an 'inert' manner and thus enable strategies to express potential therapeutic genes in BM-hMSCs.

Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.

We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

Targeting proliferating tumor cells via the transcriptional control of therapeutic genes.

We have previously reported the construction of a cell cycle-regulated HSV-1 amplicon vector (denoted as pC8-36) that confers luciferase reporter gene activities dependent on cellular divisions. However, luciferase reporter gene is well known for its relatively high sensitivity, thus, it is crucial to evaluate the therapeutic efficacy of a transcriptional targeted vector. In this report, we have engineered the FasL and FADD genes into pC8-36 and demonstrated their efficacy for the treatment of human gliomas in vitro and in vivo. Using trypan blue dye exclusion and TUNEL assay, FasL expression mediated by pC8-36 was shown to induce a significantly higher percentage of cell death in proliferating cells than those observed in the G(1)-arrested cells. The observed cell killing effect correlated well with the level of FasL protein expression when analyzed by ELISA assay. Furthermore, the incorporation of both FasL and FADD into pC8-36 resulted in the enhancement of apoptosis in the target glioma cells both in vitro and in vivo. Targeting proliferating tumor cells via the transcriptional control of therapeutic genes could potentially improve the safety and efficacy of cancer gene therapy, and thus would allow the development of strategies for more effective anticancer therapies.