Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible.

Granulosa cell tumors (GCT) constitute only ~5% of ovarian neoplasms yet have significant consequences, as up to 80% of women with recurrent GCT will die of the disease. This study investigated the effectiveness of procaspase-activating compound 1 (PAC-1), an activator of procaspase-3, in treating adult GCT (AGCT) in combination with selected apoptosis-inducing agents. Sensitivity of the AGCT cell line KGN to these drugs, alone or in combination with PAC-1, was tested using a viability assay. Our results show a wide range in cytotoxic activity among the agents tested. Synergy with PAC-1 was most pronounced, both empirically and by mathematical modelling, when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This combination showed rapid kinetics of apoptosis induction as determined by caspase-3 activity, and strongly synergistic killing of both KGN as well as patient samples of primary and recurrent AGCT. We have demonstrated that the novel combination of two pro-apoptotic agents, TRAIL and PAC-1, significantly amplified the induction of apoptosis in AGCT cells, warranting further investigation of this combination as a potential therapy for AGCT.

Traceable poly(ethylene oxide)-poly(ester) micelles were developed through chemical conjugation of a near-infrared (NIR) dye to the poly(ester) end by click chemistry. This strategy was tried for micelles with poly(ε-caprolactone) (PCL) or poly(α-benzyl carboxylate-ε-caprolactone) (PBCL) cores. The surface of both micelles was also modified with the breast cancer targeting peptide, P18-4. The results showed the positive contribution of PBCL over PCL core on micellar thermodynamic and kinetic stability as well as accumulation in primary orthotopic MDA-MB-231 tumors within 4-96 h following intravenous administration in mice. This was in contrast to in vitro studies where better uptake of PEO-PCL versus PEO-PBCL micelles by MDA-MB-231 cells was observed. The presence of P18-4 enhanced the in vitro cell uptake and homing of both polymeric micelles in breast tumors, but only at early time points. In conclusion, the use of developed NIR labeling technique provided means for following the fate of PEO-poly(ester) based nano-carriers in live animals. Our results showed micellar stabilization through the use of PBCL over PCL cores, to have a more significant effect in enhancing the level and duration of nano-carrier accumulation in primary breast tumors than the modification of polymeric micellar surface with breast tumor targeting peptide, P18-4.

Interleukin-2 (IL-2) has been used clinically for the treatment of some malignancies, but the toxicities associated with systemic IL-2 therapy are a major challenge. Here we have determined whether transcriptional targeting of IL-2 to breast cancer (BrCa) using an engineered human mammaglobin promoter/enhancer (MPE2) is a feasible option for reducing IL-2-associated toxicities while still achieving a meaningful antitumor effect. We have constructed nonreplicating adenovirus vectors encoding either a reporter gene (luciferase) or human IL-2 (hIL-2) complementary DNA under control of the MPE2 sequence, the murine cytomegalovirus immediate early (MCMV) promoter or the human telomerase reverse transcriptase (hTERT) promoter. Luciferase and hIL-2 complementary DNAs under the control of the MPE2 sequence in adenovirus vectors were expressed at high levels in BrCa cells and at lower levels in normal cells of human and murine origin. Cancer specificity of the hTERT promoter was found to be similar to that of the MPE2 promoter in cells of human origin, but reduced specificity in murine cells. The MPE2 regulatory sequence demonstrated excellent tissue specificity in a mouse tumor model. Whereas the MCMV promoter-controlled IL-2 vector generated high liver toxicity in mice, the MPE2-controlled IL-2 vector generated little or no liver toxicity. Both IL-2 vectors exerted significant tumor growth delay; however, attempts to further enhance antitumor activity of the IL-2 vectors by combining with the proapoptotic drug procaspase activating compound 1 (PAC1) were unsuccessful.

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