Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model.

In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.

Expression of the fusogenic p14 FAST protein from a replication-defective adenovirus vector does not provide a therapeutic benefit in an immunocompetent mouse model of cancer.

When injected directly into a tumor mass, adenovirus (Ad) vectors only transduce cells immediately along the injection tract. Expression of fusogenic proteins from the Ad vector can lead to syncytium formation, which efficiently spreads the therapeutic effect. Fusogenic proteins can also cause cancer cell death directly, and enhance the release of exosome-like particles containing tumor-associated antigens, which boosts the anti-tumor immune response. In this study, we have examined whether delivery of an early region 1 (E1)-deleted, replication-defective Ad vector encoding the reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein can provide therapeutic efficacy in an immunocompetent mouse tumor model. A high multiplicity of infection of AdFAST is required to induce cell fusion in mouse mammary carcinoma 4T1 cells in vitro, and FAST protein expression caused a modest reduction in cell membrane integrity and metabolic activity compared with cells infected with a control vector. Cells expressing FAST protein released significantly higher quantities of exosomes. In immunocompetent Balb/C mice harboring subcutaneous 4T1 tumors, AdFAST did not induce detectable cancer cell fusion, promote tumor regression or prolong mouse survival compared with untreated mice. This study suggests that in the context of the 4T1 model, Ad-mediated FAST protein expression did not elicit a therapeutic effect.

Programmable insect cell carriers for systemic delivery of integrated cancer biotherapy.

Due to cancer's genetic complexity, significant advances in the treatment of metastatic disease will require sophisticated, multi-pronged therapeutic approaches. Here we demonstrate the utility of a Drosophila melanogaster cell platform for the production and in vivo delivery of multi-gene biotherapeutic systems. We show that cultured Drosophila S2 cell carriers can stably propagate oncolytic viral therapeutics that are highly cytotoxic for mammalian cancer cells without adverse effects on insect cell viability or gene expression. Drosophila cell carriers administered systemically to immunocompetent animals trafficked to tumors to deliver multiple biotherapeutics with little apparent off-target tissue homing or toxicity, resulting in a therapeutic effect. Cells of this Dipteran invertebrate provide a genetically tractable platform supporting the integration of complex, multi-gene biotherapies while avoiding many of the barriers to systemic administration of mammalian cell carriers. These transporters have immense therapeutic potential as they can be modified to express large banks of biotherapeutics with complementary activities that enhance anti-tumor activity.

Phenotype of an X-linked retinitis pigmentosa family with a novel splice defect in the RPGR gene.

PURPOSE: To assess the clinical phenotype in a Swedish family with X-linked retinitis pigmentosa (XLRP) resulting from a novel splice defect in the RPGR gene. METHODS: RPGR mutation analysis was performed in one family with XLRP, and several individuals from the family were examined clinically. RESULTS: The causative mutation in the family was demonstrated to be a single base-pair change at the splice donor site in intron 7 that resulted in skipping of the complete exon 7 in the mature RPGR transcript. The aberrant mRNA is predicted to produce an RPGR protein with an in-frame deletion of 53 amino acids, corresponding to an RCC1-homology repeat. Clinical studies that included ophthalmological examination and full-field electroretinography showed that this splice mutation resulted in a comparatively less severe form of RP. CONCLUSIONS: Correlation of a causative RPGR genotype with clinical findings in hemizygotes and carrier heterozygotes is an important step toward predictive diagnosis and should assist in the development of gene-based therapies in the future.

Non-replicating rhabdovirus-derived particles (NRRPs) eradicate acute leukemia by direct cytolysis and induction of antitumor immunity.

Rhabdoviruses (RVs) are currently being pursued as anticancer therapeutics for various tumor types, notably leukemia. However, modest virion production and limited spread between noncontiguous circulating leukemic cells requires high-dose administration of RVs, which exceeds the maximum tolerable dose of the live virus. Furthermore, in severely immunosuppressed leukemic patients, the potential for uncontrolled live virus spread may compromise the safety of a live virus approach. We hypothesized that the barriers to oncolytic virotherapy in liquid tumors may be overcome by administration of high-dose non-replicating RVs. We have developed a method to produce unique high-titer bioactive yet non-replicating rhabdovirus-derived particles (NRRPs). This novel biopharmaceutical is multimodal possessing direct cytolytic and immunomodulatory activity against acute leukemia. We demonstrate that NRRP resistance in normal cells is mediated by intact antiviral defences including interferon (IFN). This data was substantiated using murine models of blast crisis. The translational promise of NRRPs was demonstrated in clinical samples obtained from patients with high-burden multidrug-resistant acute myeloid leukemia. This is the first successful attempt to eradicate disseminated cancer using a non-replicating virus-derived agent, representing a paradigm shift in our understanding of oncolytic virus-based therapies and their application toward the treatment of acute leukemia.