CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.
Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF--an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression--would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats--under cyclosporin immunosuppression--received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits.
Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ?actA/?inlB). Uptake by phagocytes in the liver results in local inflammatory responses and activation and recruitment of natural killer (NK) and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ?actA/?inlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T-cell responses against mesothelin-expressing murine tumors. These two phase I studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively.
Immunotherapeutic vaccine is potentially an effective strategy to combat cancer. Essential components of an effective vaccine must include antigens that are processed by the major histocompatibility complex class I pathway, presented by the tumor major histocompatibility complex molecules, and an effective antigen delivery platform that is capable of breaking self-tolerance. In this study, we characterized a set of ovarian cancer-specific T-cell epitopes delivered by live-attenuated recombinant Listeria monocytogenes (Lm DeltaactADeltainlB) as a vaccine vector. We present data that peptide-specific T cells recognize the human monocytic cell line THP-1 infected with recombinant Lm DeltaactADeltainlB encoding the epitopes. Furthermore, we demonstrate that recombinant L. monocytogenes (Lm)-infected antigen-presenting cells can prime and expand epitope-specific CD8 T cells in vitro and such CD8 T cells recognize not only peptide-loaded targets but also ovarian and breast tumor cells presenting endogenous epitopes. Finally, peptide-specific T cells generated using peripheral blood mononuclear cell from ovarian cancer patients recognize target cells infected with recombinant Lm DeltaactADeltainlB encoding the epitopes. Our results demonstrate that live-attenuated recombinant Lm can be used effectively as a vehicle to deliver cancer peptide antigens singly or as a multiepitope construct. Thus, the use of recombinant live-attenuated Lm strains encoding endogenously processed and presented tumor epitopes/antigens represents an attractive strategy for active cancer immunotherapy in a clinical setting.
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