Combination Of Adenoviruses Research Articles

ONCOLYSIS MEDIATED BY INFB AND ARF GENE TRANSFER ACTIVATES KEY COMPONENTS OF THE IMMUNE SYSTEM

<h3>Background</h3> The importance of the immune system for controlling neoplasms was highlighted with the success of checkpoint blockade therapy. New cancer therapeutics seek to break the immune tolerance inherent in tumor progression. To this end, some virus-mediate gene therapy approaches have been shown to induce oncolysis accompanied by emission of immunogenic cell death (ICD) factors, thus attracting and activating cells of the immune system. <h3>Objective</h3> To use combined adenovirus-mediated gene transfer of p14ARF and human interferon-β (IFNβ) to human melanoma cells to induce oncolysis, ICD and subsequent activation of immune cells. <h3>Methods</h3> Cell death was measured by PI and/or AnnexinV staining. Detection of calreticulin was performed by cytometry, while ATP and IFNβ were detected by ELISA. DC and lymphocytes from healthy donors were cultivated ex vivo. <h3>Results</h3> IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. However, detection of critical markers of ICD) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. In situ gene therapy for s.c. SK-MEL-147 tumors in Nod-Scid mice controlled tumor growth and increased animal survival in response to IFNβ alone or in combination with p14ARF. However, the immunocompromised animal model cannot demonstrate the expected immune activation. Thus, we co-cultured SK-MEL-147 transduced with our combination of adenoviruses with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors. These Mo-DCs showed increased levels of activation markers HLA-DR, CD80, and CD86. Next, we used these activated Mo-DCs to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. T cells primed in this way were co-cultured with fresh non-transduced SK-MEL-147, resulting in tumor cell killing. Currently we are expanding these studies to include three-dimensional cultures of melanoma cell lines and also the use of human melanoma samples in patient derived organoid (PDO) models. We expect to reveal the influence of oncolysis on tumor infiltrating cells, including DCs, macrophages, NK and T cells. <h3>Conclusion</h3> Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Cisplatin Relocalizes RNA Binding Protein HuR and Enhances the Oncolytic Activity of E4orf6 Deleted Adenovirus.

The combination of adenoviruses and chemotherapy agents is a novel approach for human cancer therapeutics. A meticulous analysis between adenovirus and chemotherapeutic agents can help to design an effective anticancer therapy. Human antigen R (HuR) is an RNA binding protein that binds to the AU-rich element (ARE) of specific mRNA and is involved in the export and stabilization of ARE-mRNA. Our recent report unveiled that the E4orf6 gene deleted oncolytic adenovirus (dl355) replicated for certain types of cancers where ARE-mRNA is stabilized. This study aimed to investigate whether a combined treatment of dl355 and Cis-diamminedichloroplatinum (CDDP) can have a synergistic cell-killing effect on cancer cells. We confirmed the effect of CDDP in nucleocytoplasmic HuR shuttling. In vitro and in vivo experiments showed the enhancement of cancer cell death by apoptosis induction and a significant reduction in tumor growth following combination treatment. These results suggested that combination therapy exerted a synergistic antitumor activity by upregulation of CDDP induced cytoplasmic HuR, which led to ARE mRNA stabilization and increased virus proliferation. Besides, the enhanced cell-killing effect was due to the activation of the intrinsic apoptotic pathway. Therefore, the combined treatment of CDDP and dl355 could represent a rational approach for cancer therapy.

Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

Survival and Differentiation of Adenovirus-Generated Induced Pluripotent Stem Cells Transplanted into the Rat Striatum

Induced pluripotent stem cells (iPSCs) offer certain advantages over embryonic stem cells in cell replacement therapy for a variety of neurological disorders. However, reliable procedures, whereby transplanted iPSCs can survive and differentiate into functional neurons, without forming tumors, have yet to be devised. Currently, retroviral or lentiviral reprogramming methods are often used to reprogram somatic cells. Although the use of these viruses has proven to be effective, formation of tumors often results following in vivo transplantation, possibly due to the integration of the reprogramming genes. The goal of the current study was to develop a new approach, using an adenovirus for reprogramming cells, characterize the iPSCs in vitro, and test their safety, survivability, and ability to differentiate into region-appropriate neurons following transplantation into the rat brain. To this end, iPSCs were derived from bone marrow-derived mesenchymal stem cells and tail-tip fibroblasts using a single cassette lentivirus or a combination of adenoviruses. The reprogramming efficiency and levels of pluripotency were compared using immunocytochemistry, flow cytometry, and real-time polymerase chain reaction. Our data indicate that adenovirus-generated iPSCs from tail-tip fibroblasts are as efficient as the method we used for lentiviral reprogramming. All generated iPSCs were also capable of differentiating into neuronal-like cells in vitro. To test the in vivo survivability and the ability to differentiate into region-specific neurons in the absence of tumor formation, 400,000 of the iPSCs derived from tail-tip fibroblasts that were transfected with the adenovirus pair were transplanted into the striatum of adult, immune-competent rats. We observed that these iPSCs produced region-specific neuronal phenotypes, in the absence of tumor formation, at 90 days posttransplantation. These results suggest that adenovirus-generated iPSCs may provide a safe and viable means for neuronal replacement therapies.