Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer.

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-ß cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Vaccination using melanoma cells treated with p19arf and interferon beta gene transfer in a mouse model: a novel combination for cancer immunotherapy.

Previously, we combined p19(Arf) (Cdkn2a, tumor suppressor protein) and interferon beta (IFN-ß, immunomodulatory cytokine) gene transfer in order to enhance cell death in a murine model of melanoma. Here, we present evidence of the immune response induced when B16 cells succumbing to death due to treatment with p19(Arf) and IFN-ß are applied in vaccine models. Use of dying cells for prophylactic vaccination was investigated, identifying conditions for tumor-free survival. After combined p19(Arf) and IFN-ß treatment, we observed immune rejection at the vaccine site in immune competent and nude mice with normal NK activity, but not in NOD-SCID and dexamethasone immunosuppressed mice (NK deficient). Combined treatment induced IL-15, ULBP1, FAS/APO1 and KILLER/DR5 expression, providing a mechanism for NK activation. Prophylactic vaccination protected against tumor challenge, where markedly delayed progression and leukocyte infiltration were observed. Analysis of primed lymphocytes revealed secretion of TH1-related cytokines and depletion protocols showed that both CD4(+) and CD8(+) T lymphocytes are necessary for immune protection. However, application of this prophylactic vaccine where cells were treated either with IFN-ß alone or combined with p19(Arf) conferred similar immune protection and cytokine activation, yet only the combination was associated with increased overall survival. In a therapeutic vaccine protocol, only the combination was associated with reduced tumor progression. Our results indicate that by harnessing cell death in an immunogenic context, our p19(Arf) and IFN-ß combination offers a clear advantage when both genes are included in the vaccine and warrants further development as a novel immunotherapy for melanoma.

Single nucleotide polymorphisms of the GJB2 and GJB6 genes are associated with autosomal recessive nonsyndromic hearing loss.

Single nucleotide polymorphisms (SNPs) are important markers in many studies that link DNA sequence variations to phenotypic changes; such studies are expected to advance the understanding of human physiology and elucidate the molecular basis of diseases. The DFNB1 locus, which contains the GJB2 and GJB6 genes, plays a key role in nonsyndromic hearing loss. Previous studies have identified important mutations in this locus, but the contribution of SNPs in the genes has not yet been much investigated. The aim of this study was to investigate the association of nine polymorphisms located within the DFNB1 locus with the occurrence of autosomal recessive nonsyndromic hearing loss (ARNSHL). The SNPs rs3751385 (C/T), rs7994748 (C/T), rs7329857 (C/T), rs7987302 (G/A), rs7322538 (G/A), rs9315400 (C/T), rs877098 (C/T), rs945369 (A/C), and rs7333214 (T/G) were genotyped in 122 deaf patients and 132 healthy controls using allele-specific PCR. There were statistically significant differences between patients and controls, in terms of allelic frequencies in the SNPs rs3751385, rs7994748, rs7329857, rs7987302, rs945369, and rs7333214 (P < 0.05). No significant differences between the two groups were observed for rs7322538, rs9315400, and rs877098. Our results suggest that SNPs present in the GJB2 and GJB6 genes may have an influence on ARNSHL in humans.

Guidelines for the tetra-primer ARMS-PCR technique development.

The tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS-PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl2. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS-PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.