Improving adenoviral vectors and strategies for prostate cancer gene therapy

Gene therapy has been evaluated for the treatment of prostate cancer and includes the application of adenoviral vectors encoding a suicide gene or oncolytic adenoviruses that may be armed with a functional transgene. In parallel, versions of adenoviral vector expressing the p53 gene (Ad-p53) have been tested as treatments for head and neck squamous cell carcinoma and non-small cell lung cancer. Although Ad-p53 gene therapy has yielded some interesting results when applied to prostate cancer, it has not been widely explored, perhaps due to current limitations of the approach. To achieve better functionality, improvements in the gene transfer system and the therapeutic regimen may be required. We have developed adenoviral vectors whose transgene expression is controlled by a p53-responsive promoter, which creates a positive feedback mechanism when used to drive the expression of p53. Together with improvements that permit efficient transduction, this new approach was more effective than the use of traditional versions of Ad-p53 in killing prostate cancer cell lines and inhibiting tumor progression. Even so, gene therapy is not expected to replace traditional chemotherapy but should complement the standard of care. In fact, chemotherapy has been shown to assist in viral transduction and transgene expression. The cooperation between gene therapy and chemotherapy is expected to effectively kill tumor cells while permitting the use of reduced chemotherapy drug concentrations and, thus, lowering side effects. Therefore, the combination of gene therapy and chemotherapy may prove essential for the success of both approaches

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-b 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

A incidência do acidente vascular encefálico em pacientes infectados pelo SARS- COV-2, COVID-19: uma revisão de literatura: The incidence of brain vascular accident in patients associated with SARS-COV-2, COVID-19 infection: a literature review

Objetivo: O objetivo do trabalho foi analisar a possível relação entre a covid-19 e os acidentes vascular encefálicos (AVE). Métodos: A pesquisa foi realizada nas bases eletrônicas de dados eletrônicos PubMed, Lilacs e SciELO, nas quais foram selecionados artigos originais publicados entre 2020 e 2022. Os critérios de inclusão foram os artigos originais com texto completo disponível e estudos de casos publicados que analisaram a associação entre acidente vascular encefálico com a doença Covid-19. Foram considerados critérios de exclusão os artigos duplicados e de revisão ou aqueles que não se enquadravam ao tema proposto neste estudo. Resultados: Os principais resultados dos artigos analisados demostraram intrínseca relação entre Covid-19 e AVE, uma vez que o AVE isquêmico em pacientes com covid-19 tendem em alguns casos ocorrer por meio da trombose de grandes artérias, em pacientes com envolvimento pulmonar de grau leve a moderado. Além disso, foi constatado que na maioria dos pacientes houveram a presença de marcadores, como PCR, IL-6 e d-dímero e fibrinogênio elevados, presença frequente de anticorpos antifosfolípides, geração de espécies reativas de oxigênio e trombocitopenia. Ademais, foram identificados aumento dos casos em pacientes que apresentavam alguma comorbidade, como hipertensão e diabetes. Conclusão: Portanto, como os mecanismos das complicações do Covid-19 são recentes, se faz necessário a intensificação    de pesquisas nesta área, afim de agir precocemente nas complicações que a Covid-19 pode ocasionar

Gene-based Interventions for Cancer Immunotherapy

Immunotherapy of cancer has deservedly gained much attention in the past few years and is likely to continue to advance and become a fundamental cancer treatment. While vaccines, chimeric antigen receptor (CAR) T cells and checkpoint blockade have received the lion’s share of the attention, an important direct role for gene transfer as an immunotherapy is emerging. For example, oncolytic viruses induce immunogenic cell death, thus liberating both antigens and the signals that are necessary for the activation of antigen-presenting cells, ensuring stimulation of an adaptive response. In another example, transfer of prodrug converting enzymes, such as the herpes simplex virus-thymidine kinase (HSV-tk) gene or the cytosine deaminase gene, has been shown to promote an immune response, thus functioning as immunotherapies. Alternatively, our own work involves the use of nonreplicating viral vectors for the simultaneous delivery of gene combinations that promote both cell death and an immune response. In fact, our gene transfer approach has been applied as a vaccine, immunotherapy or in situ gene therapy, resulting in immunogenic cell death and the induction of a protective immune response. Here, we highlight the development of these approaches both in terms of technical advances and clinical experience

Evaluation of the autocrine and paracrine effects of IFN gene therapy with adenovirus and its relationship with IFNAR1 in melanoma and microenvironment of the murine model

O câncer é uma doença complexa, frequentemente sem cura e que está presente em todas as populações e em todas as idades. O câncer gera enormes perdas econômicas e emocionais, sendo que seus tratamentos atuais são caros e, tipicamente, pouco efetivos em casos avançados. O melanoma é um câncer de pele agressivo na sua evolução e frequentemente mortal para os hospedeiros, que apresentam a doença metastática, essa doença ainda possui poucos tratamentos efetivos. Frente a esta situação, é necessário a criação e desenvolvimento de novas abordagens com maior eficiência no tratamento do câncer. O Interferon beta (IFN) é uma citocina imuno-estimuladora com efeito anti- tumoral conhecido, porém sua aplicação sistêmica é tóxica. Nosso grupo propõe o uso de terapia gênica utilizando vetores adenovirais portadores do transgene terapêutico IFN (Ad-mIFN), assim, proporciona a produção do IFN de modo localizado e transiente, o que pode conferir um escape do efeito tóxico sistêmico. Os dados do grupo mostram que, em modelos murinos, a terapia gênica com Ad-mIFN in vitro resulta em morte celular e no modelo in situ induz retardo na progressão tumoral do melanoma. A investigação nesta tese teve como objetivo estudar os efeitos autócrinos e parácrinos nas células do microambiente e tumorais do melanoma após terapia gênica com IFN e a influência do seu receptor IFNAR1 neste contexto. Para investigar a influência da sinalização do IFN nos efeitos autócrinos e parácrinos, realizou-se o knockout do IFNAR1 em células da B16 e do microambiente (tEnd, NIH3T3) utilizando a tecnologia de CRISPR/Cas9. Os resultados mostraram que células transduzidas pelo Ad-mIFN necessitam da produção e liberação do IFN para a ativação da via do IFN/, pela ligação com os receptores IFNAR1/IFNAR2 e finalmente induzir a célula a morte, mecanismo conhecido como autócrino. As células transduzidas pelo Ad-mIFN também induzem efeito parácrino por inibir as células próximas, pelo mecanismo parecido com o autócrino, mas direcionado a outras células próximas. As células knockout para o IFNAR1 ficaram bloqueadas de receber os efeitos autócrinos e parácrinos do Ad-mIFN e da proteína recombinante do IFN (rIFN), porém mesmo sem o IFNAR1 elas continuam produzindo o efeito parácrino. Esses dados foram observados em células tumorais e células endoteliais e de fibroblastos. Outro estudo in vitro da terapia com Ad-mIFN e Ad-p19 na angiogênese utilizando células endoteliais, descobriu uma linhagem endotelial não responsiva ao tratamento com Ad-mIFN e a proteína IFN sozinha, porém essas células sofriam inibição quando colocadas em ensaios de efeito parácrino pela linhagem de melanoma transduzida com o Ad-mIFN. A observação desse fenômeno sugere a possibilidade da existência de outros fatores além do IFN, produzidos pelas células de melanoma transduzidas com Ad-mIFN, que estariam envolvidos no efeito parácrino. Ensaios de expressão gênica da linhagem de melanoma transduzida pelo Ad-mIFN mostraram aumento significativo de TRAIL, que pode ser um candidato para estar envolvido no efeito parácrino. Juntos, estes resultados indicam que a presença de IFNAR1 não é essencial para produção dos fatores necessários para induzir o efeito parácrino, porem a presença de IFNAR1 é necessário para a célula sofrer este efeito. Futuramente, as linhagens deficientes em IFNAR1 podem ser avaliadas em ensaios in vivo para revelar o papel do IFNAR1 nos efeitos autócrinos e parácrinos entre células tumor e seu microambiente após tratamento, incluindo com Ad- mIFNCancer is a complex disease, often without cure, which is present in all populations and at all ages. Cancer generates enormous economic and emotional losses, and its current treatments are expensive and, typically, ineffective in advanced cases. Melanoma is an aggressive skin cancer in its evolution and often deadly for the hosts that present metastatic disease since few effective treatments are available. Faced with this situation, it is necessary to create and develop new approaches with greater efficacy in the treatment of cancer. Interferon beta (IFN) is an immunostimulatory cytokine with a known anti-tumor effect, but its systemic application is toxic. Our group proposes the use of gene therapy using adenoviral vectors that carry the therapeutic transgene IFN (Ad-mIFN), thus providing the production of IFN in a localized and transient way, which should avoid the systemic toxic effect. Our studies have shown that, in murine models, gene therapy with Ad-mIFN in vitro results in cell death and, in the in situ model, delays the progression of melanoma tumors. The research performed in this thesis aimed to study the autocrine and paracrine effects in the microenvironment and tumor cells of melanoma after IFN gene therapy and the role of the IFNAR1 receptor. To investigate the influence of IFN signaling on autocrine and paracrine effects, we performed IFNAR1 knockout on B16 and microenvironment cells (tEnd, NIH3T3) using the CRISPR / Cas9 technology. The results showed that cells transduced by Ad- mIFN require the production and release of IFN for activation of the IFN/ pathway, by binding with the IFNAR1/IFNAR2 receptors and finally inducing cell death, a mechanism known as autocrine. The cells transduced by Ad-mIFN also induce a paracrine effect by inhibiting nearby cells, by a mechanism similar to the autocrine but directed at other nearby cells. The cells with IFNAR1 knockout were blocked from receiving the autocrine and paracrine effects of Ad-mIFN and recombinant IFN protein (rIFN), however even without IFNAR1 they continue to produce the paracrine effect. These data were observed in tumor cells and endothelial and fibroblast cells. Another in vitro study of therapy with Ad-mIFN and Ad-p19 in angiogenesis identified an endothelial cell line unresponsive to treatment with Ad-mIFN and the IFN protein alone, however these cells suffered inhibition when tested for the paracrine effect provided by the melanoma cell line transduced with Ad-mIFN. The observation of this phenomenon suggests the existence of factors other than IFN, produced by melanoma cells transduced with Ad-mIFN, which would be involved in the paracrine effect. Gene expression assays of the melanoma lineage transduced by Ad-mIFN showed a significant increase in TRAIL, which may be a candidate for involvement in the paracrine effect. Together, these results indicate that the presence of IFNAR1 is not essential for the production of the factors necessary to induce the paracrine effect, however the presence of IFNAR1 is necessary for the cell to suffer this effect. In the future, IFNAR1-deficient strains can be evaluated in in vivo assays to reveal the role of IFNAR1 in the autocrine and paracrine effects between tumor cells and their microenvironment after treatment, including with Ad-mIF

Distinct roles of direct transduction versus exposure to the tumor secretome on murine endothelial cells after melanoma gene therapy with interferon-? and p19Arf

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliaisThe vasculature plays a central role in tumor progression and represents a therapeutic target of great interest. Inhibition of angiogenesis has the potential to slow down tumor progression and inhibit metastasis. As a result, anti-angiogenic therapies have been shown to be promising for the control of tumor growth. According to the literature, interferon ? (IFN?, activator of the innate and adaptive immune systems) and p19Arf (tumor suppressor and functional partner of p53) when studied individually alter tumor vasculature. Our group has constructed and used recombinant adenovirus vectors carrying the cDNAs of INFbeta and p19Arf and noted that the transfer of this combination of genes induced cell death and decreased tumor progression, as observed in the B16 murine model of in situ melanoma gene therapy as well as prophylactic and therapeutic vaccine approaches. In this study, we explore the idea that the combination of adenoviral vectors bearing INFbeta and p19Arf produce anti-angiogenic effects due to their impact on endothelial cells. To test this hypothesis, murine endothelial cells (tEnd) were transduced with adenoviral vectors, revealing that Ad-p19 vector confers inhibition of proliferation, tube formation, migration and induces increased expression of genes related to the p53 cell death pathway. The Ad-IFNbeta vector alone had no significant impact on these tests. Alternatively, influences on paracrine effects are evaluated on endothelial cells co-cultured with B16 cells that were previously transduced with adenoviral vectors. When the B16 cells were transduced with Ad-IFNbeta or co-transduced with Ad-IFNbeta + Ad-p19, co-culture resulted in the inhibition of proliferation of the endothelial cells. When B16 cells were transduced with Ad-p19 only, co-culture did alter endothelial cell behavior. Following the co-culture assay, we produce conditioned medium from B16 cells that were transduced with the vectors and applied the media on tEnd cells. We noted that conditioned medium derived from B16 transduced with Ad-IFN alone or in combination with Ad-19 decreased the viability and proliferation and induced cell death of tEnd. In this work, we show that inhibition of endothelial cells can be performed directly by transduction with Ad-19 or when such cells are exposed to the environment modulated by tumor cells transduced with Ad-IFNbeta. Even though the gene transfer of both IFNbeta and p19 was not found to be superior to the application of single genes, we observed that our approach may have an important impact on the inhibition of angiogenesis through endothelial cell

Improving adenoviral vectors and strategies for prostate cancer gene therapy

Gene therapy has been evaluated for the treatment of prostate cancer and includes the application of adenoviral vectors encoding a suicide gene or oncolytic adenoviruses that may be armed with a functional transgene. In parallel, versions of adenoviral vector expressing the p53 gene (Ad-p53) have been tested as treatments for head and neck squamous cell carcinoma and non-small cell lung cancer. Although Ad-p53 gene therapy has yielded some interesting results when applied to prostate cancer, it has not been widely explored, perhaps due to current limitations of the approach. To achieve better functionality, improvements in the gene transfer system and the therapeutic regimen may be required. We have developed adenoviral vectors whose transgene expression is controlled by a p53-responsive promoter, which creates a positive feedback mechanism when used to drive the expression of p53. Together with improvements that permit efficient transduction, this new approach was more effective than the use of traditional versions of Ad-p53 in killing prostate cancer cell lines and inhibiting tumor progression. Even so, gene therapy is not expected to replace traditional chemotherapy but should complement the standard of care. In fact, chemotherapy has been shown to assist in viral transduction and transgene expression. The cooperation between gene therapy and chemotherapy is expected to effectively kill tumor cells while permitting the use of reduced chemotherapy drug concentrations and, thus, lowering side effects. Therefore, the combination of gene therapy and chemotherapy may prove essential for the success of both approaches

Overhauling CAR T Cells to Improve Efficacy, Safety and Cost

Gene therapy is now surpassing 30 years of clinical experience and in that time a variety of approaches has been applied for the treatment of a wide range of pathologies. While the promise of gene therapy was over-stated in the 1990’s, the following decades were met with polar extremes between demonstrable success and devastating setbacks. Currently, the field of gene therapy is enjoying the rewards of overcoming the hurdles that come with turning new ideas into safe and reliable treatments, including for cancer. Among these modalities, the modification of T cells with chimeric antigen receptors (CAR-T cells) has met with clear success and holds great promise for the future treatment of cancer. We detail a series of considerations for the improvement of the CAR-T cell approach, including the design of the CAR, routes of gene transfer, introduction of CARs in natural killer and other cell types, combining the CAR approach with checkpoint blockade or oncolytic viruses, improving pre-clinical models as well as means for reducing cost and, thus, making this technology more widely available. While CAR-T cells serve as a prime example of translating novel ideas into effective treatments, certainly the lessons learned will serve to accelerate the current and future development of gene therapy drugs

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