Desenvolvimento de adenovírus helper de sistema adenoviral de alta capacidade capaz de expressar biomoléculas com atividade antitumoral
Data
2023-08-23
Autores
Toneto, Nicholas Pietro Agulha 
Orientadores
Tamura, Rodrigo Esaki
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Os vetores adenovirais são os mais utilizados para transferência de genes em ensaios clínicos de terapia gênica. Isso se atribuí principalmente por apresentarem alta capacidade de armazenamento e expressão gênica e menos efeitos genotóxicos. Além disso, os vetores adenovirais de alta capacidade apresentam menor imunogenicidade, o que se deve à remoção de todos os genes virais, porém dependem de um vírus helper para sintetizar a partícula viral e assim serem obtidos. No entanto, no processo de produção viral, existe a possibilidade de contaminação pelo vírus helper, que não apresenta o gene terapêutico, o que reduz a eficácia da terapia gênica. Portanto, nosso objetivo é eliminar essa contaminação, e para atingir este objetivo, o vetor adenoviral de alta capacidade e o vírus helper terão limitações de empacotamento, devido à especificidade do sorotipo da sequência de empacotamento e das proteínas envolvidas neste processo. Paralelamente, faremos a triagem de um banco de bactérias de compostagem e de extratos de origem vegetal, afim de encontrar novos compostos com atividade antitumoral contra células de câncer de próstata. Essa coleção de bactérias foi obtida no Zoológico de São Paulo durante um Projeto Temático da FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo) e os extratos vegetais foram fornecidos pelo Prof. Dr. João Lago da UFABC. Os vetores adenovirais foram clonados por arranjo de Gibson e técnicas clássicas de biologia molecular. As biomoléculas de origem bacteriana e vegetal foram avaliadas quanto à redução da viabilidade celular, e com os compostos de plantas já identificados e isolados foram realizados outros ensaios como determinação do IC50, proliferação celular e morte celular. Identificamos moléculas bioativas com atividade antitumoral, com foco principal nas células do câncer de próstata. E quanto ao vetor helper, obtivemos o vetor através de clonagens porém não conseguimos sintetizar o vírus. Dessa forma, uma perspectiva desses trabalhos será o uso do vetor adenoviral de alta capacidade com contaminação menor ou zero do vírus helper para expressar moléculas de origem bacteriana ou vegetal com atividade antitumoral
Adenoviral vectors are the most commonly used vector for gene transfer in gene therapy clinical trials. This is mainly attributed to their high storage and gene expression capacity and lower genotoxic effects. In addition, high-capacity adenoviral vectors induced reduced immunogenicity due to the removal of all viral genes, but it makes them dependent on a helper virus to synthesize the viral particle and thus be obtained. However, in the viral production process, there is the presence of helper virus contamination, which does not have the therapeutic gene, which reduces the effectiveness of gene therapy. Therefore, our goal is to eliminate this contamination, and to achieve this goal, the high-capacity adenoviral vector and the helper virus will have packaging limitations, due to the serotype specificity of the packaging sequence and the proteins involved in this process. In parallel, we will screen a bank of composted bacteria and plant extracts in order to find new compounds with antitumor activity against prostate cancer cells. This collection of bacteria was obtained at the São Paulo Zoo during a Thematic Project sponsored by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo) and the plant extracts were provided by Prof. Dr. João Lago from UFABC. Both adenoviral vectors were cloned by Gibson assembly and classical molecular biology techniques. Biomolecules of bacterial and plant origin were evaluated for the reduction of cell viability, and with the plant compounds already identified and isolated, other assays such as IC50 determination, cell proliferation and cell death were performed. We identified bioactive molecules with antitumor activity, with a main focus on prostate cancer cells. And about the helper vector, we obtained the vector through cloning but we can not synthetize the virus. As a proof of concept, the high-capacity adenoviral vector with less or zero contamination of the helper virus will be used to express molecules of bacterial or plant origin with known antitumor activity.
Adenoviral vectors are the most commonly used vector for gene transfer in gene therapy clinical trials. This is mainly attributed to their high storage and gene expression capacity and lower genotoxic effects. In addition, high-capacity adenoviral vectors induced reduced immunogenicity due to the removal of all viral genes, but it makes them dependent on a helper virus to synthesize the viral particle and thus be obtained. However, in the viral production process, there is the presence of helper virus contamination, which does not have the therapeutic gene, which reduces the effectiveness of gene therapy. Therefore, our goal is to eliminate this contamination, and to achieve this goal, the high-capacity adenoviral vector and the helper virus will have packaging limitations, due to the serotype specificity of the packaging sequence and the proteins involved in this process. In parallel, we will screen a bank of composted bacteria and plant extracts in order to find new compounds with antitumor activity against prostate cancer cells. This collection of bacteria was obtained at the São Paulo Zoo during a Thematic Project sponsored by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo) and the plant extracts were provided by Prof. Dr. João Lago from UFABC. Both adenoviral vectors were cloned by Gibson assembly and classical molecular biology techniques. Biomolecules of bacterial and plant origin were evaluated for the reduction of cell viability, and with the plant compounds already identified and isolated, other assays such as IC50 determination, cell proliferation and cell death were performed. We identified bioactive molecules with antitumor activity, with a main focus on prostate cancer cells. And about the helper vector, we obtained the vector through cloning but we can not synthetize the virus. As a proof of concept, the high-capacity adenoviral vector with less or zero contamination of the helper virus will be used to express molecules of bacterial or plant origin with known antitumor activity.