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Designing novel CAR T cells to eliminate latently HIV-1- infected cells
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Resumo(s)
O Vírus da Imunodeficiência Humana (HIV) continua a ser um problema de saúde global, com um elevado número de indivíduos infectados e de mortes por ano em todo o mundo. A terapia antirretroviral (ART) controla a infeção pelo HIV mas não erradica as células infectadas latentemente pelo HIV, também chamadas de reservatórios virais. Os agentes de reversão da latência (LRA) têm sido utilizados para reativar os reservatórios virais e promover a sua eliminação. No entanto, a utilização isolada de LRAs como estratégia não é muito eficiente, uma vez que as células T CD8+ naturais não eliminam eficazmente as células infectadas reactivadas. A utilização da terapia com células T receptoras de antigénios quiméricos (CAR) tem sido vista como promissora no campo do VIH, dada a sua maior especificidade no reconhecimento de antigénios e a sua capacidade de ultrapassar a deficiência das células T CD8 citotóxicas naturais. Este projeto propõe uma combinação de um LRA, para reativar a expressão viral, e de uma terapia com células CAR T para atingir e matar as células infectadas pelo HIV em estado latente. A geração de células CAR T será composta por células que expressam um recetor Notch sintético (synNotch) à superfície para reconhecer a proteína CD4. Este reconhecimento desencadeará a expressão de um CARtoolbox, que inclui o CAR anti-HIV, e a flagelina, que funcionará como um LRA. Começámos por isolar as células mononucleares do sangue periférico (PBMCs) por gradiente de densidade Ficoll-Paque, a partir das quais as células T e as células T CD8+ foram isoladas por seleção negativa. Estas células foram activadas e transduzidas com partículas lentivirais que transportam o sistema synNotch ou um vetor de controlo para a CARtoolboox, o BFPmCherry. A expressão das construções foi verificada através de ensaios de citometria de fluxo. Conseguimos transduzir as diferentes construções em todas as subpopulações celulares, obtendo diferentes taxas de eficiência de transdução, dependendo da construção e da população celular utilizada. Além disso, conseguimos gerar células que expressam ambas as construções. Estes resultados representam os primeiros passos para a produção de células CAR T para eliminar os reservatórios latentes do HIV.
Human Immunodeficiency Virus (HIV) is still a global health problem, with a high number of infected individuals and deaths worldwide per year. Antiretroviral therapy (ART) controls HIV infection but does not eradicate latently HIV-infected cells, also called viral reservoirs. Latent Reversal Agents (LRAs) have been used to reactivate viral reservoirs to promote their elimination. However, the singular use of LRAs as a strategy is not very efficient since natural CD8+ T cells do not efficiently eliminate the reactivated infected cells. The use of chimeric antigen receptor (CAR) T cell therapy has been seen as promising in the field of HIV given its higher antigen specificity recognition and its ability to overcome the impairment of natural cytotoxic CD8 T cells. This project proposes a combination of an LRA, to reactivate viral expression and CAR T cell therapy to target and kill latently HIV-infected cells. The generation of CAR T cells will comprise cells expressing a synthetic Notch (synNotch) receptor at the surface to recognize the CD4 protein. This recognition will trigger the expression of a CARtoolbox, comprising the anti-HIV CAR, and the flagellin, which will work as an LRA. We first isolated Peripheral Blood Mononuclear Cells (PBMCs) by Ficoll-Paque density gradient, from which T Cells and CD8+ T Cells were isolated by negative selection. These cells were activated and transduced with lentiviral particles carrying the synNotch system or a control vector for CARtoolbox, the BFPmCherry. The expression of the constructs was verified through flow cytometry assays. We could transduce the different constructs in all cell subpopulations, getting different transduction efficiency rates depending on the construct and cell population used. In addition, we successfully generated cells expressing both constructs. These results represent the first steps towards producing CAR T cells to eliminate HIV latent reservoirs.
Human Immunodeficiency Virus (HIV) is still a global health problem, with a high number of infected individuals and deaths worldwide per year. Antiretroviral therapy (ART) controls HIV infection but does not eradicate latently HIV-infected cells, also called viral reservoirs. Latent Reversal Agents (LRAs) have been used to reactivate viral reservoirs to promote their elimination. However, the singular use of LRAs as a strategy is not very efficient since natural CD8+ T cells do not efficiently eliminate the reactivated infected cells. The use of chimeric antigen receptor (CAR) T cell therapy has been seen as promising in the field of HIV given its higher antigen specificity recognition and its ability to overcome the impairment of natural cytotoxic CD8 T cells. This project proposes a combination of an LRA, to reactivate viral expression and CAR T cell therapy to target and kill latently HIV-infected cells. The generation of CAR T cells will comprise cells expressing a synthetic Notch (synNotch) receptor at the surface to recognize the CD4 protein. This recognition will trigger the expression of a CARtoolbox, comprising the anti-HIV CAR, and the flagellin, which will work as an LRA. We first isolated Peripheral Blood Mononuclear Cells (PBMCs) by Ficoll-Paque density gradient, from which T Cells and CD8+ T Cells were isolated by negative selection. These cells were activated and transduced with lentiviral particles carrying the synNotch system or a control vector for CARtoolbox, the BFPmCherry. The expression of the constructs was verified through flow cytometry assays. We could transduce the different constructs in all cell subpopulations, getting different transduction efficiency rates depending on the construct and cell population used. In addition, we successfully generated cells expressing both constructs. These results represent the first steps towards producing CAR T cells to eliminate HIV latent reservoirs.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2024, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
HIV Viral reservoirs CAR T cell therapy synNotch receptor Teses de mestrado - 2024