Estudos de validação in vivo de abordagens terapêuticas de RNA para Doenças Lisossomais de Sobrecarga
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2022-03-25
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Nos últimos anos, tem havido um avanço extraordinário no desenvolvimento de terapias inovadoras para o tratamento de doenças raras. As doenças lisossomais de sobrecarga (DLS) são um grupo de doenças hereditárias do metabolismo raras que podem originar uma patologia grave e progressiva devido a uma disfunção lisossomal específica. Atualmente, não existe cura para as DLS, embora para algumas destas doenças (apenas 11) haja estratégias de tratamento. Assim, as abordagens terapêuticas de RNA podem representar uma importante estratégia terapêutica alternativa ou adjuvante. Este trabalho tem como objetivo iniciar o processo de validação in vivo de duas abordagens terapêuticas de RNA já testadas e validadas em células de doentes que têm por base a utilização de oligonucleótidos antisense (OA) e vetores U1snRNA modificados para a correção de duas mutações causais de DLS. Para a mutação c.3503_3504delTC no gene GNPTAB, que origina a Mucolipidose II α/β (ML II α/β), foi testada uma estratégia terapêutica de exon-skipping utilizando OA. Num estudo anterior efetuado in vitro, diferentes OA foram utilizados para promover o skipping do exão 19 do gene GNPTAB em fibroblastos de doentes, tendo resultado na produção de um mRNA in-frame. Neste estudo, pretendeu-se avaliar o potencial terapêutico desta abordagem tanto in vitro (fibroblastos C57BL/6) como in vivo (murganhos C57BL/6). Primeiro, desenhamos um OA específico para a sequência Gnptab de murganho que emparelha na segunda metade do exão 19. Depois de o testar in vitro, a análise de cDNA mostrou que este OA foi capaz de promover o skipping deste exão, embora sem os níveis de eficácia desejados. Posteriormente foram efetuados ensaios in vivo, em que injetamos murganhos C57BL/6 com duas concentrações diferentes de OA testadas em dois períodos de tempo. Contudo, na análise de RT-PCR de amostras de fígado, não observamos o skipping do exão 19. Por isso, vamos testar outros OA bem como outras condições experimentais num futuro próximo. Neste trabalho, foi também dada continuidade à validação pré-clínica de uma abordagem terapêutica com um vetor U1snRNA modificado para corrigir o defeito de splicing (skipping do exão 2) causado pela mutação c.234+1G>A no gene HGSNAT que origina a Mucopolissacaridose IIIC (MPS IIIC). Num estudo anterior, foi observada a correção parcial do defeito de splicing em fibroblastos de doentes após a transfeção de um U1snRNA totalmente complementar ao 5'ss do exão 2. Nesta dissertação, o objetivo foi construir e analisar a expressão de um midigene com o alelo c.234+1G>A humano no fígado de murganhos C57BL/6 de forma a obter um modelo animal para avaliar o potencial terapêutico do vetor U1snRNA modificado específico para esta mutação. Foram construídos dois midigenes, um com a sequência wild-type de cDNA do gene HGSNAT mais parte dos intrões 1 e 2 e outro com a mesma sequência mas contendo a mutação c.234+1G>A. A análise de cDNA após a transfeção de ambos os midigenes nas linhas celulares Hep3B, COS-7 e HEK293T, revelou que estes reproduzem corretamente o padrão de splicing do exão 2 do gene HGSNAT. Também efetuamos a validação in vivo da expressão dos midigenes WT e mutado em murganhos C57BL/6. Os resultados preliminares mostraram que, pelo menos num animal, o midigene com o alelo mutado foi expresso no fígado. Embora ainda preliminares, os estudos desenvolvidos nesta dissertação contribuem para o avanço destas novas e promissoras terapias de RNA que poderão no futuro ser utilizadas no tratamento de doentes com duas patologias muito raras.
Nowadays we are seeing incredible advances in the development of medicines to treat patients with rare diseases. Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic diseases that can lead to a severe and progressive pathology due to a specific lysosomal dysfunction. Currently, there is no cure available for LSDs even though treatment strategies do exist for a small number (only 11). So, RNA-based approaches may constitute a potential alternative or an adjuvant therapeutic strategy for them. This work aims to start the process of in vivo validation of two RNA therapeutic approaches already tested and validated in patients' fibroblasts, which are based on the use of antisense oligonucleotides (AOs) and modified U1snRNA vectors to correct two LSD-causing mutations. For the frequent c.3503_3504delTC mutation in the GNPTAB gene, which causes ML II α/β, we explored the possibility of an exon-skipping therapeutic strategy based on the use of AOs. In a previous in vitro study in patients’ fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, successfully resulting in the production of an inframe mRNA. Here, our objective was to evaluate the therapeutic potential of this approach both in vitro (C57BL/6 fibroblasts) and in vivo (C57BL/6 mice). First, we designed a mousespecific AO targeting a region in the second half of exon 19. Then we tested this AO in vitro and cDNA analysis showed that it was able to promote mouse exon 19 skipping, even though not reaching the desired efficacy levels. Subsequently, we set up the in vivo experiments in which the C57BL/6 mice were injected with two different AO concentrations and during two time periods, but in the RT-PCR analyses of liver samples the exon 19 skipping was not observed. Therefore, additional AOs and experimental conditions will be tested in a near future. In this work, we also continued the preclinical validation of a modified U1snRNA therapeutic approach to correct the splicing defect (skipping of exon 2) caused by the c.234+1G>A mutation in the HGSNAT gene, which causes MPS IIIC. In a previously study in patients’ fibroblasts a U1snRNA totally complementary to the 5’ss of exon 2 was demonstrated to partially restore the splicing defect. Here, our goal was to construct and analyze the expression of a midigene cloned with the mutant allele in the liver of C57BL/6 mice to obtain an animal model to evaluate in vivo the therapeutic potential of the modified U1snRNA. For this, two full-length midigenes were generated by cloning the WT or mutated HGSNAT splicing-competent cassettes. cDNA analysis after their in vitro transfection into Hep3B, COS- 7 and HEK293T cell lines, showed that both midigenes reproduce the WT and mutant splicing patterns. The in vivo validation of both WT and mutant midigenes expression was also performed. Preliminary results showed that the mutant midigene was expressed in the liver of at least one animal. Although still preliminary, the studies developed in this dissertation contribute to pave the way of these new and promising RNA therapies that may, in the future, be used for the treatment of patients with two very rare yet life-threatening pathologies.
Nowadays we are seeing incredible advances in the development of medicines to treat patients with rare diseases. Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic diseases that can lead to a severe and progressive pathology due to a specific lysosomal dysfunction. Currently, there is no cure available for LSDs even though treatment strategies do exist for a small number (only 11). So, RNA-based approaches may constitute a potential alternative or an adjuvant therapeutic strategy for them. This work aims to start the process of in vivo validation of two RNA therapeutic approaches already tested and validated in patients' fibroblasts, which are based on the use of antisense oligonucleotides (AOs) and modified U1snRNA vectors to correct two LSD-causing mutations. For the frequent c.3503_3504delTC mutation in the GNPTAB gene, which causes ML II α/β, we explored the possibility of an exon-skipping therapeutic strategy based on the use of AOs. In a previous in vitro study in patients’ fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, successfully resulting in the production of an inframe mRNA. Here, our objective was to evaluate the therapeutic potential of this approach both in vitro (C57BL/6 fibroblasts) and in vivo (C57BL/6 mice). First, we designed a mousespecific AO targeting a region in the second half of exon 19. Then we tested this AO in vitro and cDNA analysis showed that it was able to promote mouse exon 19 skipping, even though not reaching the desired efficacy levels. Subsequently, we set up the in vivo experiments in which the C57BL/6 mice were injected with two different AO concentrations and during two time periods, but in the RT-PCR analyses of liver samples the exon 19 skipping was not observed. Therefore, additional AOs and experimental conditions will be tested in a near future. In this work, we also continued the preclinical validation of a modified U1snRNA therapeutic approach to correct the splicing defect (skipping of exon 2) caused by the c.234+1G>A mutation in the HGSNAT gene, which causes MPS IIIC. In a previously study in patients’ fibroblasts a U1snRNA totally complementary to the 5’ss of exon 2 was demonstrated to partially restore the splicing defect. Here, our goal was to construct and analyze the expression of a midigene cloned with the mutant allele in the liver of C57BL/6 mice to obtain an animal model to evaluate in vivo the therapeutic potential of the modified U1snRNA. For this, two full-length midigenes were generated by cloning the WT or mutated HGSNAT splicing-competent cassettes. cDNA analysis after their in vitro transfection into Hep3B, COS- 7 and HEK293T cell lines, showed that both midigenes reproduce the WT and mutant splicing patterns. The in vivo validation of both WT and mutant midigenes expression was also performed. Preliminary results showed that the mutant midigene was expressed in the liver of at least one animal. Although still preliminary, the studies developed in this dissertation contribute to pave the way of these new and promising RNA therapies that may, in the future, be used for the treatment of patients with two very rare yet life-threatening pathologies.
Descrição
Dissertação de Mestrado em Biologia Clínica e Laboratorial
Palavras-chave
Doenças lisossomais de sobrecarga , Murganho C57BL/6