Development of a gene therapy model for hereditary angioedema based on SERPING1 gene edition by CRISPR-Cas9 system

Abstract

Hereditary angioedema (HAE) is a rare genetic disease resulting from a deficiency of C1 inhibitor (C1-INH) or mutations in F12 gene. In both cases, the changes lead to uncontrolled activation of the kallikrein-kinin system (KKS) culminating in excessive release of bradykinin (BK). HAE profoundly affects the quality of life, treatment is expensive and often inefficient. We propose a gene therapy model based on C1-INH gene correction (SERPING1).First of all, we intend to generate mice and cell lines with the p.A297T correspondent mutation found in Brazilian HAE families using the CRISPR-Cas9 system. Then, the mutation generated in SERPING1 will be corrected using the same system. The plasmid pSpCas9 nickase (pSpCas9n) will be used to generate C1-INH deficient C57BL/6N mice through pro-nuclear microinjections. Gene editing will be evaluated by sequencing, by SURVEYOR to check indel formation and by next generation sequencing and BLESS to check off-target events. In vitro and in vivo assays will be performed in parallel. Mice characterization will be performed by C1-INH measurements and function, as well as vascular permeability evaluation. From this point, the first tests applying an in vivo SERPING1 correction protocol using hydrodinamic injection of the constructions in the tail vein of the animal will be performed.