A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach for supporting gene target validation in early drug discovery

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes ( API5, HSPB7 , and LMO2 ) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2 , there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7 , and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.