The purpose of R-FunSel was to develop a method for the identification of functionally relevant cardiomyocyte receptors through an in vivo screening method, which is unbiased in terms of a priori selection of candidates and is agnostic upon the receptor mechanism of action. R-FunSel is based on i) the generation of a library of AAV vectors (which specifically target cardiomyocytes in the heart) expressing single guide RNAs (sgRNAs) targeting 213 receptors expressed in cardiomyocytes (2 sgRNAs per gene, 426 vectors in total); ii) the development of a method for the in vivo knock-out of these receptors using cardiac-specific Cas9 expressing mice using the developed vectors; iii) the application of this technology for the functional selection in vivo after myocardial infarction to identify receptors that are beneficial or detrimental to cardiac cell survival.
Elena Chiavacci started the project by identifying, bioinformatically, the receptors expressed by cardiomyocytes, to then design 2 sgRNAs per each gene encoding these receptors. These sgRNA were cloned into AAV vectors and a library was generated. Next, she developed the R-FunSel in vivo screening procedure. In brief, the library vectors were packaged in pools of 50-100 vector each and used to transduce the heart of transgenic mice expressing Cas9 in cardiomyocytes. Then, myocardial infarction was applied as a selective stimulus: cardiomyocytes expressing sgRNAs targeting receptors exerting a negative function were positively selected, while those exerting a protective function were lost. After 3 weeks after infarction and vector administration, DNA was recovered from the transduced hearts and the sgRNA sequences used as barcodes for NGS. Frequency of each sgRNA after myocardial infarction, compared to frequency in the absence of infarction, indicated selection, either positive or negative, and thus was suggestive of a role for the receptor in myocardial infarction progression. The efficacy of the proposed approach was first validated on a pool of sgRNAs targeting genes with an expected positive or negative role, to then be extended to the screening of the whole library.
The work has proceeded according to the proposed plan until the beginning of the COVID-19 lockdown in March 2020. During the lockdown period, when experimental research with animals was suspended, Dr. Chiavacci nevertheless significantly contributed to a project aimed to identify novel drugs that block SARS-CoV-2-induced cell-cell fusion. The project ended in the identification of a drug (Niclosamide) that is very effective in this respect and of a mechanism (activation of TMEM16 proteins by Spike) that appears to have significant pathogenic relevance in COVID-19.
The R-FunSel project has resumed after the 4-month lock down period and is expected to be completed in 3-4 months from the time of writing, with no significant problems in its execution, despite the technical complexity.
The project provided Dr. Chiavacci opportunity to improve her bioinformatic skills for the selection of gene candidates and the design of single guide RNAs for CRISPR/Cas9 gene editing, promoted her advanced training in cardiovascular experimentations for myocardial infarction and generated promising targets for the development of innovative cardiac therapeutics, thus enriching her CV in view of developing her independent research.
