Periodic Reporting for period 1 - R-FunSel (In vivo functional screening via CRISPR-Cas9 to systematically identify cardiomyocyte receptors as targets for the innovative therapies for myocardial infarction and heart failure)
Periodo di rendicontazione: 2019-04-01 al 2021-03-31
There is an urgent need to develop new therapies for myocardial infarction to prevent cardiomyocyte loss, improve cardiac dysfunction and prevent eventual heart failure. All the currently available therapies are based on pato-physiological concepts developed in the mid-90’s or earlier and all drugs are small molecules. Compared to other fields in which biological drugs (in particular, monoclonal antibodies and recombinant proteins) have literally changed patients’ management (e.g. cancer and rheumatic disorders), not a single biological drug exists for any cardiac disease.
A class of particularly appealing drugs are those targeting cell surface receptors expressed in cardiomyocytes. However, scanty information is currently available on targetable/druggable receptors in these cells, and the presence of receptors themselves is, in most cases, only inferred from RNA expression studies, with limited insights into their biological role.
The R-FunSel project was aimed to identify novel therapeutic targets for myocardial infarction, the leading cause of heart failure. R-FunSel takes advantage of the CRISPR/Cas9 genome editing technology to perform in vivo systematic screenings of cardiomyocyte receptors. For this purpose, the project takes advantage of a mouse model of myocardial infarction to identify receptors necessary for cardiomyocyte survival as well as receptor playing a negative role. R-FunSel offers an unbiased way to shed light on the mechanisms regulating the outcome of myocardial infarction, contributing to the translation of basic research into novel therapeutic targets.
A class of particularly appealing drugs are those targeting cell surface receptors expressed in cardiomyocytes. However, scanty information is currently available on targetable/druggable receptors in these cells, and the presence of receptors themselves is, in most cases, only inferred from RNA expression studies, with limited insights into their biological role.
The R-FunSel project was aimed to identify novel therapeutic targets for myocardial infarction, the leading cause of heart failure. R-FunSel takes advantage of the CRISPR/Cas9 genome editing technology to perform in vivo systematic screenings of cardiomyocyte receptors. For this purpose, the project takes advantage of a mouse model of myocardial infarction to identify receptors necessary for cardiomyocyte survival as well as receptor playing a negative role. R-FunSel offers an unbiased way to shed light on the mechanisms regulating the outcome of myocardial infarction, contributing to the translation of basic research into novel therapeutic targets.
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.
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.
R-Funsel is a discovery project with no immediate practical impact on the clinic, but with the potential to unravel the mechanisms involved in cardiac protection from damage by generating systematic information of cardiomyocyte receptors and thus identifying potential targets for pharmacological development. The burden of cardiac disease is enormous (heart failure is estimated to affect over 25 million people worldwide, with a trend to increase) and consolidated evidence indicates that there is a clear correlation between the size of myocardial infarction and the probability to undergo heart dysfunction over time. Therefore, the identification of novel targets to protect the heart from damage has enormous therapeutic implications. The AAV sgRNA library generated by Dr. Chiavacci (over 400 AAV vectors) and the development of the R-FunSel method for their functional screening in vivo after myocardial infarction are important milestones towards understanding the function of cardiomyocyte receptors and developing new therapeutic strategies.