Advancing cardiac gene editing towards clinical application

This is an ambitious project aimed to develop CRISPR/Cas9 technologies for the precise correction of the genetic defects leading to inherited cardiomyopathies, including hypertrophic cardiomyopathy (HCM), which affects 1:500 individuals in the general population, and dilated cardiomyopathy (DCM) which has a prevalence of 1:2500. The advent of the CRISPR/Cas9 technology has opened the unprecedented possibility to develop a curative therapy for these conditions. However, precise gene editing in the heart remains challenging, as genetic correction must occur in a sufficiently large number of cardiomyocytes to be effective. In addition, cardiomyocytes are post mitotic cells and thus refractory to homology directed repair (HDR) and large animal models in which to test the technology are largely missing. This project will try and overcome these limitations. The project is divided into 3 main AIMS, which are correlated and bidirectional. AIM1 will fuel the project with technological tools. Besides standard saCas9 and spCas9, we will generate better gene editors with smaller size, improved efficacy and lower off-target effects, from metagenomic analysis of bacterial species and through in vitro direct evolution. AIM1 will also provide the project with cardiotropic AAV9 vectors and with lipid nanoparticles (LNPs) for cardiac delivery. The novel editors and delivery tools from AIM1 will be used in AIM2 to attempt correction of a mouse model of HCM, carrying a knock-in mutation in the MYBPC3 gene. AIM2 will also try and overcome the problem that cardiomyocytes are refractory to HDR. In one strategy, we will leverage on a series of pro-recombinogenic miRNAs we have identified. Alternatively, we will exploit Prime Editing, which allows HDR-independent gene correction. One of the challenges of cardiac gene editing is the efficiency of cardiac delivery. AIM3 will identify a most effective route of cardiac administration in pigs. We will explore LNP and/or AAV vector administration using catheterization of either the coronary arteries or the coronary sinus, which will be compared with intramyocardial injection. All these procedures are amenable to clinical translation. Finally, AIM3 will attempt gene editing in a large animal model of DCM, based on a knock-in mutation in the titin gene we developed. This is a highly integrated project among three groups with complementary expertise and converging interests, and with an extensive record of past interactions. There is a tremendous medical interest in developing therapies to prevent heart failure in inherited cardiac conditions, with relevant social and economic impact for patients and the national health system. This project has also important potential to generate IP and to attract interest from venture or pharma. The proponents have significant experience in dealing with the private sector to ensure continuity towards clinical application.

This is an ambitious project aimed to develop CRISPR/Cas9 technologies for the precise correction of the genetic defects leading to inherited cardiomyopathies, including hypertrophic cardiomyopathy (HCM), which affects 1:500 individuals in the general population, and dilated cardiomyopathy (DCM) which has a prevalence of 1:2500. The advent of the CRISPR/Cas9 technology has opened the unprecedented possibility to develop a curative therapy for these conditions. However, precise gene editing in the heart remains challenging, as genetic correction must occur in a sufficiently large number of cardiomyocytes to be effective. In addition, cardiomyocytes are post mitotic cells and thus refractory to homology directed repair (HDR) and large animal models in which to test the technology are largely missing. This project will try and overcome these limitations. The project is divided into 3 main AIMS, which are correlated and bidirectional. AIM1 will fuel the project with technological tools. Besides standard saCas9 and spCas9, we will generate better gene editors with smaller size, improved efficacy and lower off-target effects, from metagenomic analysis of bacterial species and through in vitro direct evolution. AIM1 will also provide the project with cardiotropic AAV9 vectors and with lipid nanoparticles (LNPs) for cardiac delivery. The novel editors and delivery tools from AIM1 will be used in AIM2 to attempt correction of a mouse model of HCM, carrying a knock-in mutation in the MYBPC3 gene. AIM2 will also try and overcome the problem that cardiomyocytes are refractory to HDR. In one strategy, we will leverage on a series of pro-recombinogenic miRNAs we have identified. Alternatively, we will exploit Prime Editing, which allows HDR-independent gene correction. One of the challenges of cardiac gene editing is the efficiency of cardiac delivery. AIM3 will identify a most effective route of cardiac administration in pigs. We will explore LNP and/or AAV vector administration using catheterization of either the coronary arteries or the coronary sinus, which will be compared with intramyocardial injection. All these procedures are amenable to clinical translation. Finally, AIM3 will attempt gene editing in a large animal model of DCM, based on a knock-in mutation in the titin gene we developed. This is a highly integrated project among three groups with complementary expertise and converging interests, and with an extensive record of past interactions. There is a tremendous medical interest in developing therapies to prevent heart failure in inherited cardiac conditions, with relevant social and economic impact for patients and the national health system. This project has also important potential to generate IP and to attract interest from venture or pharma. The proponents have significant experience in dealing with the private sector to ensure continuity towards clinical application.