Title: Prime editing toolkit development to enhance precise gene correction of human inherited cardiomyopathies

Abstract:

Dilated cardiomyopathy (DCM) is a genetic heart disease which progressively worsen the contractility of the myocardium, leading to left ventricular wall dilation, ultimately compromising organ function over time. Since most cases are characterised by mutations inherited in a dominant fashion, gene editing is considered one of the most suitable strategies to find a cure. Prime editing, a novel and versatile technique which do not rely on double strand break formation, could be effective in cardiomyocytes. Moreover, being the editor size not compatible with single AAV delivery, non-viral delivery methods based on RNA encapsulation in lipid nanoparticles (LNPs) could specifically target the heart and overcome the need to use dual AAV vectors.

To find possible cell modifiers able to increase prime editing efficiency we used a U2OS stable cell line expressing a mutated form of the EGFP (Y66S) to detect prime edited cells and performed a high throughput screening, testing the effect of a library of 2602 human microRNAs mimics. The screening identified a miRNA family composed of hsa-miR-301a-3p and hsa-miR-454-3p able to significantly increase prime editing efficiency by 2.7-fold over control in both plasmid and RNA delivery fashion. By transcriptomic analysis of cells treated with miR-301a-3p, we identified a selected group of genes that we evaluated through a siRNA sub-screening linking downregulation to prime editing efficiency. Our results demonstrate that CAV2 and c-MET are miRNAs targets and that their down-regulation has incremental effect on prime editing performance. Interestingly, we demonstrated a connection between c-MET/CAV2 silencing and DNA repair highlighting how the regulation of these two genes can affect PCNA stability. Consequently, we showed that direct downregulation of PCNA with siRNAs can increased prime editing efficiency in cardiomyocytes and in other cell lines. Aiming to link enhanced efficiency with increased safety for trials in vivo, we developed a prime editing RNA tool kit packaged in LNPs optimised for cardiomyocytes delivery. We successfully proved the co-encapsulation of prime editing enhancing siRNAs coupled with the prime editor mRNA and a targeting pegRNA (e-pegRNA). Finally, we demonstrated both efficient delivery and editing in vitro in neonatal cardiomyocytes derived from a DCM mouse model characterised by a humanised mutation in exon 9 (R636Q) of RBM20, a gene which is frequently mutated in human patients.

Our study revealed that prime editing efficiency can be enhanced by specific siRNAs and that stable LNPs formulations can effectively package prime editing RNA tool kit and siRNAs while safely target cells in vitro. These results will contribute to the evolution of RNA-based editing technologies in the cardiovascular field, defining features of promising tools to correct inherited mutations using a non-viral delivery method.

Speaker: Luca Venditti

Biography:

Luca Venditti graduated in Biological Sciences from “La Sapienza” University of Rome (Italy) and completed a Master of Science in Functional Genomics at University of Trieste (Italy) under the supervision of Prof. Oscar Burrone (ICGEB) in the molecular virology laboratory. Here, he developed a method to advance rotavirus reverse genetics platform and study the rotaviral cycle using a CRISPR RNA endonuclease. In January 2020 he moved to the UK in Alexander Borodavka’s laboratory at Cambridge University (Biochemistry Department) as Research Assistant to work on the assembly mechanism of dsRNA viral segments in the encapsulation of new rotaviral particles. In October 2020 he started a BHF funded PhD programme (1+3) based in James Black Centre (BHF centre of research excellence) here at King’s College London in Mauro Giacca’s laboratory. His PhD project spans from identifying and using small RNA adjuvants to increase prime editing efficiency in cardiomyocytes and optimising lipid nanoparticle formulation for cardiac gene editing.

This hybrid event is both online and in person.