Clin Res Cardiol (2021) DOI DOI https://doi.org/10.1007/s00392-021-01843-w

Amenability of Porcine Cardiomyocyte Compartment to rAAV-Delivered CRISPR Genome Editing

T. Bozoglu1, F. Giesert2, A. Bähr1, B. Rieblinger3, A. Schnieke3, W. Wurst2, C. Kupatt1

1Klinik und Poliklinik für Innere Medizin I, Klinikum rechts der Isar Technischen Universität München, München; 2Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg; 3Biotechnologie der Nutztiere, Technische Universität München, Freising-Weihenstephan;

“Clustered regularly interspaced short palindromic repeats” (CRISPR) and associated endonucleases (CAS) have already revolutionized many areas of biomedical research. Combined with adeno-associated viruses (AAV), with its FDA-approved efficacy and safety features, as the delivery medium, CRISPR genome editing is an obvious choice for somatic editing of tissues for which tropism of an AAV serotype exists. Thanks to the high levels of cardiac tropism of AAV9, such a combination has been used for cardiomyocyte editing in different models including mice and pigs. However, inconsistency in efficacy of editing between different loci has been reported. Here, we report our investigation of factors affecting amenability of porcine cardiac genome editing. As a baseline, we have used AAV2/9 serotype, coated with G2 PAMAM nanoparticles, encoding CMV driven CRE recombinase systemically delivered to tdTomato reporter pigs. All animals underwent testing for AAV9 neutralizing antibodies, with a titer of 1/4 or less as inclusion criterion for this study. Efficiency of editing was measured by the shift of fluorescence from red to green as a result of recombination, as well as western blots against tdRFP and GFP. CRISPR editing efficacy was evaluated using a porcine model of Duchenne muscular dystrophy, where double delivery of AAVs encoding N- and C- terminal parts of intein mediated split CAS9 along with sgRNAs targeting introns 50 and 51 of dystrophin gene resulted in restoration of open reading frame via exon skipping. Efficacies of delivery and editing was determined by dystrophin protein levels, western blot for N- and C- terminal halves of CAS9 and full length ligated CAS9, as well as qPCR analysis of sgRNA transcripts. To single out efficacy of editing independent of delivery and kinetics of CAS9 expression, we have made use of Rosa26-CAS9 knock-in pigs. We generated self-complementary AAVs encoding heterologous sgRNA expression cassettes targeting ERCC1 and MYBPC3 genes and a truncated-CMV driven mCherry marker and delivered locally to left ventricle. Viable ex vivo LV sections under pacing were also transduced, eliminating inhibitory effects of immune system. Our results demonstrate markedly lower efficiency of Crispr-mediated editing in comparison to CRE-mediated recombination (decreasing from up to 100% for CMV-CRE to <10%). Moreover, CAS9 levels of AAV9-transduced tissues are comparable to knock-in animals, rendering the AAV transduction as the critical step. Strikingly, we have observed that upon in cis delivery of sgRNAs excision of loci by flanking double strand breaks is heavily favored in comparison to indel formation upon repair of single double-strand breaks. Thus, systemic application of AAV9 encoding for gRNAs with CAS9 either co-delivered by AAVs or expressed in transgenic animals yields detectable but low gene editing efficacy. Local transduction and optimization of AAV9 uptake may be required for high transduction rates in target regions of the heart in vivo. Furthermore, characterization of cardiac epigenomic landscape and its correlation with amenability to editing of loci is underway.

https://dgk.org/kongress_programme/jt2021/aP611.html