Abstract 13223: Split-Intein Mediated Adeno Associated Virus Delivery of CRISPR/dHFCas9-TET3CD as Antifibrotic Therapy

Introduction: Cardiac fibrosis is characterized by excessive deposition of extracellular matrix for which no specific therapy is available as of yet. Gene methylation plays an important role in the progression of cardiac fibrosis. We previously demonstrated that high-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression of fibrosis suppressor genes and attenuates renal fibrosis. Its efficacy in treating cardiac fibrosis, however, has not been examined. Hypothesis: While AAV-mediated delivery of CRISPR/Cas9 holds great therapeutic potential to treat cardiac fibrosis, several challenges still need to be overcome. We here aimed to circumvent the limiting viral packaging capacity of AAVs, and to identify the best AAV serotype to target mouse cardiac fibroblasts. Methods: We utilized split-intein mediated protein-splicing to divide dHFCas9-TET3CD (dHFCas9 fused to the catalytic domain of TET3) into two parts in order to perform AAV-mediated gene-specific demethylation of a fibrosis suppressor gene. Moreover, we explored the efficacy of 9 different serotypes in targeting mouse cardiac fibroblasts. To explore the antifibrotic efficacy of the fused dHFCas9-TET3CD in vivo, the Angiotensin-II induced cardiac fibrosis mouse model was used. Results: We identified that AAV9-SLRSPPS has a tropism for mouse cardiac fibroblasts with an efficiency of 72% in vitro and 51% and 32% in vivo after 7 days and 4 weeks, respectively. We showed that upon AAV transduction, the fused dHFCas9-TET3CD protein leads to gene-specific demethylation and attenuates cardiac fibrosis in the Angiotensin-II induced cardiac fibrosis mouse model . Conclusions: We demonstrate the use of a Cas9 derivative (dHFCas9-TET3CD) in combination with the split-intein technology and the AAV9-SLRPPS serotype to perform targeted demethylation both in vitro and in vivo which results in amelioration of cardiac fibrosis in vivo. This new technology holds promise not only for cardiac disease but for any disease associated with hypermethylation as well as for other applications where CRISPR/Cas9 in combination with AAV is useful.