Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes

Abstract

Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

Improving the efficiency of liver targeting rAAV-mediated homologous recombination using ribonucleotide reductase inhibitors

“…It should be noted most of in vivo hepatocytes are in G0, which is considerably different from cultured cells and could contribute to the discordance 46 . Recently, it was reported that AAV-HR in cardiomyocytes occurred independently of S phase entry, which is in line with our present data 47 . This lack of S phase progression in HR+ hepatocytes prompted the hypothesis that udarabine may have effects on non-proliferating hepatocytes.…”

Targeted Gene Delivery: Where to Land

“…Gene-specific strategy Limited to gene body mutations Urnov et al, 2005;Lombardo et al, 2007;Li et al, 2011;Genovese et al, 2014;Voit et al, 2014;Dever et al, 2016;Hubbard et al, 2016;Schiroli et al, 2017;Sweeney et al, 2017;Kuo et al, 2018;Wang et al, 2019Wang et al, , 2020aRai et al, 2020 (Hubbard et al, 2016;Kuo et al, 2018), and Wiskott-Aldrich Syndrome (Rai et al, 2020). Beside HSC and terminally differentiated blood cells, like B and T cells (Wang et al, 2016;Hung et al, 2018), AAV and nucleases have been the preferred method to achieve targeted transgene integration in many tissues in vivo (Suzuki et al, 2019;Kohama et al, 2020;Nishiguchi et al, 2020), especially the liver.…”

DGK and DZHK position paper on genome editing: basic science applications and future perspective

“…Viral delivery systems such as vectors based on adeno-associated virus (AAV), adenovirus, or lentivirus are able to transduce non-dividing cells [ 84 ]. For cardiomyocytes, AAV vector delivery enables HDR in murine adult heart tissues and human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSCs) independently of the cell cycle stage [ 73 ]. In contrast to AAV, for adenovirus (AdV) vectors, genomic integration is rather an exception.…”

“…In contrast to AAV, for adenovirus (AdV) vectors, genomic integration is rather an exception. In cultured neonate cardiomyocytes, however, some limited integration was observed for cells which had entered S-phase [ 73 ]. Viral vector systems vary in their packaging capacity, the genetic material (DNA/RNA), and the vector genome form.…”