ESR12: CRISPR/Cas9 gene editing for excision and resue of deep-intronic mutations in ABCA4
Partner
Eberhard Karls University Tubingen, Germany (www.eye-tuebingen.de/wissingerlab) Supervisor Dr. S. Kohl |
Pietro De Angeli
My research background is on molecular biology and genome engineering. Over my bachelor’s and part of my master’s studies, I contributed to the development of an innovative protocol for the treatment of a rare genetic metabolic disease, Guanidinoacetate methyltransferase deficiency. The final purpose was to validate a scalable enzyme-replacement-therapy-based protocol to administered recombinant enzymes via a patented delivery device to patients affected by the aforementioned autosomal recessive disorder. During my Master thesis, I then spent six months at the Sanger Institute (UK), in Leopold Part’s team, as a visiting worker where I collaborated to genome editing projects that involved CRISPR-Cas9 high-throughput screens. I am now pursuing my PhD thesis in Wissinger Lab at the Universitätsklinikum Tübingen, within the “StarT” innovative training network. The aim of my PhD project is to implement CRISPR-Cas9 approaches to rescue some Stargardt-causing-deep-intronic mutations in ABCA4 gene, and to further investigate the possibility of harnessing the genome editing potential as a promising technology for the treatment of genetic conditions. Also a few words about my interpersonal profile: I regard myself as highly motivated, inquisitive, dynamic, collaborative, a good reader (considering scientific literature) and one who is truly “addicted” to the field of science. These capabilities, along with my practical expertise, allow me to learn quickly and contribute immediately to the state of science – whether within a team or independently |
Abstract
Deep-intronic mutations represent a considerable fraction of disease alleles in ABCA4-IRD. Since ABCA4 is difficult to target by AAV-based gene supplementation therapy, alternative therapeutic approaches are needed. Here, we propose CRISPR/Cas9 gene editing to target cryptic splice sites and make use of the default non-homologous end joining (NHEJ)-based repair pathway to delete or excise illegitimate exons or the critical splice donor/acceptor sites to eventually rescue normal splicing of transcripts and re-establish normal gene function. The advantages of such an approach are the loosened need for precision in gene editing, the once-forever intervention strategy and the circumvention of all dose-related/overdosing issues in standard approaches. First proof-of-concept of this strategy has been obtained by P3-EKUT in an in vitro assay of an OPA1 deep-intronic mutation. ESR12 will use this strategy to target two clusters of deep-intronic mutations in intron 30 (V4 cluster) and 36 (V1 cluster) of ABCA4 by a duplex CRISPR/Cas9 nuclease or quadruplex sgRNA/Cas9 nickase inducing double-strand breaks up- and downstream of the deep-intronic mutation and resulting in small deletions due to intrinsic NHEJ repair. Efficacy of the approach and its validation will be tested in minigene assays in HEK293 cells and in retinoblastoma Y79 cell clones in which common deep-intronic mutations will be introduced by CRISPR/Cas9 homology-directed repair.
Deep-intronic mutations represent a considerable fraction of disease alleles in ABCA4-IRD. Since ABCA4 is difficult to target by AAV-based gene supplementation therapy, alternative therapeutic approaches are needed. Here, we propose CRISPR/Cas9 gene editing to target cryptic splice sites and make use of the default non-homologous end joining (NHEJ)-based repair pathway to delete or excise illegitimate exons or the critical splice donor/acceptor sites to eventually rescue normal splicing of transcripts and re-establish normal gene function. The advantages of such an approach are the loosened need for precision in gene editing, the once-forever intervention strategy and the circumvention of all dose-related/overdosing issues in standard approaches. First proof-of-concept of this strategy has been obtained by P3-EKUT in an in vitro assay of an OPA1 deep-intronic mutation. ESR12 will use this strategy to target two clusters of deep-intronic mutations in intron 30 (V4 cluster) and 36 (V1 cluster) of ABCA4 by a duplex CRISPR/Cas9 nuclease or quadruplex sgRNA/Cas9 nickase inducing double-strand breaks up- and downstream of the deep-intronic mutation and resulting in small deletions due to intrinsic NHEJ repair. Efficacy of the approach and its validation will be tested in minigene assays in HEK293 cells and in retinoblastoma Y79 cell clones in which common deep-intronic mutations will be introduced by CRISPR/Cas9 homology-directed repair.
