Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina

• 成果类型: Article
• 署名作者: Kumar, Satheesh; Hsiao, Yi - Wen; Wong, Vickie H. Y.; Aubin, Deborah; Wang, Jiang - Hui; Lisowski, Leszek; Rakoczy, Elizabeth P.; Li, Fan; Martinez, Luis Alarcon -; Cordero, Anai Gonzalez -; Bui, Bang V.; Liu, Guei-Sheung
• 署名单位: Royal Victorian Eye & Ear Hospital; Centre for Eye Research Australia; University of Melbourne; University of Tasmania; Menzies Institute for Medical Research; University of Melbourne; University of Sydney; Children's Medical Research Institute - Australia; University of Sydney; Children's Medical Research Institute - Australia; University of Sydney; Military Institute of Aviation Medicine; Children's Medical Research Institute - Australia; NSW Health; Sydney Childrens Hospitals Network; University of Western Australia; Sun Yat Sen University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10639
• DOI: 10.1073/pnas.2408345121
• 发表日期: 2024-11-05
• 关键词: vegf; disease; glia

摘要

CRISPR-Cas13 nucleases are programmable RNA- targeting effectors that can silence gene expression in a transient manner. Recent iterations of Cas13 nucleases are compact for adeno- associated virus (AAV) delivery to achieve strong and persistent expression of various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all- in- one AAV gene therapy with Cas13bt3 can effectively silence VEGFA mRNA in human retinal organoids and humanized VEGF transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)- derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 by AAV2.7m8 can efficiently transduce mouse retinal cells for specific knockdown of human VEGFA in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity, and establish the Cas13bt3 RNA editing system as a potential anti-VEGF agent that can achieve significant control of VEGFA for the treatment of retinal neovascularization.