Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina

• Published in: Molecular therapy. Nucleic acids Vol. 35; no. 4; p. 102349
• Main Authors: Pulman, Juliette, Botto, Catherine, Malki, Hugo, Ren, Duohao, Oudin, Paul, De Cian, Anne, As, Marie, Izabelle, Charlotte, Saubamea, Bruno, Forster, Valerie, Fouquet, Stéphane, Robert, Camille, Portal, Céline, El-Amraoui, Aziz, Fisson, Sylvain, Concordet, Jean-Paul, Dalkara, Deniz
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 10-12-2024; American Society of Gene & Cell Therapy; Elsevier
• Subjects: base editor; CRISPR/Cas9; gene editing; gene therapy; inherited retinal dystrophy; MT: Delivery Strategies; Original; photoreceptor; retina; RPE; CRISPR/Cas9; base editor; retina; RPE; inherited retinal dystrophy; photoreceptor; gene therapy; MT: Delivery Strategies; gene editing
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2024.102349

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo. However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and retinal pigment epithelium cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future.
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Pulman and colleagues demonstrate that transient delivery of Cas9 or adenine-base editor ribonucleoprotein complexes can enter photoreceptors in vivo without carrier compounds, achieving around 10% editing efficiency, a level comparable to the highest tested dose of AAV. Editing rates vary based on guide RNA efficacy and gene expression levels.