Wei Spencer C, Cantor Aaron J, Walleshauser Jack, Mepani Rina, Melton Kory, Bans Ashil, Khekare Prachi, Gupta Suhani, Wang Jonathan, Soares Craig, Kiwan Radwan, Lee Jieun, McCawley Shannon, Jani Vihasi, Leong Weng In, Shahi Pawan K, Chan Jean, Boivin Pierre, Otoupal Peter, Pattnaik Bikash R, Gamm David M, Saha Krishanu, Gowen Benjamin G, Haak-Frendscho Mary, Janatpour Mary J, Silverman Adam P
Spotlight Therapeutics, Hayward, CA, USA.
McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2025 Jun 24;20(6):e0317387. doi: 10.1371/journal.pone.0317387. eCollection 2025.
Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of BE-eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in preclinical studies for the development of genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.
包括CRISPR/Cas技术在内的基因药物，在解决遗传性视网膜疾病和其他适应症中尚未满足的医疗需求方面展现出巨大前景；然而，治疗药物的开发仍面临挑战。在此，我们通过使用小鼠和猪动物模型进行视网膜下给药，在体内评估工程化Cas9核糖核蛋白（eRNP）介导的基因组编辑。视网膜下注射腺嘌呤碱基编辑器和诱导双链断裂的Cas9核酸酶eRNP在这两种物种中均介导基因组编辑。编辑发生在视网膜色素上皮（RPE）和光感受器细胞中，两种物种均具有良好的耐受性。使用转基因报告菌株，我们确定编辑主要发生在给药部位附近，即在与视网膜下注射相关的气泡区域内。我们的结果表明，在小鼠中视网膜下注射BE-eRNP介导的碱基编辑可达整个神经视网膜的12%，在测试的最高剂量下观察到的平均编辑率为7%。相比之下，在小型猪中观察到的编辑效率要低得多；即使仅直接量化处理区域，观察到的最大碱基编辑率为1.5%，平均编辑率<1%。我们的数据强调了在针对眼睛的基因药物开发的临床前研究中考虑物种因素的重要性，并提供了一个在视网膜下注射Cas9 eRNP的情况下，小动物模型和大动物模型之间缺乏转化的例子。
