Ross M, Obolensky A, Averbukh E, Desrosiers M, Ezra-Elia R, Honig H, Yamin E, Rosov A, Dvir H, Gootwine E, Banin E, Dalkara D, Ofri R
Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Rehovot, Israel.
Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Gene Ther. 2022 Nov;29(10-11):624-635. doi: 10.1038/s41434-021-00306-1. Epub 2021 Dec 2.
Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4-23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.
携带突变型CNGA3基因的绵羊视锥细胞功能减退，提供了一种自然发生的全色盲大型动物模型。向视网膜下注射携带CNGA3转基因的载体可使这些绵羊的视锥细胞功能和明视觉长期恢复。目前正在开展研究以开发有效的载体，实现更安全的转基因递送，同时避免视网膜下注射的潜在缺点。本研究评估了两种改良载体，腺相关病毒2-7m8（AAV2-7m8）和AAV9-7m8。在视锥细胞特异性启动子控制下，向野生型绵羊玻璃体内注射携带增强型绿色荧光蛋白的AAV2-7m8，可导致中等程度的光感受器转导，而向视网膜周边下递送AAV9-7m8则导致载体在注射点以外呈放射状扩散。向突变型绵羊玻璃体内注射携带人CNGA3的AAV2-7m8导致轻度光感受器转导，但未实现明视觉的临床挽救，而接受视网膜下注射治疗的昼盲绵羊则出现明视觉功能恢复。玻璃体内注射治疗的眼睛视网膜中转基因信使RNA水平相当于内源性CNGA3水平的4%-23%，表明需要>23%的表达水平才能实现临床挽救。总体而言，我们的结果表明，玻璃体内注射AAV2.7m8可转导绵羊光感受器，但效率不足以在CNGA3突变型绵羊中实现临床挽救。
