Lu Yi, Ai Jianzhong, Gessler Dominic, Su Qin, Tran Karen, Zheng Qiang, Xu Xun, Gao Guangping
1 Department of Ophthalmology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiaotong University , Shanghai, China.
2 Shanghai Key Laboratory of Fundus Disease , Shanghai, China.
Hum Gene Ther. 2016 Aug;27(8):598-608. doi: 10.1089/hum.2015.167. Epub 2016 May 16.
Corneal disease is one of the leading causes of blindness worldwide. Gene therapy is an attractive therapeutic strategy for corneal diseases, but currently underdeveloped. Recombinant adeno-associated viral (rAAV) vectors have emerged as a highly promising gene therapy platform. This study aims to identify rAAV vectors that can efficiently transduce corneal stroma for potential applications in studying pathophysiology of corneal diseases and therapeutic development. We characterized 14 rAAV serotypes expressing enhanced green fluorescent protein (EGFP), for cell specificity and transduction efficiency after either intrastromal injection or topical administration in mouse corneas in vivo. Our results show that intrastromal injections of rAAVrh.8, rAAVrh.10, rAAVrh.39, and rAAVrh.43 efficiently transduce mouse corneal stroma in vivo, and that topical administrations of rAAVrh.10 and rAAVrh.39 subsequent to epithelial scraping generate detectable transgene expression. In vivo imaging analysis revealed that transgene expression became detectable by 1 week postadministration, peaked at 2 weeks, and lasted for the duration of the study (i.e., 4 weeks). Both rAAVrh.10 and rAAVrh.39 transduced more than 50% of keratocytes, the major cell type in the corneal stroma, by intrastromal injection and 30% by topical administration. Histopathology indicated that rAAV transduction of cornea caused no morphological adverse effects. Overall, our findings suggest that some rAAV serotype vectors can efficiently transduce corneal stroma in vivo, constituting a potentially powerful and safe gene delivery platform for gene therapy of corneal diseases.
角膜疾病是全球失明的主要原因之一。基因治疗是一种针对角膜疾病颇具吸引力的治疗策略，但目前尚不完善。重组腺相关病毒（rAAV）载体已成为一种极具前景的基因治疗平台。本研究旨在鉴定能够有效转导角膜基质的rAAV载体，以用于角膜疾病病理生理学研究和治疗开发的潜在应用。我们对14种表达增强型绿色荧光蛋白（EGFP）的rAAV血清型进行了表征，以研究其在小鼠角膜基质内注射或局部给药后在体内的细胞特异性和转导效率。我们的结果表明，在体内向小鼠角膜基质内注射rAAVrh.8、rAAVrh.10、rAAVrh.39和rAAVrh.43能够有效转导角膜基质，并且在上皮刮除后局部给予rAAVrh.10和rAAVrh.39可产生可检测到的转基因表达。体内成像分析显示，给药后1周可检测到转基因表达，2周时达到峰值，并在研究期间（即4周）持续存在。通过基质内注射，rAAVrh.10和rAAVrh.39均可转导超过50%的角膜基质主要细胞类型角膜细胞，通过局部给药则可转导30%。组织病理学表明，rAAV对角膜的转导未引起形态学不良反应。总体而言，我们的研究结果表明，一些rAAV血清型载体能够在体内有效转导角膜基质，构成了一种用于角膜疾病基因治疗的潜在强大且安全的基因递送平台。
