Department of Biochemistry and Molecular Biology, Nippon Medical School.
Department of Ophthalmology, Nippon Medical School.
J Nippon Med Sch. 2021;88(2):103-108. doi: 10.1272/jnms.JNMS.2021_88-203.
The adeno-associated virus (AAV) vector is a promising vector for ocular gene therapy. Surgical internal limiting membrane peeling before AAV vector administration is useful for efficient retinal transduction. However, no report has investigated localization of AAV vectors after administration into a post-vitrectomy eye. This study investigated the effects of vitrectomy surgery on intravitreal-injected AAV vector-mediated gene expression in the anterior segment and examined the presence of neutralizing antibodies (NAbs) in serum before and after AAV vector administration.
Of six eyes from three female cynomolgus monkeys, four were vitrectomized (Group VIT) and two were non-vitrectomized (Group IV). All eyes were injected with 50 μL of triple-mutated self-complementary AAV2 vector (1.9 × 10 v.g./mL) encoding green fluorescent protein (GFP). NAbs in the serum were examined before administration and at 2 and 6 weeks after administration. GFP expression was analyzed at 19 weeks after administration.
Immunohistological analysis showed no GFP expression in the trabecular meshwork in any eye. The GFP genome copy in two slices of the anterior segment was 2.417 (vector genome copies/diploid genome) in Group VIT and 4.316 (vector genome copies/diploid genome) in group IV. The NAb titer was 1:15.9 (geometric mean) before administration, 1:310.7 at 2 weeks after administration, and 1:669.4 at 6 weeks after administration.
Previous vitrectomy surgery did not affect gene expression in the anterior segment after intravitreal injection of AAV vectors.
腺相关病毒（AAV）载体是一种很有前途的眼科基因治疗载体。在 AAV 载体给药前进行外科内界膜剥离有助于提高视网膜转导效率。然而，目前尚无研究报道 AAV 载体给药后在玻璃体切除眼中的定位。本研究旨在探讨玻璃体切除术对玻璃体内注射 AAV 载体介导的基因表达在前节中的影响，并在 AAV 载体给药前后检测血清中的中和抗体（NAb）。
三只雌性食蟹猴的六只眼，其中四只进行玻璃体切除术（VIT 组），两只未进行玻璃体切除术（IV 组）。所有眼均注射 50μL 三重突变自互补 AAV2 载体（1.9×10 v.g./mL），编码绿色荧光蛋白（GFP）。给药前和给药后 2 周和 6 周检测血清中的 NAb。给药后 19 周分析 GFP 表达。
免疫组织化学分析显示，任何眼的小梁网均无 GFP 表达。VIT 组前节的两个切片中的 GFP 基因组拷贝数为 2.417（载体基因组拷贝/二倍体基因组），IV 组为 4.316（载体基因组拷贝/二倍体基因组）。给药前 NAb 滴度为 1:15.9（几何均数），给药后 2 周为 1:310.7，给药后 6 周为 1:669.4。
玻璃体内注射 AAV 载体前节的基因表达不受先前玻璃体切除术的影响。
