Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7041, USA.
Invest Ophthalmol Vis Sci. 2010 Jan;51(1):236-48. doi: 10.1167/iovs.09-3847. Epub 2009 Aug 13.
AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals.
Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays.
No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys.
The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.
AAV 载体在许多组织中产生稳定的转基因表达并引起低免疫反应。AAV 已成为眼部基因治疗的首选载体，特别是视网膜。scAAV 是经过修饰的 AAV，可绕过所需的第二条链 DNA 合成，以实现转基因的转录。目的是研究 AAV 载体将转基因长期、安全递送至活体动物小梁网的能力。
单次剂量的 AAV2.GFP 和 AAV2.RGD.GFP/Ad5.LacZ 通过前房内（IC）注射入大鼠（n = 28 只眼）。单次剂量的 scAAV.GFP 通过 IC 注射入大鼠（n = 72 只眼）和食蟹猴（n = 3 只）。通过荧光、免疫组织化学和非侵入性房角镜评估 GFP 表达。使用校准眼压计（大鼠）和 Goldmann 眼压计（猴子）测量眼内压（IOP）。通过微阵列确定 scAAV 感染的人小梁细胞（HTM）的差异表达。通过 ELISA 和外周血增殖测定评估体液和细胞介导的免疫反应。
注射 AAV 后 27 天内，在前节组织中未观察到 GFP 转导。相比之下，scAAV2 非常有效地转导小梁网，起效迅速（4 天）。眼睛保持透明，未观察到不良反应。在大鼠中，转基因表达持续时间超过 3.5 个月，在猴子中持续时间超过 2.35 年。
scAAV 病毒载体在大鼠和猴子的小梁网中提供了长期和安全的转导。这种载体的稳定表达和安全特性可能有助于开发用于青光眼基因治疗的小梁网药物。
