Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA.
Department of Ophthalmology and Visual Science, Medical College of Wisconsin, Milwaukee, WI, USA.
Transl Vis Sci Technol. 2022 Aug 1;11(8):28. doi: 10.1167/tvst.11.8.28.
Retinal pericytes play a vital role in maintaining retinal homeostasis, and their dysfunction underlies pathogenesis in such vascular eye diseases as diabetic retinopathy and wet age-related macular degeneration. Consequently, retinal pericytes are attractive therapeutic targets for gene therapy, but effectively targeting pericytes with recombinant adeno-associated virus (rAAV) vectors remains a challenge.
We introduced genetic modifications into the surface-exposed variable regions of the rAAV2/2 capsid to generate a complex library (>1 × 107) of capsid mutants that were then screened for preferential tropism toward retinal pericytes. Using the Tg(Cspg4-DsRed.T1)1Akik/J reporter mouse model, which has red fluorescent pericytes that can be isolated via flow cytometry in order to recover vector genomes, we performed three rounds of screening and identified seven putative mutants capable of transducing retinal pericytes.
Following intravitreal administration of mutant vectors packaging ubiquitously expressing green fluorescent protein reporters and postmortem flow cytometry of enzymatically digested retinae, two mutants in particular, Peri-E and Peri-G, demonstrated significantly greater transduction of retinal pericytes than unmodified rAAV2/2 (1.4-fold and 2.8-fold, respectively).
Although difficult to characterize the effect of each point mutation in the context of multiple amino acid variations from the wild-type AAV2 sequence, we identified several point mutations that may play critical roles in limiting HSPG binding, evading neutralization by murine A20 monoclonal antibodies, modulating antigenicity, and evading ubiquitination to ultimately improve transduction efficiency of retinal pericytes.
Identification of novel retinal pericyte targeting rAAV vectors enables the development of new, long-lasting gene therapies for retinal diseases such as diabetic retinopathy and wet age-related macular degeneration.
视网膜周细胞在维持视网膜内环境稳定方面发挥着重要作用，它们的功能障碍是糖尿病视网膜病变和湿性年龄相关性黄斑变性等血管性眼病发病机制的基础。因此，视网膜周细胞是基因治疗的有吸引力的治疗靶点，但有效地用重组腺相关病毒 (rAAV) 载体靶向周细胞仍然是一个挑战。
我们在 rAAV2/2 衣壳的表面暴露可变区引入遗传修饰，生成一个 (>1×107) 衣壳突变体的复杂文库，然后对其进行筛选，以确定对视网膜周细胞的优先趋向性。使用 Tg(Cspg4-DsRed.T1)1Akik/J 报告小鼠模型，该模型具有红色荧光周细胞，可通过流式细胞术分离，以回收载体基因组，我们进行了三轮筛选，鉴定出了 7 种能够转导视网膜周细胞的潜在突变体。
在玻璃体腔注射包装普遍表达绿色荧光蛋白报告基因的突变体载体后，通过酶消化视网膜的死后流式细胞术，两种突变体，Peri-E 和 Peri-G，与未修饰的 rAAV2/2 相比，对视网膜周细胞的转导明显增加（分别为 1.4 倍和 2.8 倍）。
尽管难以在野生型 AAV2 序列的多个氨基酸变异的背景下描述每个点突变的影响，但我们确定了几个点突变，这些突变可能在限制 HSPG 结合、逃避鼠 A20 单克隆抗体中和、调节抗原性和逃避泛素化方面发挥关键作用，最终提高视网膜周细胞的转导效率。
鉴定出新型视网膜周细胞靶向 rAAV 载体，为糖尿病视网膜病变和湿性年龄相关性黄斑变性等视网膜疾病的新的、持久的基因治疗方法的发展提供了可能。
