Gene Therapy Department, Science Center for Translational Medicine, Sirius University of Science and Technology, 354340 Sirius, Russia.
Molecular Virology Laboratory, First Moscow State Medical University (Sechenov University), 119991 Moscow, Russia.
Int J Mol Sci. 2023 Dec 22;25(1):197. doi: 10.3390/ijms25010197.
Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 () as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human . Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type ) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes' transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4.
遗传性视网膜疾病（IRD）已成为基因治疗研究的主要焦点，因为腺相关病毒为基础的疗法（voretigene neparvovec-rzyl）治疗 2 型莱伯先天性黑蒙（LCA2）取得了成功。数十种单基因 IRD 可以通过类似的方法用腺相关病毒（AAV）将功能性基因转移到视网膜中得到治疗。在这里，我们展示了设计、生产和体外测试结果，使用携带优化密码子（co）的芳香烃受体相互作用蛋白样 1（AIPL1）的腺相关病毒 9 型（AAV9）载体作为 LCA4 潜在治疗方法。pAAV-AIPL1co 能够成功转导视网膜色素上皮细胞（ARPE-19）并启动人 AIPL1 的表达。有趣的是，与转导 AAV9-AIPL1wt（野生型）的细胞相比，转导 AAV9-AIPL1co 的细胞显示出较低的抗病毒反应。转导 AAV9-AIPL1co 的 ARPE-19 细胞的转录组测序（RNA-seq）分析表明，参与先天免疫反应的基因表达存在显著差异。相比之下，AAV9-AIPL1wt 诱导了多个干扰素刺激基因的显著激活。可能的调节分子机制的关键部分是 dsRNA 反应性抗病毒寡聚腺苷酸合成酶的激活，以及 RNA-seq 数据中代表性较高的组蛋白编码基因转录物水平的显著增加（即 H1、H2A、H2B、H3 和 H4）。RNA-seq 数据表明，与 AAV9-AIPL1wt 相比，AAV9-AIPL1co 表现出较低的免疫原性，可用于使用相关动物模型进行效力测试，以开发用于治疗 LCA4 的未来疗法。
