PDF(Pubmed)
Abstract：
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 (AIPL1) 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 AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) 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.
摘要：
自从基于腺相关病毒的疗法（voretigeneneparvovovec-rzyl）成功治疗Leber先天性黑蒙2型（LCA2）以来，遗传性视网膜疾病（IRD）已成为基因治疗研究的主要重点。使用腺相关病毒（AAV）将功能基因转移到视网膜中的类似方法，可以潜在地治疗数十种单基因IRD。这里,我们介绍了设计的结果，生产,和体外测试携带密码子优化的（co）拷贝的芳香烃受体相互作用蛋白样1（AIPL1）作为LCA4的可能治疗的AAV血清型9（AAV9）载体。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，用AAV9-AIPL1co转导的细胞显示出比转导的AAV9-AIPL1wt（野生型AIPL1）少得多的抗病毒反应。用AAV9-AIPL1co转导的转分化ARPE-19细胞的RNA测序（RNA-seq）分析显示了与先天免疫反应有关的基因表达的显着差异。相比之下,AAV9-AIPL1wt诱导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激活dsRNA反应性抗病毒寡腺苷酸合成酶，以及在RNA-seq数据中过度代表的组蛋白编码基因转录物的水平显着增加(即，H1,H2A,H2B,H3和H4）。RNA-seq数据表明，AAV9-AIPL1co表现出比AAV9-AIPL1wt更低的免疫原性可用于效力测试，使用相关动物模型开发LCA4的未来疗法。
