应用具有酪氨酸突变的新型AAV转基因载体通过玻璃体腔注射来探讨治疗全色盲动物模型的新途径

The current ongoing RPE65 Leber congenital amaurosis (LCA2) clinical trial performed single or multiple subretinal injections to treat more paramacular and peripheral retinal area. Although the vision originated from the macular area comprises 90% of the entire vision, subretinal injection of vector to this most important area is almost impossible due to the potential injection-related damage since the cone structure in human macula, especially the central fovea is very fine and sensitive to any invasive procedure. Although it can transduce larger area of the inner retina without causing retinal detachment, intravitreal injection with conventional vectors cannot penetrate the inner retina and transduce the rod or cone cells in outer retinal layers. This is an unmet need currently in the field and a challenge for conventional vectors. Recently it was reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK)-mediated tyrosine phosphorylation of exposed residues of the AAV capsid promotes ubiquitination and subsequent proteasomal degradation of AAV particles, and that this process decreases AAV vector transduction efficiency. Site-directed tyrosine to phenylanine (Y-F) mutagenesis of selected tyrosine residues in AAV2 was shown to protect vector particles from proteasomal degradation and significantly increase the transduction efficiency of these mutant AAV vectors relative to the wild-type AAV vectors. Recently we found that AAV8 has a higher transduction efficiency in photoreceptors than AAV2 or 5，and serotype 8 AAV containing two point-mutations in a surface-exposed tyrosine residue confers stronger transgene expression in photoreceptor cells than standard AAV8 and tranduced photoreceptor cells following intravitreal injection. Another key aspect of this endeavor is to employ an animal model with a genetically and phenotypically well characterized cone dysfunction model that could response to gene based therapy. We have therefore chosen to focus on the cpfl5 mouse that exhibits complete cone dysfunction due to a recessive cnga3 defect in order to fully assess the generality of our hypotheses. We will test the hypothesis that multiple Y-F mutant capsid AAV8 vectors when delivered to the vitreous can achieve rescue in cpfl5 cones that is similar to conventional vector delivered subretinally. We propose to explore ways to restore and/or maintain retinal cone structure, function and vision dependent behavior in a mouse model of human achromatopsia with cnga3 mutation using multiple Y-F mutant capsid AAV vectors as the gene delivery vehicles. Our overall hypothesis is that appropriately designed gene-based therapies following intravitreal injection will be clinically useful for a wide variety of retinal diseases with cone dysfunctions.

最近国外用基因疗法治疗眼科视网膜遗传病已进入临床试验阶段,但目前使用的传统腺相关病毒(AAV)等载体通过玻璃体腔注射后很难穿透内层视网膜到达感光细胞层，必须通过视网膜下腔注射才能转染外层靶细胞。可是由于黄斑部特别是中心凹的结构极其精细，视网膜下腔注射造成的视网膜脱离能对锥细胞造成损害，导致中心视力下降。最近发现将两点突变的AAV8载体携带示踪基因，通过简单易行的玻璃体腔注射后发现AAV8能转染感光细胞全层。应用锥细胞特异的具有两点酪氨酸突变的AAV8载体将人类cnga3基因通过玻璃体腔注射转入cnga3缺失小鼠锥细胞后，不但能短期阻止蓝锥视蛋白的变性，还能部分恢复锥细胞功能。本项目将对AAV8载体造成酪氨酸多点突变并筛选出穿透能力更强的相应AAV8载体，评价及确认通过玻璃体腔注射的基因疗法对全色盲动物的长期疗效及安全性。该新技术如获成功，那么目前阻碍黄斑部疾病基因治疗的主要问题将得到解决。

最近国外用基因疗法治疗眼科视网膜遗传病已进入临床试验阶段，但目前使用的传统AAV载体转染及穿透力不够，必须将载体注射到视网膜色素上皮（RPE）和光感受器（PR）之间的视网膜下腔才能转染这两层细胞。虽然视网膜下腔注射造成的暂时性网脱对周边部视网膜影响不大，但对视锥细胞集中的灵长类黄斑部特别是中心凹的精细结构影响较大，可造成感光细胞层变薄；临床上对患者造成中心视力损害，而非提高。玻璃体腔注射的方法虽然操作简单、相对安全，且能转染较大面积的视网膜组织，但是目前常用的AAV载体，很难由玻璃体腔一侧穿越玻璃体和视网膜之间的膜性结构，从而有效转染黄斑区的RPE或PR细胞并表达足够的功能蛋白。研究发现，AAV衣壳蛋白多点酪氨酸突变，可降低其磷酸化和泛素化水平，从而有效抑制病毒载体被靶细胞的蛋白酶体降解。本研究中，我们证实新型载体（两点酪氨酸突变的AAV8）较传统2、5和8型AAV的转染效率明显提高，目前为止是尝试玻璃体腔注射首选的转基因载体。. 人类全色盲2型的主要病变部位是在视网膜黄斑中心凹，该处只有视锥细胞，而没有视杆细胞。我们选择了Cnga3-/-/Nrl-/-小鼠作为研究对象，该模型是在全色盲的基础上进一步敲除与视杆细胞感光功能相关的基因。Cnga3-/-/Nrl-/-小鼠的视网膜与外核层只有3%视锥细胞的全色盲2型cpfl5小鼠相比，更加接近人类黄斑部的结构。玻璃体腔注射AAV8 (Y447,733F)-IRBP/GNAT2-Cnga3后，新型载体能穿越小鼠玻璃体和视网膜之间的膜性结构以及内层视网膜组织，进入视锥细胞后使其携带的鼠源Cnga3基因得到充分表达。CNGA3蛋白恢复后，视锥细胞中与感光功能密切相关的中波长敏感视蛋白和短波长敏感视蛋白也得以保持正常。功能方面，明适应ERG反应达到野生型小鼠正常水平的（50～60）%，而传统AAV载体玻璃体腔注射则几乎无效。上述基因干预除获得长期疗效外，实验未见基因治疗引起蛋白过量表达从而导致视网膜肿瘤的发生，也初步提示新型AAV8载体介导的转基因表达在小鼠视网膜上是安全的，本项目可以为国内外全色盲2型基因治疗的临床试验提供参考。

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