Directed Evolution of Adeno-Associated Virus for Retinal Gene Therapy

描述(申请人提供)：基因疗法在治疗几种导致失明的单基因缺陷方面越来越成功。特别是，多项成功的Leber先天性黑色素2型(LCA2)临床试验利用了一种已有25年历史的以腺相关病毒(AAV)2型为基础的病毒载体，将RPE65基因的功能副本传递到视网膜色素上皮(RPE)。这些试验在增强30多名患者的视觉功能方面取得了里程碑式的进展，这一成功建立了概念证明，即如果在一组患者中能识别出致病基因，就可以用AAV包装并安全地输送功能替代基因。然而，随着视网膜退行性疾病的大多数突变现已被识别，人们已经清楚地看到，几乎所有的突变都编码光感受器特异的转录本，从而将光感受器确立为视网膜基因治疗的主要靶点。此外，这些突变中的许多是常染色体显性的，因此基因替换策略不适用。为了在LCA2试验成功的基础上再接再厉，至少必须克服两个阻碍视网膜基因治疗更广泛应用的主要障碍。首先，基于自然AAV变异体的载体需要载体的视网膜下介导基因传递到光感受器或RPE，并伴随着视网膜脱离，在光感受器和潜在的RPE之间产生“水泡”。这一过程会损害视网膜，可能会加剧视网膜退化，并会导致反应性胶质细胞增厚。此外，视网膜下注射将治疗效果限制在TH水泡区域，AAV不会扩散到该区域。从玻璃体运送基因将大大减少创伤，并将提供泛视网膜转导的潜力，这两项都将是重大进展。由于在小鼠或非人类灵长类动物(NHP)模型中没有天然的AAV血清型可以从玻璃体转导光感受器，我们开发并实施了定向进化方法，正如我们最近发表的那样，已经产生了一种能够从小鼠玻璃体中转导光感受器的新型AAV，并在一定程度上能够在NHP视网膜中转导。我们现在建议在这一成功的基础上，设计AAV变体，以便将最佳的治疗性基因输送到NHP视网膜。光感受器基因治疗的第二个问题是，许多视网膜变性是常染色体显性遗传的。虽然RNAi可以产生病理等位基因的部分击倒，但完全去除这些基因将是可取的。最近在开发能够敲除靶基因的位点特异性DNA核酸酶方面取得了进展，我们将在这些进展的基础上敲除视网膜变性背后的主导等位基因。因此，我们提出了分子病毒学、蛋白质工程和具有翻译重要性的动物模型的独特结合，以设计用于治疗人类视网膜疾病的增强型遗传递送系统和货物。

DESCRIPTION (provided by applicant): Gene therapy has been increasingly successful in treating several single-gene defects that cause blindness. In particular, multiple successful clinical trials for Leber's congenital amaurosis type 2 (LCA2) have utilized a 25 year-old viral delivery vehicle, based on adeno-associated virus (AAV) serotype 2, to deliver a functional copy of the rpe65 gene to the retinal pigment epithelium (RPE). These trials have taken landmark strides in enhancing visual function in over 30 patients, success that has established the proof of concept that if a causative gene can be identified in a group of patients, a functional replacement gene can be packaged and safely delivered with AAV. However, as the majority of mutations underlying retinal degenerative diseases have now been identified, it has become clear that almost all encode photoreceptor-specific transcripts, establishing photoreceptors as the primary target for retinal gene therapy. Furthermore, many of these mutations are autosomal dominant, such that gene replacement strategies are not suitable. To build upon the LCA2 trial successes, at least two major hurdles that impede broader application of retinal gene therapy must thus be overcome. First, vectors based on natural AAV variants require a subretinal of the vector to mediate gene delivery to photoreceptors or RPE, with accompanying retinal detachment with the creation of a "bleb" between the photoreceptors and underlying RPE. This procedure damages the retina, may exacerbate the retinal degeneration, and can induce reactive gliosis. In addition, subretinal injection limits the therapeutic effect to the area of th bleb, beyond which the AAV does not spread. Gene delivery from the vitreous would be considerably less traumatic and would offer the potential for pan-retinal transduction, both of which would represent significant advances. Since no natural AAV serotypes can transduce the photoreceptors from the vitreous in either murine or non-human primate (NHP) models, we developed and implemented a directed evolution approach that, as we have recently published, has yielded a novel AAV capable of photoreceptor transduction from the vitreous in the murine and to an extent in the NHP retina. We now propose to build upon this success and engineer AAV variants for optimal therapeutic gene delivery to the NHP retina. A second problem with photoreceptor gene therapy is that many retinal degenerations are autosomal dominant. While RNAi can yield a partial knockdown of pathological alleles, a full ablation of such genes would be desirable. There have been recent advances in the development of site-specific DNA nucleases that can knock out target genes, and we will build upon these advances to knock out dominant alleles that underlie retinal degeneration. We thus propose a unique blend of molecular virology, protein engineering, and a translationally important animal model to engineer enhanced genetic delivery systems and cargo for treating human retinal disease.