Directed Evolution of Adeno-Associated Virus for Retinal Gene Therapy

用于视网膜基因治疗的腺相关病毒的定向进化

基本信息

批准号：
9130224
负责人：
John Gerard Flannery
金额：
$ 41.07万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9130224
关键词：
Ablation Address Alleles Anatomy Animal Disease Models Animal Model Apical Area Blindness Bulla Canis familiaris Cell Death Choroideremia Clinical Clinical Trials Cone Data Deoxyribonucleases Dependovirus Development Directed Molecular Evolution Disease Engineering Evolution Eye Gene Delivery Gene Targeting Gene Transfer Genes Genetic Genetic Engineering Glaucoma Gliosis Goals Health Human In Vitro Injection of therapeutic agent Inner Limiting Membrane Knock-out Knowledge Leber&apos s amaurosis Location Macular degeneration Mediating Methods Minor Modeling Molecular Virology Mus Mutate Mutation Nature Neurons Patients Photoreceptors Primates Procedures Process Protein Engineering Publishing RNA Interference Retina Retinal Retinal Cone Retinal Degeneration Retinal Detachment Retinal Diseases Retinitis Pigmentosa Risk Rodent Rodent Model Serotyping Single-Gene Defect Site Structure of retinal pigment epithelium Structure-Activity Relationship System Technology Testing Therapeutic Effect Toxic effect Toxicity Tests Transcript Translations Variant Vertebrate Photoreceptors Viral Viral Vector Virus Vision Vitreous humor Work achromatopsia adeno-associated viral vector autosomal dominant mutation base design fovea centralis gene replacement gene therapy genetic variant genome editing improved improved functioning inherited retinal degeneration knock-down macula nonhuman primate novel nuclease null mutation pressure retinal rods retinol isomerase subretinal injection success therapeutic gene vector

项目摘要

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.

描述（由申请人提供）：基因治疗在治疗几种引起失明的单基因缺陷方面越来越成功。特别是，基于腺相关病毒（AAV）血清型2的Leber先天性症型2型（LCA2）的多项成功临床试验已使用25年历史的病毒递送车，以将RPE65基因的功能副本传递给视网膜颜料上皮（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意义，成功证明了一个概念证明，如果可以在一组患者中鉴定出病因基因，则可以将功能替代基因包装并用AAV安全地递送。然而，由于现在已经鉴定出了视网膜退行性疾病的大多数突变，因此很明显，几乎所有编码光感受器特异性转录本已确立了光感受器作为视网膜基因治疗的主要靶标。此外，这些突变中的许多是常染色体显性的，因此基因置换策略不合适。为了建立LCA2试验成功，必须克服至少两个阻碍视网膜基因疗法应用的主要障碍。首先，基于天然AAV变体的矢量需要载体的视网膜下介导基因递送到感光器或RPE，并伴随视网膜脱离，并在感光体和基础RPE之间创建“ BLEB”。此过程会损害视网膜，可能加剧视网膜变性，并可能诱导反应性神经胶质变性。此外，视网膜下注射将治疗作用限制为TH BLEB区域，除了AAV不扩散。从玻璃体中递送的基因递送将减少创伤性，并提供泛视网膜转导的潜力，这两者都代表了重大进展。由于没有天然的AAV血清型可以在鼠或非人类灵长类动物（NHP）模型中从玻璃体中转导感光器，因此我们开发并实施了一种定向进化方法，正如我们最近发表的那样，该方法产生了一种新颖的AAV，能够从Merine和Nhp Retnian中的玻璃体中产生一种具有光感受器转导的AAV。现在，我们建议以这种成功和AAV工程型变种为基础，以最佳的治疗基因输送到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症。尽管RNAi可以部分敲除病理等位基因，但希望完全消融这种基因。最近在位点特异性DNA核酸酶的发展中取得了进步，这些核酸酶可以敲除靶基因，我们将基于这些进步，以淘汰视网膜变性的主要等位基因。因此，我们提出了一种分子病毒学，蛋白质工程以及翻译重要的动物模型的独特混合物，以增强遗传递送系统和用于治疗人类视网膜疾病的货物。