Non-viral gene delivery platforms for the treatment of Usher Syndrome Type 2A.

项目概要/摘要： 我们的目标是推进我们目前的玻璃体内基因治疗平台，包括DNA纳米颗粒（DNA- NPs）/透明质酸纳米球（HA-NS）递送大基因，以开发安全/有效的疗法 Usher综合征2A型（USH 2A）的视力丧失。目前还没有USH 2A的治疗方法。我们 在纳米制剂和视觉和听觉的分子生物学/生理学方面的互补专业知识 系统将促进我们的非病毒治疗的进步，以挽救与大多数疾病相关的视力丧失。 usherin中常见的USH 2A突变，c.2299delG。我们的平台包括：1）CK 30 PEG DNA-NPs， 有效抑制光感受器（PR），多次注射后不产生毒性作用， 在小鼠和狒狒模型中的视网膜下空间。重要的是，这些DNA-NP提供了 在多种小鼠视网膜疾病模型中的结构/功能拯救。2)HA-NS由透明质酸制成， 容纳DNA或DNA-NP用于受控长期DNA释放。HA-NS能够在 玻璃体内递送。3)磺基酪氨酸（ST），这是一种无毒的小分子，可增强视网膜渗透 玻璃体内注射HA-NS。4)具有基因组元件的工程化DNA载体，以携带和促进 大的治疗基因的表达。开发USH 2A的有效治疗方法一直具有挑战性， 其大编码序列（15.8kb）已经阻止了其使用标准方法的递送， 存在功能尚未完全了解的多种亚型。我们已经克隆了两个usherin 使用我们的创新平台（DNA-NP/HA-NS/ST）测试同种型，以安全地推进基因治疗， USH2A。我们将使用三个先前生成的敲入小鼠模型来了解更多关于usherin转录本的信息， 同种型及其在PR中的作用，以帮助开发针对USH 2A的精心设计的疗法。这些模式是：1）UNYFP， 其中具有3XFlag标签的YFP插入在Ush 2a的N-末端的分泌序列之后; 2） UCmKate，其中具有3XMyc标签的mKATE 2插入在跨膜结构域之前的C-末端 和3）ush 2aG/G，其中引入小鼠等效的人c.2299delG突变， 通过人20个氨基酸的延伸，随后是3XFlag标签。在目的1中，我们将评估视网膜中的Ush 2a亚型， 与耳蜗，在ush 2aG/G模型中的听觉和视觉缺陷的机制，以及在体外测试的最 用于基因治疗的合适的Ush 2a同种型。在目标2中，我们将研究转导效率， 在ush 2aG/G模型中玻璃体内递送的含Ush 2a的DNA-NP/HA-NS/ST的治疗功效。 首先，我们将进行短期研究，以评估玻璃体内与视网膜下递送usherin的能力， 在ush 2aG/G视网膜的PR中产生基因表达。第二，我们将长期评估 从第I部分鉴定的平台/载体/同种型的治疗功效，以提供持久的表型拯救， ush 2aG/G模型。总之，这一建议将为理解 因此，我们需要研究每种usherin同种型，并开发有效的基因治疗平台来治疗USH 2A相关的视觉缺陷。

Project Summary/Abstract: Our goal is to advance our current intravitreal gene therapy platform consisting of DNA nanoparticles (DNA- NPs)/hyaluronic acid nanospheres (HA-NSs) to deliver large genes in order to develop safe/effective therapies for visual loss in Usher Syndrome type 2A (USH2A). Currently, there are no treatments for USH2A. Our complementary expertise in nanoformulation and in molecular biology/physiology of the visual and auditory systems will facilitate the advancement of our non-viral therapy to rescue vision loss associated with the most common USH2A mutation in usherin, c.2299delG. Our platform consists of: 1) CK30PEG DNA-NPs that efficiently transfect photoreceptors (PRs), exert no toxic effects after multiple injections, and are distributed throughout the subretinal space in both mouse and baboon models. Critically, these DNA-NPs provide structural/functional rescue in multiple mouse retinal disease models. 2) HA-NS made of hyaluronic acid which house DNA or DNA-NPs for controlled long-term DNA release. HA-NSs are capable of reaching the PRs after intravitreal delivery. 3) Sulfotyrosine (ST), which is a non-toxic small molecule that enhances retinal penetration of intravitreally delivered HA-NS. 4) Engineered DNA vectors with genomic elements to carry and promote expression of large therapeutic genes. Developing an effective treatment for USH2A has been challenging due to its large coding sequence (15.8 kb) that has precluded its delivery using standard approaches and the presence of multiple isoforms with functions that are not fully understood. We have already cloned two usherin isoforms to be tested with our innovative platform (DNA-NP/HA-NS/ST) to safely advance gene therapy for USH2A. We will use three previously generated knockin mouse models to learn more about usherin transcripts, isoforms, and their role in PRs to help develop well-designed therapies for USH2A. The models are: 1) UNYFP, where YFP with a 3XFlag tag was inserted after the secretory sequence at the N-terminus of Ush2a; 2) UCmKate, where mKATE2 with a 3XMyc tag was inserted at the C-terminus before the transmembrane domain of Ush2a; and 3) ush2aG/G, where the mouse-equivalent of human c.2299delG mutation was introduced followed by the human 20 aa extension followed by 3XFlag tag. In aim 1, we will evaluate Ush2a isoforms in the retina vs cochlea, the mechanism of auditory and visual defects in the ush2aG/G model, and in vitro testing of the most suitable Ush2a isoform(s) for gene therapy. In aim 2, we will investigate the transduction efficiency and therapeutic efficacy of Ush2a-containing intravitreally delivered DNA-NPs/HA-NSs/ST in the ush2aG/G model. First, we will perform short-term studies to evaluate the ability of intravitreal vs subretinally delivered usherin to generate gene expression in PRs in the ush2aG/G retina. Second, we will longitudinally assess long-term therapeutic efficacy of the platform/vectors/isoform identified from Part I to provide lasting phenotypic rescue in the ush2aG/G model. In summary, this proposal will provide a solid foundation for understanding the function of each usherin isoform and develop an effective gene therapy platform to treat USH2A associated visual defects.