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

用于治疗 2A 型亚瑟综合症的非病毒基因递送平台。

• 批准号: 10578428
• 负责人: Muna I. Naash
• 金额: $ 40.08万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578428
• 关键词: Affect; Age; Ankle; Auditory; Auditory system; Basement membrane; Binding; Biochemical; Blindness; Cells; Cilia; Cochlea; Code; Complementary DNA; Complex; DNA; DNA delivery; Defect; Development; Disease; Disease model; Electroretinography; Elements; Encapsulated; Engineering; Eye diseases; Formulation; Foundations; GRK1 gene; Gene Delivery; Gene Expression; Genes; Genomics; Goals; Hair Cells; Hemagglutinin; Hereditary Disease; Histology; Human; Hyaluronic Acid; Immunohistochemistry; Injections; Inner Limiting Membrane; Investigation; Knock-in; Knock-in Mouse; Knowledge; Lead; Learning; Light; Link; Measures; Membrane; Modeling; Molecular Biology; Morphology; Mus; Mutation; N-terminal; Nanosphere; Ocular Physiology; Outcome; Outcome Study; Papio; Pattern; Penetration; Phenotype; Photoreceptors; Play; Process; Protein Isoforms; Proteins; Quantitative Reverse Transcriptase PCR; Retina; Retinal Diseases; Retinitis Pigmentosa; Role; Route; Safety; Saline; Severities; Signal Transduction; Solid; Structure; Symptoms; Testing; Toxic effect; Transcript; Transfection; Transmembrane Domain; Treatment Efficacy; USH2A gene; Usher Proteins; Usher Syndrome; Usher Syndrome Type 2A; Vision; Visual; Visual Acuity; Visual System; Western Blotting; autosome; controlled release; deafness; delivery vehicle; effective therapy; extracellular; follow-up; fundus imaging; gene therapy; hearing impairment; in vitro testing; innovation; intravitreal injection; link protein; mouse model; mutant; nanoformulation; nanoparticle; non-viral gene delivery; non-viral gene therapy; nonhuman primate; promoter; protein transport; rational design; small molecule; therapeutic DNA; therapeutic development; therapeutic gene; therapy design; transduction efficiency; tyrosine O-sulfate; vector

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.

项目摘要/摘要： 我们的目标是促进我们当前的玻璃体内基因治疗平台，该平台由DNA纳米颗粒组成（DNA-- NPS）/透明质酸纳米球（HA-NSS）提供大基因，以开发安全/有效的疗法 对于Usher综合征2A型（USH2A）的视觉丧失。目前，没有对USH2A的治疗方法。我们的 视觉和听觉的纳米制剂和分子生物学/生理学方面的互补专业知识 系统将促进我们的非病毒疗法的发展，以挽救与最大相关的视力丧失 usherin中常见的USH2A突变，C.2299delg。我们的平台包括：1）CK30PEG DNA-NP 有效转染光感受器（PR），多次注射后无毒作用，并分布 在小鼠和狒狒模型中的视网膜下空间中。至关重要的是，这些DNA-NP提供 多种小鼠视网膜疾病模型中的结构/功能救援。 2）由透明质酸制成的HA-NS 用于控制的长期DNA释放的房屋DNA或DNA-NP。 ha-nss能够在 玻璃体内递送。 3）磺胺酪氨酸（ST），它是一种无毒的小分子，可增强视网膜渗透率 玻璃体内交付的ha-ns。 4）具有基因组元素的工程DNA向量，以携带和促进 大型治疗基因的表达。为USH2A开发有效的治疗 对于其大型编码序列（15.8 kb），该序列无法使用标准方法和 具有未完全理解的功能的多种同工型的存在。我们已经克隆了两个usherin 使用我们的创新平台（DNA-NP/HA-NS/ST）测试的同工型，以安全地推进基因疗法 USH2A。我们将使用三种先前生成的敲蛋白鼠标模型来了解有关usherin转录本的更多信息， 同工型及其在PRS中的作用，以帮助开发为USH2A开发精心设计的疗法。模型是：1）UNYFP， 其中在USH2A的N末端分泌序列后插入了带有3XFLAG标签的YFP； 2） UCMKATE，其中Mkate2在跨膜域之前插入了C-terminus的Mkate2 ush2a; 3）ush2ag/g，其中引入了人类c.2299delg突变的小鼠等效物 通过人类20 AA扩展，然后是3xflag标签。在AIM 1中，我们将评估视网膜中的USH2A同工型 VS耳蜗，USH2AG/G模型中听觉和视觉缺陷的机制，以及最多的体外测试 适用于基因治疗的合适USH2A同工型。在AIM 2中，我们将调查转导效率和 在USH2AG/G模型中，含有USH2A的玻璃体内的DNA-NPS/HA-NSS/ST的治疗功效。 首先，我们将进行短期研究，以评估玻璃体内与下re递送的Usherin的能力 在USH2AG/G视网膜中的PR中产生基因表达。其次，我们将纵向评估长期 平台/矢量/同工型从第一部分确定的治疗功效，以提供持久的表型救援 USH2AG/G模型。总而言之，该建议将为理解的功能提供稳固的基础 每种USHERIN同工型，并开发一个有效的基因治疗平台来治疗USH2A相关的视觉缺陷。