Overcoming gene delivery barriers to the back of the eye

克服眼后部的基因传递障碍

• 批准号: 10058049
• 负责人: Gaurav Sahay
• 金额: $ 23.81万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058049
• 关键词: Affect; Affinity; Back; Bar Codes; Binding; Bioinformatics; Biological; Blindness; Cells; Characteristics; Charge; Cholesterol; Clinical; DNA; Data; Development; Diffusion; Encapsulated; Endosomes; Evaluation; Eye; Family; Gene Delivery; Genes; Goals; Immunohistochemistry; Incidence; Individual; Inner Limiting Membrane; Label; Lead; Libraries; Lipids; Macaca mulatta; Maps; Mediating; Messenger RNA; Modeling; Mus; Neural Retina; Non-Viral Vector; Nucleic Acids; Operative Surgical Procedures; Outpatients; Patients; Penetration; Peptide Library; Peptide Phage Display Library; Peptides; Permeability; Phospholipids; Photoreceptors; Population; Procedures; Property; Proteins; Randomized; Reporter Genes; Research Priority; Retina; Retinal Degeneration; Retinal gene therapy; Route; Structure; Structure of retinal pigment epithelium; Surface; System; Techniques; Toxic effect; Transfection; Translations; Variant; Vertebrate Photoreceptors; Viral; Vision; Vitreous Chamber; Vitreous humor; aged; cell type; cellular targeting; clinically relevant; design; gene delivery system; gene therapy; genome editing; inherited retinal degeneration; intravitreal injection; knock-down; lipid nanoparticle; nanocarrier; nanoparticle; next generation sequencing; nonhuman primate; novel; nucleic acid delivery; peptide structure; protein expression; restoration; screening; subretinal injection; therapy development; vector

ABSTRACT Gene therapy strategies including augmentation, editing or knockdown, can lead to restoration of vision for patients suffering from inherited retinal degenerations (IRDs). Viral and non-viral vectors efficiently deliver genes through the subretinal route of administration, but this requires a surgical procedure that detaches the fragile degenerating retina. Intravitreal injections offer a safer ocular route for gene delivery and is a widely used outpatient procedure, but significant biological barriers limit the transport of carriers from the vitreous chamber to the back of the eye. The vehicle, once delivered in the vitreous chamber, will have to move through the vitreous humor, which is composed of many proteins that restrict diffusion. If intact, the vehicle must then penetrate an inner limiting membrane functioning as a sieve and restricting permeability of substances into the retina. Finally, once the vehicle hits the target cell population, it must get internalized and then escape the endosome to allow for cytosolic delivery of nucleic acids. After subretinal injection, clinically approved lipid nanoparticles (LNPs) carrying mRNA can mediate protein expression in the retinal pigment epithelium (RPE) and photoreceptors, the cell types mainly afflicted in IRDs. However, our preliminary data show that these LNPs have limited expression post-intravitreal injection. There is a critical need to develop carriers that can traverse the retinal barriers after intravitreal injection. Our long-term goal is to develop novel, peptide targeted LNP systems that can deliver genes to the outer retina after an intravitreal injection with limited toxicity. We hypothesize that nanoparticle physicochemical properties like surface charge, size, stability, and ionizability will be the critical determinants that enhance permeation towards the retina. We posit that peptides can penetrate and target specific cell types within the outer retina. Our main objectives are to 1) evaluate the physicochemical parameters of nanoparticles that are a prerequisite for gene delivery to the outer retina and 2) identify peptides and their structural features that allow for cell-specific delivery. Individually evaluating each physicochemical characteristic of LNPs and all peptide moieties available is arduous. Thus, we will generate a diverse DNA barcoded LNP library and use a phage display peptide library to identify optimal nanocarriers and peptides, respectively. Most nanocarriers have had limited translation potential due to species-specific difference in ocular barriers. Therefore, we are evaluating our nanocarriers in an ex-vivo non-human primate (NHP) model for rapid screening of multiple vectors after intravitreal delivery. Overall, this application will 1) identify novel peptides and structural features of LNPs that enable gene delivery to the outer retina post- intravitreal administration, and 2) generate a translational, transfection efficiency platform that can be widely used for the evaluation of gene delivery systems.

摘要 基因治疗策略，包括增强，编辑或敲除，可以导致视力恢复， 患有遗传性视网膜变性（IRD）的患者。病毒和非病毒载体有效地递送 基因通过视网膜下途径的管理，但这需要一个外科手术，分离 脆弱退化的视网膜玻璃体内注射为基因递送提供了一种更安全的眼部途径，并且是一种广泛应用的方法。 使用门诊手术，但显著的生物屏障限制了载体从玻璃体的转运 眼睛后面的房间。载体一旦进入玻璃体腔， 玻璃体液，由许多限制扩散的蛋白质组成。如果完好无损，车辆必须 穿透起筛子作用并限制物质渗透进入的内界膜。 视网膜。最后，一旦载体击中靶细胞群，它必须被内化，然后逃离靶细胞群。 在一些实施方案中，核内体可以是核内体，以允许核酸的胞质递送。视网膜下注射后，临床批准的脂质 携带mRNA的纳米颗粒（LNPs）可以介导视网膜色素上皮（RPE）中的蛋白质表达 以及感光细胞，这是主要受IRD影响的细胞类型。然而，我们的初步数据显示， LNP在玻璃体内注射后具有有限的表达。迫切需要开发能够 在玻璃体内注射后穿过视网膜屏障。我们的长期目标是开发新的，肽靶向 LNP系统可以在玻璃体内注射后以有限的毒性将基因递送到外视网膜。我们 假设纳米粒子物理化学性质如表面电荷、尺寸、稳定性和电离性将 是增强向视网膜渗透的关键决定因素。我们认为肽可以穿透 并针对视网膜外层的特定细胞类型。我们的主要目标是：1）评估 纳米颗粒的物理化学参数是基因递送到外视网膜的先决条件，以及2） 鉴定允许细胞特异性递送的肽及其结构特征。分别评价每 LNP和所有可用的肽部分的物理化学特性是艰巨的。因此，我们将生成一个 不同的DNA条形码LNP文库，并使用噬菌体展示肽文库来鉴定最佳纳米载体， 肽，分别。由于物种特异性，大多数纳米载体具有有限的翻译潜力。 眼屏障的差异。因此，我们正在体外非人灵长类动物中评估我们的纳米载体 (NHP)用于玻璃体内递送后快速筛选多种载体的模型。总体而言，该应用程序将1） 鉴定新的肽和LNP的结构特征，其能够使基因递送到后视网膜外层， 玻璃体内给药，和2）产生一种翻译的转染效率平台，其可以广泛地 用于评价基因递送系统。