Overcoming gene delivery barriers to the back of the eye

摘要 基因治疗策略，包括增强、编辑或敲除，可以帮助患者恢复视力 患有遗传性视网膜变性（IRD）的患者。病毒和非病毒载体有效传递 基因通过视网膜下给药途径，但这需要通过外科手术来分离 脆弱的退化视网膜。玻璃体内注射为基因传递提供了更安全的眼部途径，是一种广泛应用的方法。 使用门诊手术，但显着的生物屏障限制了携带者从玻璃体的运输 腔室到眼睛后部。车辆一旦进入玻璃体室，就必须通过 玻璃体液，由许多限制扩散的蛋白质组成。如果完好无损，则车辆必须 穿透内界膜，充当筛子并限制物质渗透进入 视网膜。最后，一旦载体击中目标细胞群，它必须被内化，然后逃离 内体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 (LNP) 可以介导视网膜色素上皮 (RPE) 中的蛋白质表达 和光感受器，这些细胞类型主要受 IRD 影响。然而，我们的初步数据显示，这些 LNP 在玻璃体内注射后表达有限。迫切需要开发能够 玻璃体内注射后可穿过视网膜屏障。我们的长期目标是开发新型肽靶向药物 LNP 系统可以在玻璃体内注射后将基因传递到外视网膜，且毒性有限。我们 假设纳米颗粒的物理化学性质（如表面电荷、尺寸、稳定性和电离性）将 是增强视网膜渗透性的关键决定因素。我们假设肽可以渗透 并针对外视网膜内的特定细胞类型。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传递到外视网膜的先决条件；2) 识别允许细胞特异性递送的肽及其结构特征。单独评估每个 LNP 和所有可用肽部分的理化特性非常困难。因此，我们将生成一个 多样化的 DNA 条形码 LNP 文库，并使用噬菌体展示肽文库来识别最佳纳米载体和 肽，分别。由于物种特异性，大多数纳米载体的翻译潜力有限 眼屏障的差异。因此，我们正在离体非人类灵长类动物中评估我们的纳米载体 (NHP) 模型，用于玻璃体内递送后快速筛选多个载体。总的来说，该应用程序将 1) 识别新的肽和 LNP 的结构特征，使基因能够在 玻璃体内给药，2) 生成可广泛应用的转化、转染效率平台 用于评估基因传递系统。

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.