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地传递 基因通过视网膜下给药途径，但这需要外科手术来分离 脆弱的退化视网膜。玻璃体内注射为基因输送提供了一种更安全的眼部途径，是一种广泛的 使用门诊手术，但显著的生物障碍限制了携带者从玻璃体的运输 眼球后方的腔室。这辆车一旦被送进玻璃室，就必须通过 玻璃体体液，由许多限制扩散的蛋白质组成。如果完好无损，车辆必须 穿透起筛子作用的内部限制膜，并限制物质进入 视网膜。最后，一旦车辆到达目标细胞群，它必须被内化，然后逃脱 允许胞液输送核酸的内体。视网膜下注射后，临床批准的脂质 携带信使核糖核酸的纳米颗粒可介导视网膜色素上皮(RPE)中蛋白质的表达 和光感受器，这些细胞类型主要见于红斑狼疮。然而，我们的初步数据显示，这些 玻璃体内注射后，LNPs的表达有限。迫切需要开发能够 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、有针对性的多肽 LNP系统，可以在玻璃体内注射后将基因输送到外部视网膜，毒性有限。我们 假设纳米粒子的物理化学性质，如表面电荷、大小、稳定性和电离性将 是促进视网膜渗透的关键决定因素。我们假设多肽可以穿透 并针对外部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，这是基因传递到视网膜外部的先决条件和2) 确定允许细胞特异性递送的多肽及其结构特征。分别评估每一项 LNPs和所有可用的多肽部分的物理化学特性是困难的。因此，我们将生成一个 不同的DNA条码LNP文库，并使用噬菌体展示肽库来鉴定最佳的纳米载体和 分别为多肽。由于物种的特殊性，大多数纳米载体的翻译潜力有限 眼部障碍的差异。因此，我们正在评估我们的纳米载体在体外非人类灵长类动物中的作用。 (NHP)模型，用于玻璃体内注射后快速筛选多个载体。总体而言，此应用程序将1) 识别新的多肽和LNPs的结构特征，使基因能够在后视网膜外输送 玻璃体内给药，以及2)产生翻译的、可广泛应用的转基因效率平台 用于评价基因传递系统。

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.