DNA nanoparticle formulations for optimal ocular gene delivery

描述(申请人提供)：该计划的目标是推进当前的DNA纳米颗粒(NP)传递和表达技术，以开发安全有效的治疗影响光感受器(PR)和视网膜色素上皮(RPE)细胞的重要眼病的方法。该计划将把专家与分子生物工程、视觉科学、物理和化学相结合，以加快产生有效的眼部非病毒基因治疗的关键步骤。DNA纳米粒由单分子DNA与赖氨酸-聚乙二醇聚阳离子结合而成，最小直径为8-11 nm。它们的体积很小，再加上一种特殊的摄取机制，可以有效地将NPs输送到细胞核并绕过溶酶体降解系统，这可能是NPs强大地转染有丝分裂后分化细胞的能力的原因。配方时使用的反离子决定了 NP形状和杆状和椭圆形的NPs都显示出强大的眼细胞转染性，包括RPE、PR和视网膜神经节细胞(RGCs)。此外，小鼠RDS NPs在RDS单倍体功能不全的视网膜色素变性小鼠模型中显示出部分表型纠正。在之前展示了这些纳米粒子在眼睛中有效使用的原理证明之后，在本应用中，我们将采取必要的步骤来优化用于临床眼部使用的颗粒。首先，我们将优化用于PR和RPE特异性基因转移的NP配方(NP形状、大小和化学成分)(目标1)。其次，我们将设计适合临床的DNA载体(目标2)，以产生持久的、高水平的转基因表达。第三，我们将在非人类灵长类动物模型(狒狒)中测试这些NPs靶向黄斑的能力(目标3)，包括评估非侵入性玻璃体内注射NPs是否可以感染黄斑/中心凹视锥。对于这项技术的临床应用来说，这是一个新的和关键的发展，因为许多视网膜退行性病变都是针对黄斑的。此外，由于啮齿动物的视网膜中没有斑点，这是一个无法由老鼠系统模拟的步骤。我们还将在狒狒身上进行毒理学和DNA生物分布研究，包括详细评估大脑视觉通路。基于现有NP配方能够穿透视网膜深层的已知能力，我们假设，通过目标1中详细介绍的NP配方优化程序，可以提高这一效率，从而实现强大的中心凹锥体基因转移。综上所述，这一应用的结果将有助于眼部基因输送的DNA纳米粒的临床前试验评估。 与公众健康相关：该计划旨在推动我们的紧凑型DNA纳米技术的发展，以促进其未来用作眼病的临床基因治疗。临床前研究计划为将这项技术用作临床可行的眼部疾病(如视网膜色素变性(RP)、各种黄斑营养不良(MD)和糖尿病视网膜病变)的基因输送系统奠定基础。我们的NPs的最小直径(8-11 nm)比任何病毒载体都小得多，可以实现唯一有效和安全的玻璃体内给药，将基因输送到RPE细胞和中心凹视锥。

DESCRIPTION (provided by applicant): The goal of this program is to advance current DNA nanoparticle (NP) delivery and expression technologies to develop safe and effective therapies for important ocular disorders affecting the photoreceptor (PR) and retinal pigment epithelial (RPE) cells. The program will merge experts with molecular bioengineering, vision science, physics, and chemistry to accelerate essential steps for the generation of effective ocular non-viral gene therapy. The DNA NPs consist of single molecules of DNA compacted with lysine-PEG polycations and have a minimum diameter of 8-11 nm. Their small size, coupled with a specific uptake mechanism that efficiently traffics the NPs to the nucleus and bypasses the lysosomal degradation system, likely accounts for the ability of the NPs to robustly transfect post-mitotic, differentiated cells. The counter-ion used at the time of formulation determines the NP shape and both rod-like and ellipsoidal NPs show robust transfection of ocular cells, including RPE, PRs and retinal ganglion cells (RGCs). Moreover, murine RDS NPs have demonstrated partial phenotypic correction in a retinitis pigmentosa mouse model of RDS haploinsufficiency. Having previously shown proof-of-principle for the effective use of these NPs in the eye, in this application we will take the necessary steps to optimize the particles for clinical ocular use. First, we will optimize the NP formulation (NP shape, size, and chemical composition) for PR- and RPE-specific gene transfer (Aim #1). Second, we will engineer clinically-appropriate DNA vectors (Aim #2) to generate persistent, high levels of transgene expression. Thirds, we will test the ability of these NPs to target the macula in a non-human primate model (baboon) (Aim #3) including an assessment of whether non-invasive intravitreal delivery of NPs can transfect macular/foveal cones. This is a novel and critical development for clinical application of this technology, as many retinal degenerations target the macula. Furthermore, it is a step that cannot be modeled by a mouse system due to the absence of a macula in the rodent retina. We will also conduct toxicology and DNA biodistribution studies in baboons, including a detailed evaluation of brain visual pathways. Building on the documented ability of the current NP formulation to penetrate deep retinal layers, we hypothesize that this efficiency can be improved by the NP formulation optimization program detailed in Aim #1, enabling robust foveal cone gene transfer. In summary, results from this application will facilitate preclinical trial evaluations of DNA NPs for ocular gene delivery. PUBLIC HEALTH RELEVANCE: This program is designed to advance our compacted DNA nanotechnology to facilitate its future use as a clinical gene therapy treatment for ocular diseases. Preclinical studies are planned to lay the foundation for use of this technology as a clinically viable gene delivery system for ocular diseases such as retinitis pigmentosa (RP), various macular dystrophies (MD), and diabetic retinopathy. The minimum diameter (8-11 nm) of our NPs is much smaller than any viral vector, and may permit uniquely effective and safe intravitreal ocular dosing to deliver genes into RPE cells and foveal cones.