DNA nanoparticle formulations for optimal ocular gene delivery

用于最佳眼部基因传递的 DNA 纳米颗粒配方

• 批准号: 8734431
• 负责人: Muna I. Naash
• 金额: $ 40.03万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734431
• 关键词: Accounting; Affect; Animals; Automobile Driving; Biodistribution; Biologic Characteristic; Biomedical Engineering; Blindness; Brain; Bypass; Caliber; Cell Nucleus; Cell membrane; Cells; Characteristics; Chemicals; Chemistry; Clinic; Clinical; Cone; Coupled; DNA; Data; Development; Diabetic Retinopathy; Disease; Dose; Drug Formulations; Elements; Engineering; Enhancers; Enzyme-Linked Immunosorbent Assay; Epithelial; Evaluation; Excipients; Exhibits; Eye; Face; Fluorescence; Foundations; Future; GRB10 gene; Gene Delivery; Gene Expression; Gene Transfer; Generations; Genes; Goals; Human; Injection of therapeutic agent; Interphase Cell; Ions; Length; Longevity; Luciferases; Lung; Lysine; Matrix Attachment Regions; Mediating; Methodology; Methods; Mitotic; Modeling; Molecular; Mus; Nanotechnology; Papio; Parkinson Disease; Pathway interactions; Penetration; Peptides; Performance; Photoreceptors; Physics; Plasmids; Polyethylene Glycols; Polylysine; Primates; Procedures; Proteins; Research; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinal Ganglion Cells; Retinal Pigments; Retinitis Pigmentosa; Retinol Binding Proteins; Rodent; Route; Safety; Shapes; Solvents; Structure of retinal pigment epithelium; System; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicology; Transfection; Treatment Efficacy; Vertebral column; Viral Vector; Visual Pathways; Vitelliform macular dystrophy; clinical application; cystic fibrosis patients; density; design; effective therapy; gene delivery system; gene therapy; improved; intravitreal injection; mRNA Expression; macula; macular dystrophy; minimally invasive; mouse model; nanoparticle; non-viral gene therapy; nonhuman primate; novel; particle; polycation; postnatal; preclinical study; prevent; programs; promoter; protein expression; ranpirnase; response; retinal rods; single molecule; therapeutic gene; trafficking; transgene expression; uptake; vector; vision science

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.

描述（由申请人提供）：该项目的目标是推进当前的 DNA 纳米颗粒 (NP) 递送和表达技术，为影响光感受器 (PR) 和视网膜色素上皮 (RPE) 细胞的重要眼部疾病开发安全有效的疗法。 该计划将融合分子生物工程、视觉科学、物理学和化学领域的专家，以加快产生有效的眼部非病毒基因疗法的关键步骤。 DNA NP 由用赖氨酸-PEG 聚阳离子压实的单分子 DNA 组成，最小直径为 8-11 nm。 它们的小尺寸，加上有效地将纳米颗粒运输到细胞核并绕过溶酶体降解系统的特定摄取机制，可能解释了纳米颗粒能够稳健地转染有丝分裂后分化细胞的能力。 配制时使用的抗衡离子决定了 NP 形状以及杆状和椭圆形 NP 对眼部细胞（包括 RPE、PR 和视网膜神经节细胞 (RGC)）具有强大的转染作用。 此外，鼠类 RDS NP 在 RDS 单倍体不足的视网膜色素变性小鼠模型中表现出部分表型校正。 之前已经证明了这些纳米粒子在眼中的有效使用的原理证明，在本次应用中，我们将采取必要的步骤来优化临床眼部使用的颗粒。 首先，我们将优化 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、高水平的转基因表达。 第三，我们将测试这些 NP 在非人类灵长类动物模型（狒狒）中靶向黄斑的能力（目标#3），包括评估 NP 的非侵入性玻璃体内递送是否可以转染黄斑/中心凹锥体。 对于该技术的临床应用来说，这是一项新颖且关键的进展，因为许多视网膜变性都以黄斑为目标。 此外，由于啮齿动物视网膜中不存在黄斑，因此该步骤无法通过小鼠系统进行建模。 我们还将对狒狒进行毒理学和 DNA 生物分布研究，包括对大脑视觉通路的详细评估。 基于现有 NP 制剂穿透视网膜深层的能力，我们假设可以通过目标 #1 中详述的 NP 制剂优化程序来提高这种效率，从而实现稳健的中心凹锥体基因转移。 总之，该应用的结果将有助于对用于眼部基因递送的 DNA NP 进行临床前试验评估。