Compacted DNA Nanoparticles for Ocular Therapy

用于眼部治疗的压缩 DNA 纳米颗粒

• 批准号: 8504140
• 负责人: Muna I. Naash
• 金额: $ 37万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8504140
• 关键词: 220kDa rod outer segment rim protein; Accounting; Biology; Biomedical Engineering; Blindness; Brain; Bypass; Caliber; Cell Nucleus; Cells; Chemistry; Chromatin; Clinical; Codon Nucleotides; Complement; Complementary DNA; Coupled; DNA; Defect; Development; Disease; Disease model; Dose; Ectopic Expression; Elements; Engineering; Epigenetic Process; Evaluation; Exhibits; Eye; Foundations; Funding; Future; Gene Delivery; Gene Expression; Gene Silencing; Generations; Genes; Goals; Hour; Human; Immune response; Injection of therapeutic agent; Knock-in Mouse; Leber' s amaurosis; Lung; Lysine; Lysosomes; Matrix Attachment Regions; Mediating; Methylation; Mitotic; Modeling; Molecular; Mus; Mutation; Nanotechnology; Nuclear; Papio; Patients; Phenotype; Photoreceptors; Physics; Physiological; Physiology; Plasmids; Protein Isoforms; Proteins; Regulation; Retinal; Retinitis Pigmentosa; Safety; Site; Stargardt' s disease; Structural Protein; Structure of retinal pigment epithelium; System; Technology; Testing; Therapeutic; Tissues; Toxic effect; Transfection; Usher Syndrome; Vertebral column; Viral; Vision; Visual Pathways; Wild Type Mouse; base; clinically significant; design; disease-causing mutation; effective therapy; gene delivery system; gene therapy; hearing impairment; improved; loss of function mutation; model development; mouse model; nanoparticle; non-viral gene therapy; nonhuman primate; polycation; prevent; programs; promoter; public health relevance; ranpirnase; scaffold; single molecule; therapeutic gene; therapeutic target; tool; trafficking; treatment strategy; uptake; vector; 220kDa rod outer segment rim protein; Accounting; Biology; Biomedical Engineering; Blindness; Brain; Bypass; Caliber; Cell Nucleus; Cells; Chemistry; Chromatin; Clinical; Codon Nucleotides; Complement; Complementary DNA; Coupled; DNA; Defect; Development; Disease; Disease model; Dose; Ectopic Expression; Elements; Engineering; Epigenetic Process; Evaluation; Exhibits; Eye; Foundations; Funding; Future; Gene Delivery; Gene Expression; Gene Silencing; Generations; Genes; Goals; Hour; Human; Immune response; Injection of therapeutic agent; Knock-in Mouse; Leber' s amaurosis; Lung; Lysine; Lysosomes; Matrix Attachment Regions; Mediating; Methylation; Mitotic; Modeling; Molecular; Mus; Mutation; Nanotechnology; Nuclear; Papio; Patients; Phenotype; Photoreceptors; Physics; Physiological; Physiology; Plasmids; Protein Isoforms; Proteins; Regulation; Retinal; Retinitis Pigmentosa; Safety; Site; Stargardt' s disease; Structural Protein; Structure of retinal pigment epithelium; System; Technology; Testing; Therapeutic; Tissues; Toxic effect; Transfection; Usher Syndrome; Vertebral column; Viral; Vision; Visual Pathways; Wild Type Mouse; base; clinically significant; design; disease-causing mutation; effective therapy; gene delivery system; gene therapy; hearing impairment; improved; loss of function mutation; model development; mouse model; nanoparticle; non-viral gene therapy; nonhuman primate; polycation; prevent; programs; promoter; public health relevance; ranpirnase; scaffold; single molecule; therapeutic gene; therapeutic target; tool; trafficking; treatment strategy; uptake; vector

DESCRIPTION (provided by applicant): The goal of this program is to advance current DNA nanoparticle (NP) delivery system and expression technologies to develop safe and effective therapies targeting important photoreceptor-associated ocular disorders caused by defects in large genes. These NPs have demonstrated efficient gene expression with vectors up to 20 kbp in the lung and 14 kbp in the eye (the largest sizes tested) which make them an ideal complement to AAVs especially for delivery of large genes. The program will merge experts with backgrounds in molecular bioengineering, eye biology/physiology, 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 (bypassing lysosomes), likely accounts for their ability to transfect post-mitotic, differentiated cells. We have shown that NP treatment leads to efficient transfection of ocular cells including photoreceptors (PRs), exerts no toxic effects on the eye even after multiple injections, distributes throughout the subretinal space, and mediates appreciable structural and functional rescue in mouse models of retinitis pigmentosa (RP, Rds+/-), Leber's congenital amaurosis (LCA, Rpe65-/-), and Stargardt's disease (STGD1, Abca4-/-). Effective gene expression without toxicity has also been demonstrated in baboons. These proof-of-principle studies confirmed the potential clinical significance of this technology for treating blindness in patients and highlighted the value of a large capacity delivery vehicle, but also highlighted the need for improvements in PR gene expression levels. Our main goal here is therefore to develop NPs and vectors capable of providing long-term gene expression at levels high enough to mediate full phenotypic rescue in models of ocular diseases associated with large genes. We propose to accomplish this by first studying the epigenetic regulation of the pEPi-ABCA4 vector to understand the mechanisms that underlie gene silencing (Aim 1), then implement targeted vector engineering to enhance NP entry into the cell, promote stability in the nucleus, prevent epigenetic silencing, and increase gene expression levels (Aim 2). Subsequently, we will test these optimized vectors for their ability to mediate full phenotypic rescue in large gene disease models; specifically the Abca4-/- model of STGD1 and two models (Ush2a-/- and Ush2a c2299delG knock-in) associated with usher syndrome type 2 (USH2) (Aim 3). USH2A is a very large gene which cannot be accommodated by traditional vectors, and as a result development of targeted therapeutics for Usher syndrome has lagged. In summary, results from this application will facilitate the advancement of DNA NPs for ocular diseases associated with large genes.

描述（由申请人提供）：该程序的目的是推动当前的DNA纳米颗粒（NP）输送系统和表达技术，以开发针对重要的光感受器相关的眼部疾病的安全有效疗法，由大基因中的缺陷引起。这些NP表现出有效的基因表达，肺中最多20 kbp和14 kbp的载体（已测试的最大尺寸）使其成为AAV的理想补充，尤其是用于大基因的递送。该计划将与专家合并具有分子生物工程，眼睛生物学/生理学，物理学和化学的背景，以加快生成有效的眼科非病毒基因治疗的基本步骤。 DNA NP由用赖氨酸-PEG多阳离子压实的DNA的单分子组成，最小直径为8-11 nm。它们的尺寸很小，再加上特定的摄取机制，该机制有效地将NPS运输到细胞核（绕过溶酶体），可能是它们转染后有丝质后分化细胞的能力。 We have shown that NP treatment leads to efficient transfection of ocular cells including photoreceptors (PRs), exerts no toxic effects on the eye even after multiple injections, distributes throughout the subretinal space, and mediates appreciable structural and functional rescue in mouse models of retinitis pigmentosa (RP, Rds+/-), Leber's congenital amaurosis (LCA, Rpe65-/-), and Stargardt病（STGD1，ABCA4 - / - ）。在狒狒中也证明了没有毒性的有效基因表达。这些原则研究证实了该技术在治疗患者失明的潜在临床意义，并强调了大容量输送工具的价值，但也强调了需要改善PR基因表达水平的需求。因此，我们的主要目标是开发能够在足够高的水平上提供长期基因表达的NP和向量，以介导与大基因相关的眼部疾病模型中的全部表型救助。我们建议通过首先研究PEPI-ABCA4载体的表观遗传调节，以了解基因沉默构成的机制（AIM 1），然后实施靶向载体工程，以增强NP进入细胞，促进核中的稳定性，促进核中的稳定性，防止表观遗传沉积和增加基因表达水平（AIM 2）。随后，我们将测试这些优化的向量，以介导大基因疾病模型中的全表型救援的能力。特别是与USHER综合征2型（USH2）相关的STGD1和两个模型（USH2A - / - 和USH2A C2299DELG敲入）的ABCA4 - / - 模型（AIM 3）。 USH2A是一个非常大的基因，无法容纳传统的载体，因此，针对Usher综合征的靶向治疗剂的发展落后。总而言之，该应用的结果将促进与大基因相关的眼部疾病的DNA NP的进步。