Compacted DNA Nanoparticles for Ocular Therapy

用于眼部治疗的压缩 DNA 纳米颗粒

基本信息

批准号：
8204931
负责人：
Muna I. Naash
金额：
$ 35.16万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8204931
关键词：
Acetates Acute Address Adult Aftercare Biological Biomedical Engineering Bypass CMV promoter Caliber Cations Cell Nucleus Cell membrane Cells Charge Chemistry Chickens Circular DNA Clinical Complementary DNA DNA Defect Development Disease Disease model Drops Drug Formulations Effectiveness Endocytosis Engineering Epithelial Cells Eye diseases Future GRB10 gene Gene Delivery Gene Expression Gene Transduction Agent Gene Transfer Gene Transfer Techniques Generations Genes Goals Human Immune response In Vitro Injection of therapeutic agent Interphase Cell Knock-out Lead Leber&apos s amaurosis Left Longevity Lysine Measures Mitotic Modeling Molecular Mus Nanotechnology Non-Viral Vector Nuclear Envelope Opsin Pattern Pharmacologic Substance Phenotype Photoreceptors Physical condensation Physics Plasmid Cloning Vector Plasmids Play Polyethylene Glycols Polymers Procedures RPE65 protein Radial Retina Retinal Retinal Degeneration Role Safety Specificity Stargardt&apos s disease Structure Structure of retinal pigment epithelium Technology Testing Therapeutic Tissues Toxic effect Transduction Gene Universities Vertebral column Viral Vector Vitelliform macular dystrophy Wild Type Mouse Work abstracting beta Actin clinical application computer science design early onset efficacy testing gene delivery system gene therapy human TFRC protein improved in vivo inflammatory marker nanoparticle non-viral gene delivery non-viral gene therapy novel nucleolin overexpression particle plasmid DNA postnatal pre-clinical programs promoter ranpirnase receptor retinal progenitor cell retinal rods retinol binding protein 3, interstitial, human subretinal injection trafficking transgene expression uptake vector

项目摘要

Project Summary/Abstract: The goal of this program is to advance the current compacted DNA nanoparticle based gene therapy technology to enable efficient and long-lasting gene delivery to dividing and non-dividing cells. The program will merge experts with molecular bioengineering, physics, chemistry, and computer science backgrounds at OUHSC, Stanford University and Copernicus Therapeutics, Inc, to accelerate essential preclinical steps for effective non-viral gene therapy. The plan is to engineer DNA vectors with efficient uptake and transport through the plasma membrane that can provide persistent transgene expression without toxicity. This technology can unimolecularly compact DNA with lysine polymers substituted with polyethylene glycol (PEG) into neutral charge nanoparticles with radii of less than 18 nm. These particles can penetrate the cell membrane via nucleolin receptor associated endocytosis and cross the nuclear membrane pore to the nucleus within 15 minutes. The DNA condensation formulation will compact either linear or circular DNA enabling us to eliminate plasmid backbone sequences known to play a significant role in inhibiting gene expression. The potential scientific and clinical benefits of these enhancements are substantial. While our ultimate aim is to use gene transfer to treat human ocular disease, we plan to address basic biological questions that will be important for rational design of vectors for gene therapy applications. Given the dangers inherent in the use of viral vectors, our strategy will enable us to access the favorable aspects of viral vectors while providing the safety and pharmaceutical qualities inherent in non-viral gene delivery systems. Towards this goal, we are working on developing new non-viral vectors for gene transfer to ocular tissues and establishing the cellular and molecular mechanisms involved in gene transduction. Three aims are proposed to optimize, mechanistically assess, and test our nanoparticle technology. Aim 1 will generate and compare the efficiency and longevity of EGFP expression between standard circular plasmid vectors and linear or minicircle constructs lacking the vector backbone sequence. The aim will also combine two novel gene therapy technologies, compacted DNA nanoparticles and pEPI-1 vector containing S/MAR sequence to develop an efficient and persistent gene transfer strategy in vivo. The effect of different vector sequences on promoter specificity will be assessed with two commonly used promoters in retinal gene therapy trials. To direct specific rod photoreceptor expression we will use the mouse opsin promoter (MOP) and to direct expression in the retinal pigment epithelium, we will use the vitelliform macular dystrophy 2 (VMD2) promoter. The constructs will be compacted and subretinally injected into WT mice during development at postnatal day 5 (P5) and in adults (P30). Injections at P5 will evaluate the efficacy of the nanoparticles in transfecting dividing retinal progenitor cells, and results will be relevant for the treatment of early onset eye diseases. Injections in adults will evaluate the efficacy of the nanoparticles in post-mitotic cells which is an appropriate experimental paradigm for treating late onset ocular diseases. Aim 2 will assess potential barriers to clinical vector application by evaluating particles uptake, trafficking, mechanisms of vector silencing, and in vivo safety. Aim 3 will test the efficacy of the vectors in rescuing the phenotypes in two well-known disease models: RPE65-/- (Leber's congenital amaurosis) and ABCR-/- (Stargardt's macular dystrophy).

项目摘要/摘要：该计划的目标是推进当前基于压缩 DNA 纳米颗粒的基因治疗能够高效、持久地将基因传递到分裂和非分裂细胞的技术。该计划将汇集具有分子生物工程、物理、化学和计算机科学背景的专家 OUHSC、斯坦福大学和 Copernicus Therapeutics, Inc 将加速重要的临床前步骤有效的非病毒基因治疗。该计划旨在设计具有高效摄取和运输能力的 DNA 载体通过质膜，可以提供持久的转基因表达而无毒性。这技术可以用聚乙二醇 (PEG) 取代的赖氨酸聚合物单分子压缩 DNA 成半径小于18 nm的中性电荷纳米粒子。这些颗粒可以穿透细胞通过核仁素受体相关的内吞作用进入细胞膜并穿过核膜孔到达细胞核 15 分钟内。 DNA 浓缩配方将压缩线性或环状 DNA，使我们能够消除已知在抑制基因表达中起重要作用的质粒主链序列。这这些增强功能的潜在科学和临床效益是巨大的。虽然我们的最终目标是利用基因转移来治疗人类眼部疾病，但我们计划解决基本问题对于基因治疗应用载体的合理设计非常重要的生物学问题。给定考虑到使用病毒载体所固有的危险，我们的战略将使我们能够获得有利的方面病毒载体，同时提供非病毒基因传递固有的安全性和药物品质系统。为了实现这一目标，我们正在致力于开发新的非病毒载体，用于将基因转移到眼部组织并建立参与基因转导的细胞和分子机制。三个目标是建议优化、机械评估和测试我们的纳米颗粒技术。目标 1 将生成比较标准环状质粒载体和线性质粒载体之间 EGFP 表达的效率和寿命或缺少载体主链序列的小环构建体。该目标还将结合两个新基因治疗技术、压缩 DNA 纳米颗粒和含有 S/MAR 序列的 pEPI-1 载体开发有效且持久的体内基因转移策略。不同载体序列对的影响将使用视网膜基因治疗试验中两种常用的启动子来评估启动子特异性。到直接特异性视杆光感受器表达，我们将使用小鼠视蛋白启动子（MOP）并直接为了在视网膜色素上皮中表达，我们将使用卵黄样黄斑营养不良 2 (VMD2) 启动子。该结构将在出生后一天的发育过程中被压实并注射到 WT 小鼠的视网膜下 5 (P5) 和成人 (P30)。 P5 时的注射将评估纳米颗粒在转染中的功效分裂视网膜祖细胞，其结果将与治疗早发性眼病相关。成人注射将评估纳米粒子在有丝分裂后细胞中的功效，这是一个适当的方法治疗迟发性眼部疾病的实验范例。目标 2 将评估临床应用的潜在障碍通过评估颗粒摄取、运输、载体沉默机制和体内安全性来评估载体应用。目标 3 将测试载体在两种众所周知的疾病模型中拯救表型的功效： RPE65-/-（莱伯先天性黑蒙）和 ABCR-/-（斯塔加特黄斑营养不良）。