Compacted DNA Nanoparticles for Ocular Therapy

用于眼部治疗的压缩 DNA 纳米颗粒

• 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
• 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' 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' 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

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目前基于致密dna纳米颗粒的基因治疗。 能够高效和持久地将基因传递给分裂和非分裂细胞的技术。该计划 将把具有分子生物工程、物理、化学和计算机科学背景的专家合并到 OUHSC、斯坦福大学和哥白尼治疗公司，以加快基本的临床前步骤 有效的非病毒基因治疗。该计划是设计具有高效摄取和运输功能的DNA载体 通过质膜可以提供持续的转基因表达，而不会产生毒性。这 用聚乙二醇单分子取代赖氨酸聚合物可以使DNA紧凑 形成半径小于18纳米的中性电荷纳米粒子。这些颗粒可以穿透细胞。 核膜通过核素受体相关的内吞作用，穿过核膜孔进入细胞核 在15分钟内。DNA缩合配方将使线状或环状DNA紧凑，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骨架序列。这个 这些增强技术的潜在科学和临床益处是巨大的。 虽然我们的最终目标是使用基因转移来治疗人类眼病，但我们计划解决基本的 生物学问题，这对于合理设计基因治疗应用的载体将是重要的。vt.给出 使用病毒载体固有的危险，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所固有的安全性和药学性质 系统。为此，我们正致力于开发新的非病毒载体，用于眼部基因转移。 并建立参与基因转导的细胞和分子机制。三个目标是 建议对我们的纳米颗粒技术进行优化、机械评估和测试。目标1将生成和 比较标准环状表达载体和线性载体表达绿色荧光蛋白的效率和寿命 或缺少载体主干序列的小环结构。该目标还将结合两个新基因 治疗技术、致密DNA纳米粒和含有S/MAR序列的PEPI-1载体 开发一种有效和持久的体内基因转移策略。不同载体序列对DNA序列的影响 在视网膜基因治疗试验中，将用两种常用的启动子来评估启动子的特异性。至 我们将利用小鼠视蛋白启动子(MOP)来直接引导杆状感光细胞表达 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。 这些构建物将被压实并在出生后一天的发育过程中注射到WT小鼠的视网膜下 5(P5)和成人(P30)。在P5注射将评估纳米颗粒在转染中的效果 分裂视网膜祖细胞，结果将与早发性眼病的治疗相关。 成人注射将评估纳米颗粒在有丝分裂后细胞中的效果，这是一种合适的 治疗迟发性眼病的实验范式。AIM 2将评估临床上的潜在障碍 通过评估颗粒的摄取、运输、媒介沉默的机制和体内安全性来评估媒介的应用。 目标3将测试载体在挽救两个众所周知的疾病模型中的表型的效果： RPE65-/-(Leber先天性黑色素沉着症)和ABCR-/-(Stargardt黄斑营养不良症)。