Compacted DNA Nanoparticles for Ocular Therapy

用于眼部治疗的压缩 DNA 纳米颗粒

• 批准号: 8007344
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007344
• 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; 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

DESCRIPTION (provided by applicant): 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).

描述（由申请人提供）：该计划的目标是推进当前基于压缩 DNA 纳米粒子的基因治疗技术，以实现高效且持久的基因递送至分裂和非分裂细胞。该项目将汇集来自 OUHSC、斯坦福大学和 Copernicus Therapeutics, Inc 的具有分子生物工程、物理、化学和计算机科学背景的专家，以加快有效非病毒基因治疗的重要临床前步骤。该计划是设计能够通过质膜有效摄取和运输的 DNA 载体，从而提供持久的转基因表达而无毒性。该技术可将聚乙二醇（PEG）取代的赖氨酸聚合物单分子地将DNA压实成半径小于18 nm的中性电荷纳米颗粒。这些颗粒可以通过核仁素受体相关的内吞作用穿透细胞膜，并在15分钟内穿过核膜孔到达细胞核。 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发挥重要作用的质粒主链序列。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的生物学问题，这对于基因治疗应用载体的合理设计非常重要。考虑到使用病毒载体固有的危险，我们的策略将使我们能够获得病毒载体的有利方面，同时提供非病毒基因传递系统固有的安全性和药物品质。为了实现这一目标，我们正在开发新的非病毒载体，用于将基因转移到眼组织，并建立基因转导涉及的细胞和分子机制。提出了三个目标来优化、机械评估和测试我们的纳米颗粒技术。目标 1 将生成并比较标准环状质粒载体和缺乏载体主链序列的线性或小环状构建体之间 EGFP 表达的效率和寿命。该项目还将结合两种新型基因治疗技术，即压缩 DNA 纳米粒子和含有 S/MAR 序列的 pEPI-1 载体，以开发有效且持久的体内基因转移策略。将使用视网膜基因治疗试验中两种常用的启动子来评估不同载体序列对启动子特异性的影响。为了指导特定视杆光感受器的表达，我们将使用小鼠视蛋白启动子 (MOP)，为了指导视网膜色素上皮中的表达，我们将使用卵黄样黄斑营养不良 2 (VMD2) 启动子。该结构将在出生后第 5 天 (P5) 和成年 (P30) 发育过程中被压实并注射到 WT 小鼠体内。 P5 时的注射将评估纳米颗粒转染分裂中的视网膜祖细胞的功效，其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功效，这是治疗迟发性眼部疾病的适当实验范例。目标 2 将通过评估颗粒摄取、运输、载体沉默机制和体内安全性来评估临床载体应用的潜在障碍。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-/-（莱伯氏先天性黑蒙）和 ABCR-/-（斯塔加特黄斑营养不良）。