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; 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）：该程序的目的是推进目前的基于压实的DNA纳米粒子基因治疗技术，以使有效且持久的基因递送到分裂和非分散细胞。该计划将在OUHSC，斯坦福大学和哥白尼Therapeutics，Inc合并专家与分子生物工程，物理，化学和计算机科学背景，以加速有效的非病毒基因疗法的基本临床前步骤。该计划是针对有效摄取和通过质膜运输的DNA载体，该媒介可以提供持续的转基因表达而无需毒性。该技术可以用用聚乙烯乙二醇（PEG）取代的赖氨酸聚合物（PEG）将其非分子紧凑型DNA中中性电荷纳米颗粒，而半径小于18 nm。这些颗粒可以通过核仁受体相关的内吞作用穿透细胞膜，并在15分钟内将核膜孔穿过核孔。 DNA缩合配方将紧凑线性或圆形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。这些增强的潜在科学和临床益处是可观的。虽然我们的最终目的是使用基因转移来治疗人眼疾病，但我们计划解决基本生物学问题，对于基因治疗应用的合理设计至关重要。鉴于使用病毒载体固有的危险，我们的策略将使我们能够进入病毒载体的有利方面，同时提供非病毒基因递送系统固有的安全性和药物质量。为了实现这一目标，我们正在努力开发新的非病毒载体，以转移到眼组织，并建立涉及基因转导的细胞和分子机制。提出了三个目的，以优化，机械地评估和测试我们的纳米颗粒技术。 AIM 1将产生和比较标准圆形质粒向量与线性或小圆形构建体之间缺乏矢量骨架序列之间的EGFP表达的效率和寿命。该目标还将结合两种新型的基因疗法技术，压实的DNA纳米颗粒和含有S/MAR序列的PEPI-1载体，以在体内开发有效且持续的基因转移策略。在视网膜基因治疗试验中，将用两个常用的启动子评估不同矢量序列对启动子特异性的影响。为了指导特定的杆感光体表达，我们将使用小鼠OPSIN启动子（MOP）并直接在视网膜色素上皮中表达，我们将使用Vitellistion Masular Basular Drophytrophy 2（VMD2）启动子。在产后第5天（P5）和成年人（P30）的发育期间，该构建体将被压缩并从下续注入WT小鼠中。 P5处的注射将评估纳米颗粒在转染视网膜祖细胞转染中的功效，结果将与治疗早期发作眼病的治疗有关。成人的注射将评估纳米颗粒在有丝分裂细胞中的功效，这是一种适合治疗晚期发作眼部疾病的实验范式。 AIM 2将通过评估颗粒吸收，贩运，向量沉默的机制和体内安全性来评估临床向量应用的潜在障碍。 AIM 3将测试向量在两个众所周知的疾病模型中拯救表型的功效：RPE65 - / - （Leber的先天性amaurisos）和Abcr - / - （Stargardt的黄斑营养不良）。