Compacted DNA Nanoparticles for Ocular Therapy

描述(申请人提供)：该计划的目标是推进当前基于致密DNA纳米颗粒的基因治疗技术，使高效和持久的基因输送到分裂和非分裂细胞。该计划将合并OUHSC、斯坦福大学和哥白尼治疗公司的具有分子生物工程、物理、化学和计算机科学背景的专家，以加快有效的非病毒基因治疗的基本临床前步骤。该计划是设计DNA载体，使其能够通过质膜有效地摄取和运输，从而提供无毒性的持续转基因表达。这项技术可以用聚乙二醇单分子取代赖氨酸聚合物制成半径小于18 nm的中性电荷纳米颗粒。这些颗粒可以通过与核仁素受体相关的内吞作用穿透细胞膜，并在15分钟内穿过核膜孔进入细胞核。DNA缩合配方将使线形或环状DNA紧凑，使我们能够消除已知在抑制基因表达方面发挥重要作用的质粒骨架序列。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，这些问题对于合理设计用于基因治疗的载体将是重要的。考虑到使用病毒载体固有的危险，我们的战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递送系统固有的安全性和药学特性。为了实现这一目标，我们正致力于开发新的非病毒载体，用于眼组织的基因转移，并建立参与基因转导的细胞和分子机制。提出了优化、机械评估和测试我们的纳米颗粒技术的三个目标。目的1建立并比较标准环状表达载体和缺少载体骨架序列的线状或小环状构建体之间的EGFP表达效率和寿命。该目标还将结合两种新的基因治疗技术，即致密的DNA纳米颗粒和含有S/MAR序列的PEPI-1载体，以开发一种高效和持久的体内基因转移策略。在视网膜基因治疗试验中，将用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。为了指导特定视杆感受器的表达，我们将使用小鼠视蛋白启动子(MOP)，为了在视网膜色素上皮中直接表达，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，并在出生后第5天(P5)和成年(P30)发育期间注射到WT小鼠的视网膜下。在P5注射将评估纳米颗粒在转染分裂中的视网膜前体细胞方面的效果，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疗效，这是治疗迟发性眼病的合适的实验范例。目的2将通过评估颗粒的摄取、运输、载体沉默的机制和体内安全性来评估临床载体应用的潜在障碍。目的3将测试载体在挽救两种著名疾病模型中的表型的效果：RPE65-/-(Leber‘s先天性黑素瘤)和ABCR-/-(Stargardt’s黄斑营养不良)。

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).