Compacted DNA Nanoparticles for Ocular Therapy

描述（由申请人提供）：该计划的目标是推进目前基于紧凑DNA纳米颗粒的基因治疗技术，以实现对分裂和非分裂细胞的有效和持久的基因递送。该计划将合并OUHSC，斯坦福大学和Copernicus Therapeutics，Inc的分子生物工程，物理，化学和计算机科学背景的专家，以加速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，使其通过质膜有效吸收和转运，从而可以提供持久的转基因表达而没有毒性。该技术可以将聚乙二醇（PEG）取代的赖氨酸聚合物与DNA单分子压缩成半径小于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缩合制剂将压缩线性或环状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骨架序列。这些增强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部疾病，我们计划解决基本的生物学问题，这将是重要的基因治疗应用载体的合理设计。考虑到使用病毒载体的固有危险，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，并建立基因转导的细胞和分子机制。提出了三个目标，以优化，机械评估，并测试我们的纳米粒子技术。目的1将产生并比较标准环状质粒载体和缺乏载体骨架序列的线性或小环构建体之间EGFP表达的效率和寿命。该目标还将结合联合收割机两种新的基因治疗技术，紧凑的DNA纳米颗粒和含有S/MAR序列的pEPI-1载体，以开发一种有效和持久的体内基因转移策略。不同载体序列对启动子特异性的影响将用视网膜基因治疗试验中两种常用的启动子进行评估。为了指导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），并且为了指导在视网膜色素上皮中的表达，我们将使用卵黄状黄斑营养不良2（VMD 2）启动子。在出生后第5天（P5）和成年（P30）发育期间，将构建体压实并视网膜下注射到WT小鼠中。在P5时注射将评估纳米颗粒在抑制分裂的视网膜祖细胞中的功效，并且结果将与早发性眼部疾病的治疗相关。在成人中的注射将评估纳米颗粒在有丝分裂后细胞中的功效，这是用于治疗迟发性眼部疾病的适当实验范例。目标2将通过评价颗粒摄取、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。目的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：RPE 65-/-（Leber先天性黑蒙）和ABCR-/-（Stargardt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).