Compacted DNA Nanoparticles for Ocular Therapy

项目总结 我们的目标是推进我们目前的DNA纳米颗粒(NP)传递平台和基因表达，以开发安全的 以及针对重要的光感受器相关眼病的有效治疗方法 大基因。我们正在融合分子生物工程、纳米配方、材料科学、眼科等领域的知识 生物/生理和化学加速产生有效的非病毒基因的基本步骤 眼科疾病投放平台。NPs由单分子DNA与多聚赖氨酸组成- 聚乙二醇阳离子，直径8-11 nm。它们的小尺寸加上细胞通过细胞表面的摄取 核仁能有效地将NPs输送到细胞核，这是它们有能力感染有丝分裂后的能力的原因 光感受器。使用NPs导致了大基因的高效表达，这是 瞄准难以挽救的视网膜疾病。尽管基因大小上限尚未确定， 在肺中测试的最大尺寸为20kb，在眼睛中测试的最大尺寸为~14kb，这使得NPs成为理想的替代品 用于传递大基因的AAVs。我们已经证明，NP治疗导致了有效的眼部转基因 细胞，包括光感受器和视网膜色素上皮，即使在 多次注射，分布在视网膜下间隙，并调节明显的结构/功能 小鼠视网膜色素变性、Leber‘s先天性黑色素、Stargardt’s和糖尿病模型的抢救 视网膜病变。在狒狒眼睛中也实现了无毒性的有效基因表达。这些证明- 原则研究证实了这项技术的潜在临床意义，并强调了 大容量，但强调需要长期高水平的基因表达。我们的主要目标是 增强光感受器对NPs的摄取以达到长期治疗水平的大基因表达 完全的表型救援。我们建议通过有针对性的载体工程来促进表达 与NP-递送平台一起，增强其从玻璃体到光感受器的递送，实现 泛视网膜分布，促进核内上体稳定，防止表观遗传沉默。 随后，我们将测试这些优化的NP/交付平台(S)是否具有调节完全表型的能力 大基因疾病模型中的救援；特别是与缺乏基因相关的Stargardt的ABCA4-/-模型 ABCA4基因。ABCA4是一个大基因，传统的病毒载体还没有完全挽救它，作为一种 结果Stargardt‘s的靶向治疗发展滞后。我们计划在AIM 1中设计出载体 能够在光感受器中达到治疗性水平的表达，并在目标2中开发有效的NP- 提供平台，以加强光感受器摄取来自玻璃体的NPs。目标3将测试 最有效的NPs的最佳递送平台，用于治疗有效的表达水平 ABCA4-/-小鼠发病前后的光感受器。总而言之，这一结果 应用将促进DNA纳米粒在与大基因相关的眼部疾病中的应用。

PROJECT SUMMARY We aim to advance our current DNA nanoparticle (NP) delivery platform and gene expression to develop safe and effective therapies targeting important photoreceptor-associated ocular disorders caused by defects in large genes. We are merging knowledge in molecular bioengineering, nanoformulation, material science, eye biology/physiology and chemistry to accelerate essential steps for the generation of effective non-viral gene delivery platform for eye diseases. The NPs consist of single molecules of DNA compacted with poly-lysine- PEG polycation and have a diameter of 8-11 nm. Their small size coupled with cellular uptake via cell-surface nucleolin, which efficiently traffics the NPs to the nucleus, accounts for their ability to transfect post-mitotic photoreceptors. Using NPs has led to efficient expression of large genes, an essential prerequisite for targeting hard-to-rescue diseases of the retina. Although upper gene size limitation has not been established, the largest size tested in the lung was 20 kb and in the eye was ~14 kb making NPs an ideal alternative to AAVs for delivery of large genes. We have showed that NP treatment led to efficient transfection of ocular cells including photoreceptors and retinal pigment epithelium, exerted no toxic effects on the eye even after multiple injections, distributed throughout the subretinal space, and mediated appreciable structural/functional rescue in mouse models of retinitis pigmentosa, Leber’s congenital amaurosis, Stargardt’s and diabetic retinopathy. Effective gene expression without toxicity has also been achieved in baboon eyes. These proof- of-principle studies confirms the potential clinical significance of this technology and highlights the value of a large capacity, but emphasized the need for prolonged high levels of gene expression. Our main goal is to enhance photoreceptor uptake of NPs to achieve long-term therapeutic levels of expression of large genes for full phenotypic rescue. We propose to accomplish this by targeted vector engineering to boost expression levels along with NP-delivery platform to enhance their delivery from the vitreous to photoreceptors, achieve pan retinal distribution, promote episomal stability in the nucleus and prevent epigenetic silencing. Subsequently, we will test these optimized NPs/delivery platform(s) for their ability to mediate full phenotypic rescue in a large gene disease model; specifically the Abca4-/- model of Stargardt’s associated with the lack of ABCA4 gene. ABCA4 is a large gene which has not been fully rescued by traditional viral vectors and as a result development of targeted therapeutics for Stargardt’s has lagged. We plan in aim1 to engineer vectors that can achieve therapeutic levels of expression in photoreceptors and in aim 2 to develop effective NP- delivery platforms to enhance photoreceptor uptake of NPs from the vitreous. Aim 3 will test the efficacy of the best delivery platform of the most effective NPs for therapeutically-effective levels of expression in photoreceptors of Abca4-/- mice before and after the onset of the disease. In summary, results from this application will facilitate the advancement of DNA NP use for ocular diseases associated with large genes.