Compacted DNA Nanoparticles for Ocular Therapy

描述(申请人提供)：该计划的目标是推进当前的DNA纳米颗粒(NP)传递系统和表达技术，以开发针对由大基因缺陷引起的重要光感受器相关眼病的安全有效的治疗方法。这些NPs已经证明了高效的基因表达，其载体在肺中高达20kbp，在眼睛中高达14kbp(测试的最大尺寸)，这使它们成为AAVs的理想补充，特别是在运送大基因方面。该计划将合并具有分子生物工程、眼生物学/生理学、物理学和化学背景的专家，以加快产生有效的眼部非病毒基因治疗的关键步骤。DNA纳米粒由单分子DNA与赖氨酸-聚乙二醇聚阳离子结合而成，最小直径为8-11 nm。它们的体积很小，再加上一种特殊的摄取机制，可以有效地将NPs输送到细胞核(绕过溶酶体)，这可能是它们能够转染有丝分裂后分化细胞的原因。在视网膜色素变性(RP，Rds/-)、Leber‘s先天性黑色素症(LCA，RPE65-/-)和Stargardt病(STGD1，ABCA4-/-)的小鼠模型中，NP治疗导致了包括光感受器(PR)在内的眼细胞的有效转基因，即使在多次注射后对眼睛也没有毒性作用，分布在视网膜下间隙，并介导了明显的结构和功能挽救。无毒性的有效基因表达也已在狒狒身上被证明。这些原则验证研究证实了这项技术在治疗患者失明方面的潜在临床意义，并强调了大容量递送工具的价值，但也强调了提高PR基因表达水平的必要性。因此，我们的主要目标是开发能够在足够高的水平上提供长期基因表达的NPs和载体，以在与大基因相关的眼部疾病模型中介导完全的表型拯救。我们建议通过首先研究Pepi-ABCA4载体的表观遗传调控来了解基因沉默的机制(目标1)，然后实施靶向载体工程来促进NP进入细胞，促进核内的稳定性，防止表观遗传沉默，并提高基因表达水平(目标2)。随后，我们将测试这些优化的载体在大基因疾病模型中介导完全表型拯救的能力；特别是STGD1的ABCA4-/-模型和与Usher综合征2型(USH2)相关的两个模型(Ush2a-/-和Ush2a c2299delG)(目标3)。USH2A是一个非常大的基因，不能被传统的载体所容纳，因此针对Usher综合征的靶向治疗的发展滞后。综上所述，这一应用的结果将促进与大基因相关的眼部疾病的DNA NPs的进展。

DESCRIPTION (provided by applicant): The goal of this program is to advance current DNA nanoparticle (NP) delivery system and expression technologies to develop safe and effective therapies targeting important photoreceptor-associated ocular disorders caused by defects in large genes. These NPs have demonstrated efficient gene expression with vectors up to 20 kbp in the lung and 14 kbp in the eye (the largest sizes tested) which make them an ideal complement to AAVs especially for delivery of large genes. The program will merge experts with backgrounds in molecular bioengineering, eye biology/physiology, physics, and chemistry to accelerate essential steps for the generation of effective ocular non-viral gene therapy. The DNA NPs consist of single molecules of DNA compacted with lysine-PEG polycations and have a minimum diameter of 8-11 nm. Their small size, coupled with a specific uptake mechanism that efficiently traffics the NPs to the nucleus (bypassing lysosomes), likely accounts for their ability to transfect post-mitotic, differentiated cells. We have shown that NP treatment leads to efficient transfection of ocular cells including photoreceptors (PRs), exerts no toxic effects on the eye even after multiple injections, distributes throughout the subretinal space, and mediates appreciable structural and functional rescue in mouse models of retinitis pigmentosa (RP, Rds+/-), Leber's congenital amaurosis (LCA, Rpe65-/-), and Stargardt's disease (STGD1, Abca4-/-). Effective gene expression without toxicity has also been demonstrated in baboons. These proof-of-principle studies confirmed the potential clinical significance of this technology for treating blindness in patients and highlighted the value of a large capacity delivery vehicle, but also highlighted the need for improvements in PR gene expression levels. Our main goal here is therefore to develop NPs and vectors capable of providing long-term gene expression at levels high enough to mediate full phenotypic rescue in models of ocular diseases associated with large genes. We propose to accomplish this by first studying the epigenetic regulation of the pEPi-ABCA4 vector to understand the mechanisms that underlie gene silencing (Aim 1), then implement targeted vector engineering to enhance NP entry into the cell, promote stability in the nucleus, prevent epigenetic silencing, and increase gene expression levels (Aim 2). Subsequently, we will test these optimized vectors for their ability to mediate full phenotypic rescue in large gene disease models; specifically the Abca4-/- model of STGD1 and two models (Ush2a-/- and Ush2a c2299delG knock-in) associated with usher syndrome type 2 (USH2) (Aim 3). USH2A is a very large gene which cannot be accommodated by traditional vectors, and as a result development of targeted therapeutics for Usher syndrome has lagged. In summary, results from this application will facilitate the advancement of DNA NPs for ocular diseases associated with large genes.