Compacted DNA Nanoparticles for Ocular Therapy

描述（由申请人提供）：该计划的目标是推进当前的DNA纳米颗粒（NP）递送系统和表达技术，以开发针对由大基因缺陷引起的重要光感受器相关眼部疾病的安全有效的疗法。这些NP已经证明了在肺中高达20 kbp的载体和在眼睛中高达14 kbp的载体（测试的最大尺寸）的有效基因表达，这使它们成为AAV的理想补充，特别是用于递送大基因。该计划将合并具有分子生物工程，眼部生物学/生理学，物理学和化学背景的专家，以加速有效的眼部非病毒基因治疗的产生的重要步骤。DNA NP由用赖氨酸-PEG聚阳离子压实的DNA单分子组成，并且具有8-11 nm的最小直径。它们的小尺寸，加上有效地将NP运输到细胞核（绕过溶酶体）的特定摄取机制，可能是它们抑制有丝分裂后分化细胞的能力的原因。我们已经表明，NP治疗导致包括光感受器（PR）在内的眼细胞的有效转染，即使在多次注射后也不会对眼睛产生毒性作用，分布在整个视网膜下空间，并且在视网膜色素变性小鼠模型中介导明显的结构和功能拯救（RP，Rds+/-），Leber先天性黑蒙（LCA，Rpe 65-/-），和Stargardt病（STGD 1，Abca 4-/-）。在狒狒中也证明了有效的基因表达而没有毒性。这些原理验证研究证实了该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载体的价值，但也强调了改善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与大基因相关的眼部疾病模型中以足够高的水平提供长期基因表达以介导完全表型拯救的NP和载体。我们建议通过首先研究pEPi-ABCA 4载体的表观遗传调控来实现这一目标，以了解基因沉默的机制（目标1），然后实施靶向载体工程以增强NP进入细胞，促进细胞核中的稳定性，防止表观遗传沉默，并增加基因表达水平（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STGD 1的Abca 4-/-模型和与2型usher综合征（USH 2）相关的两种模型（Ush 2a-/-和Ush 2a c2299 delG敲入）（目的3）。USH 2A是一个非常大的基因，不能被传统载体容纳，因此Usher综合征的靶向治疗的发展已经滞后。总之，来自该应用的结果将促进用于与大基因相关的眼部疾病的DNA NP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.