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.
This program is designed to expand the benefits of our compacted DNA nanotechnology for large genes to facilitate its future use as a clinical gene therapy treatment for their associated ocular diseases. Studies are planned to lay the foundation for use of this technology as a clinically viable gene delivery system for ocular diseases associated with large genes such as those associated with Stargardt's and Usher syndrome. The size (8-11 nm in diameter) and positive gene expression/safety profiles of our NPs may permit uniquely effective and safe dosing options to facilitate the widespread use of this technology.
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