Abstract

Purpose: Mutations in photoreceptor and retinal pigment epithelium (RPE) genes cause a wide range of blinding diseases including Leber’s congenital amaurosis (LCA), Stargardt’s macular dystrophy, retinitis pigmentosa (RP), and a variety of other forms of macular dystrophy. Therefore, we have been exploring non-viral gene replacement therapy with highly engineered vectors delivered in compacted DNA nanoparticles (NPs). Our recent work has focused on comparison of vectors containing only cDNA vs. vectors containing genomic DNA elements (gDNA) such as introns.

Methods: NPs are composed of polyethylene-glycol conjugated polylysine and DNA, and contained multiple different vectors, including those containing either rhodopsin cDNA or gDNA (under the control of the native rhodopsin promoter). They are subretinally delivered to neonatal mice such as the rhodopsin knockout (RKO). Follow-up at multiple timepoints includes structural analysis, functional analysis, and biochemical analysis.

Results: We demonstrated that the NPs are well-tolerated and safe for use in the eye and can mediate long-term, (often for the life of the animal) phenotypic improvements in mouse models of RP (rho^-/-, rds^+/-), Stargardt’s dystrophy (abca4^-/-), and LCA (rpe65^-/-). Specifically, NPs carrying rhodopsin gDNA but not cDNA can mediate structural and functional improvements in the rho^-/- phenotype that persist for up to 8 months post-injection. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at timepoints when cones in the RKO are degenerated. These data suggest that vector engineering strategies incorporating genomic DNA elements can significantly improve outcomes compared to traditional cDNA constructs.

Conclusions: These positive results suggest that optimized vectors coupled with an effective delivery approach can be successful in the treatment of retinal degenerative disease. However, further improvements in levels and distribution of expression are needed. To assess ways to improve distribution, we have expanded testing in large animal models, and we are also testing the efficacy of a variety of additional DNA elements to prevent silencing and down-regulation of expression. With these advancements, compacted DNA NPs have the potential to become clinically relevant treatments for a variety of inherited ocular diseases.