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.

Public Health Relevance

Our program is designed to expand the benefits of compacted DNA nanotechnology for large genes to facilitate its future use in clinical gene therapy treatment for retinal degenerative disorders. Proposed studies will lay the foundation for use of this technology as a clinically viable gene delivery system for retinal diseases associated with mutations in large genes such as those linked to Stargardt?s. The small size of this therapeutic platform, coupled with uptake via cell-surface nucleolin that facilitates active transport to the nucleus, accounts for their ability to transfect post-mitotic, differentiated cells like the photoreceptors.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY018656-11
Application #
9616719
Study Section
Nanotechnology Study Section (NANO)
Program Officer
Neuhold, Lisa
Project Start
2008-01-01
Project End
2022-08-31
Budget Start
2018-09-01
Budget End
2019-08-31
Support Year
11
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Houston
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
036837920
City
Houston
State
TX
Country
United States
Zip Code
77204
Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I (2018) Retbindin Is Capable of Protecting Photoreceptors from Flavin-Sensitized Light-Mediated Cell Death In Vitro. Adv Exp Med Biol 1074:485-490
Ikelle, Larissa; Naash, Muna I; Al-Ubaidi, Muayyad R (2018) Role of Fibulins 2 and 5 in Retinal Development and Maintenance. Adv Exp Med Biol 1074:275-280
Zulliger, Rahel; Watson, Jamie N; Al-Ubaidi, Muayyad R et al. (2018) Optimizing Non-viral Gene Therapy Vectors for Delivery to Photoreceptors and Retinal Pigment Epithelial Cells. Adv Exp Med Biol 1074:109-115
Agbaga, Martin-Paul; Merriman, Dana K; Brush, Richard S et al. (2018) Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors. J Lipid Res 59:1586-1596
Stuck, Michael W; Conley, Shannon M; Naash, Muna I (2016) PRPH2/RDS and ROM-1: Historical context, current views and future considerations. Prog Retin Eye Res 52:47-63
Mitra, Rajendra N; Conley, Shannon M; Naash, Muna I (2016) Therapeutic Approach of Nanotechnology for Oxidative Stress Induced Ocular Neurodegenerative Diseases. Adv Exp Med Biol 854:463-9
Mitra, Rajendra Narayan; Nichols, Chance A; Guo, Junjing et al. (2016) Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy. J Control Release 236:31-7
Chakraborty, Dibyendu; Conley, Shannon M; Zulliger, Rahel et al. (2016) The K153Del PRPH2 mutation differentially impacts photoreceptor structure and function. Hum Mol Genet 25:3500-3514
Conley, Shannon M; Whalen, Patrick; Lewin, Alfred S et al. (2016) Characterization of Ribozymes Targeting a Congenital Night Blindness Mutation in Rhodopsin Mutation. Adv Exp Med Biol 854:509-15
Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I (2015) Retbindin is an extracellular riboflavin-binding protein found at the photoreceptor/retinal pigment epithelium interface. J Biol Chem 290:5041-52

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