Currently, few options are available to prevent or treat blindness associated with retinitis pigmentosa (RP), and there is no cure. Therefore, options for gene therapy have been developed. Most current gene targeting strategies use cDNA; however, evidence has shown that endogenous non-coding sequences (e.g., UTRs and introns) in the genomic loci (gDNA), can preserve the stability of the message, promote normal gene regulation, and improve translational yield in a real time manner. However, testing the utility of genomic sequences to improve gene expression levels and therapeutic efficacy of ocular gene therapies has been hindered by size limitations of traditional delivery vehicles. To circumvent this limitation, we use large capacity compacted DNA nanoparticles (NPs), which have proven to be safe and effective for use in the eye, making them an excellent platform for delivering large expression cassettes. More importantly, recently, we have shown that NPs carrying intron-containing rhodopsin DNA efficiently targeted photoreceptors and improved visual function in a rhodopsin knockout mouse (RKO) model up to 8 months (duration of the study) post-injection compared with the intron- less cDNA-based system. We further demonstrated that transgene-mediated silencing was associated with the cDNA vector, but not the intron-containing intermediate form genomic DNA. All of these results indicate that endogenous genetic regulatory elements have a strong advantage over cDNA and whenever possible inclusion in the therapeutic transgene should achieve optimal functional rescue. Our exciting peer reviewed results highlight the potential clinical significance of this approach for treating blindness in RP, particularly when combined with knockdown technology to combat dominant mutations. To further capitalize on these findings, we propose to use our NP system to deliver a complete rhodopsin genomic locus (15 kb), without any bacteria backbone, to treat two murine RP models: an autosomal recessive RP (arRP) of RKO and an autosomal dominant RP (adRP) of P23H knockin. We selected these models because rhodopsin mutations are the most common cause of dominant RP in patients, making it a clinically important target, and because both over- and underexpression of rhodopsin are deleterious to photoreceptors, so promoting proper regulation is critical for therapeutic efficacy. We hypothesize that a key factor that controls gene expression is the arrangement of the genomic locus, which contain the endogenous elements necessary for stable and regulated expression. We plan to accomplish these approaches by NP mediated rhodopsin gDNA (both human and mouse) delivery to RKO mouse eyes to test therapeutic efficacy and physiological stability (Aim 1), and to determine if these treatments will cause epigenetic changes (Aim 2). The best vector selected from Aims 1 & 2 will then be used to restore normal rod function in the adRP P23H mouse model to gain insight into its therapeutic effect of this technology (Aim 3). The proposed project is a practical extension of our ongoing studies. Our goal is to continue to develop novel strategies for the treatment of RP using NP-mediated gDNA delivery.
The development of a stable and regulated gene expression system at physiological levels is critical for targeted ocular gene therapy. A feasible and effective approach is to transfer the full-length genomic DNA of the gene, which contains all the regulatory sequences similar to those in the body, to the deficient cell; yet, the strategy is hampered by the large size (e.g., more than 10,000 base pair) of the gene, which exceeds the capacity of viral vectors. Our lab is developing a method that uses novel nanoparticles that have the ability to transfer larger genes to overcome these limitations.
| Mitra, Rajendra Narayan; Zheng, Min; Weiss, Ellen R et al. (2018) Genomic form of rhodopsin DNA nanoparticles rescued autosomal dominant Retinitis pigmentosa in the P23H knock-in mouse model. Biomaterials 157:26-39 |
| Wang, Kai; Mitra, Rajendra Narayan; Zheng, Min et al. (2018) Nanoceria-loaded injectable hydrogels for potential age-related macular degeneration treatment. J Biomed Mater Res A 106:2795-2804 |
| Asokan, Priyadarsini; Mitra, Rajendra N; Periasamy, Ramesh et al. (2018) A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies. Invest Ophthalmol Vis Sci 59:746-756 |
| Wang, Kai; Han, Zongchao (2017) Injectable hydrogels for ophthalmic applications. J Control Release 268:212-224 |
| Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A et al. (2016) Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model. FASEB J 30:1076-86 |
| Mitra, Rajendra Narayan; Zheng, Min; Han, Zongchao (2016) Nanoparticle-motivated gene delivery for ophthalmic application. Wiley Interdiscip Rev Nanomed Nanobiotechnol 8:160-74 |
| 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 |
