Development of Chitosan Nanoparticles for PRPF31 Gene Delivery in the Treatment of Retinitis Pigmentosa

INTRODUCTION: Retinitis Pigmentosa (RP), an inherited retinal disease, characterized by a progressive loss of photoreceptor cells, leading to vision impairment. Mutations in the pre-mRNA processing factor gene (PRPF31) major cause of autosomal dominant RP. While viral vectors like AAV are commonly used in gene therapy, they are limited by low DNA cargo capacity and immunogenicity. We aimed to develop a chitosan-based NPs to deliver PRPF31 plasmid DNA (pDNA) into the ARPE-19 retinal cell line. METHODS: The Complex coacervation method was used to prepare CS NPs loaded with PRPF31. The NPs were characterized for particle size, polydispersity index (PDI), and zeta potential using a zetasizer. Surface morphology of CS-based NPs was examined using SEM. Additionally, the loading capacity, encapsulation efficiency, protective effect of CS on the pDNA, in vitro transfection efficiency, and cytotoxicity were evaluated in ARPE-19 cell lines Results: The particle size, PDI, and zeta potential of chitosan nanoparticles were 245.19 ± 21.27nm, 0.265 ± 0.023, and +20.5 ± 5.63 mV, respectively. Scanning Electron Microscopy (SEM) images confirmed the formation of NPs. The pDNA encapsulation efficiency was 66.80%± 4.69 and a loading efficiency of 21.93%± 1.08. The transfection efficiency of naked pDNA in ARPE-19 cells was 24.40% while that of CS pDNA NP was 48.30%. Cells transfected with pDNA using the standard transfecting agent Lipofectamine showed a higher transfection efficiency of 65.24% as compared to both naked pDNA and CS-pDNA NPs. DISCUSSION: Cs- pDNA particles demonstrated moderate transfection efficiency and lower cytotoxicity in the retinal cell line. In vivo research is required to realize its future therapeutic potential. CONCLUSION: These CS-pDNA NPs may have potential as a non-viral delivery vehicle in gene therapy in RP, however, further research in animal models is needed to validate the in vitro findings and for their potential therapeutic applications.