Abstract
Purpose :
Retinitis Pigmentosa (RP), an inherited retinal disease, is characterized by a progressive loss of photoreceptor cells, leading to vision impairment. Current treatments, including retinoids and vitamin A supplements, primarily offer symptomatic relief without addressing the genetic root causes of this disorder. Mutations in the PRPF31 gene are key genetic contributors to the pathology of autosomal dominant RP. Since RP often results from specific genetic anomalies, gene therapy represents an ideal treatment approach. However, despite advancements in gene therapy for RP, effective delivery remains a significant challenge. Chitosan (CS) nanoparticles (NPs) offer a promising solution due to their biocompatibility and potential for gene delivery. This study aims to develop a chitosan NP-based delivery system for the PRPF31 gene.
Methods :
The plasmid DNA (pDNA) was cloned and extracted according to the manufacturer's protocol. The CS NPs loaded with pDNA were prepared through a complex coacervation process. The NPs were characterized for particle size, zeta potential, and surface morphology. Additionally, the loading capacity and encapsulation efficiency of the nanoparticles, as well as their protective effect on the plasmid, were evaluated.
Results :
The average particle size, polydispersity index, and zeta potential were 284.52 ± 9.46 nm, 0.316 ± 0.08, and +45.47 ± 4.58 mV, respectively. Scanning Electron Microscopy images confirmed the formation of nanoparticles and their spherical morphology. The pDNA encapsulation efficiency was 98.43 ± 0.27%, with a loading efficiency of 23.58 ± 1.52%. Gel electrophoresis results indicated that the chitosan nanoparticles effectively protected the pDNA.
Conclusions :
We have successfully prepared chitosan nanoparticles for PRPF31 gene delivery in nano-size range. Gel electrophoresis affirmed the similarity between the cloned plasmid and the original plasmid. The gel retardation assay helped determine the optimal chitosan-to-plasmid ratio for effective DNA encapsulation. The integrity of encapsulated plasmids within NPs was confirmed by their intact recovery following the enzymatic cleavage of the NPs. The high encapsulation efficiency emphasizes the effectiveness of this nanoparticle formulation in entrapping genetic material.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.