(M1230-04-21) Self-Assembling β-cyclodextrin siRNA Nanoparticle Complex in Silencing Mutant Huntingtin Protein (mHtt)

Purpose: Huntington's Disease (HD) is a hereditary condition inherited in an autosomal dominant pattern, stemming from an expansion of CAG trinucleotide repeats within the HTT gene situated on chromosome 4. It is characterized by a progressive onset of motor, cognitive, and psychiatric impairments. Presently, there are no known methods to cure or halt the advancement of HD, although various medications have been utilized to manage the abnormal movements associated with the disease. This project aimed to assess the efficacy of cationic β-cyclodextrin -anti-HD siRNA (CD-siRNA) nanoparticles in reducing the expression of mutant Huntingtin protein (mHtt) within PC-12 cell lines engineered to express mutant HTT protein consisting of 73 polyglutamines.

Methods: Cationic β-cyclodextrin nanoparticles were synthesized through a one-step condensation polymerization process involving β-cyclodextrin (β-CD), epichlorohydrin (EP), and choline chloride (CC) at molar ratios of (1/5/2), (1/15/1), and (1/15/2) (Fig 1). We isolated the unmodified β-CD monomers via dialysis over 24 hours. The β-CD nanoparticles were characterized for their surface charge, particle size, percent yield, and structural polymeric substitution through proton nuclear magnetic resonance (1H NMR). Complex formation between CD and siRNA was confirmed through gel retardation assays utilizing siRNA-HDex1 (5’ GCCUUCGAGUCCCUCAAGUCC-3’). Cell viability studies based on MTT assays were conducted with naked siRNA, blank CD nanoparticles, and β-CD-siRNA complexes using PC-12 cells, wherein mutant Huntingtin (mHtt) formation was chemically induced. The expression of Huntingtin protein was evaluated through western blot analysis both before and after cell treatment with naked siRNA and CD-siRNA formulations.

Results: In our comprehensive assessment, the molar ratio of 1:5:2 yielded notably smaller particle dimensions (147 nm) following sonication for two minutes, contrasting with the molar ratios of 1:15:1 (239 nm) and 1:15:2 (315 nm), which did not exhibit commensurate reductions with extended sonication durations (Fig 2). Consequently, the 1:5:2 ratio was selected for further investigation. Subsequent to the formation of CD-siRNA complexes, the average surface charge of the synthesized nanoparticles underwent reduction to +15.2 mV. Proton nuclear magnetic resonance (1H NMR) spectra conclusively affirmed the presence of functional polymeric end groups and the successful introduction of cationic moieties into the β-CD backbone. Upon normalization against the media control, data from the MTT assay revealed comparable or superior cell viability outcomes for free siRNA, drug-free CD nanoparticles, and CD-siRNA nanoparticle complexes, thus corroborating the non-toxic nature of the formulations under scrutiny. Lastly, Western blot analysis underscored the efficacy of the synthesized β-CD-siRNA nanoparticle complexes in effecting a substantial knockdown of huntingtin protein expression subsequent to 72-hour treatment (Fig 3).

Conclusion: The successful formulation of β-CD-siRNA nanoparticle complexes represents a significant achievement, demonstrating their capacity to effectively modulate the expression levels of mutant huntingtin (mHTT) protein within PC-12 cells. Comparative analysis revealed a notable reduction in mHTT protein levels when utilizing β-CD-siRNA nanoparticle complexes in contrast to the application of naked siRNA alone. This outcome underscores the potential therapeutic utility of these complexes in attenuating the expression of mHTT protein, thus offering promise for the management and treatment of Huntington's Disease.

References: Mantha N, Das SK, Das NG. RNAi-based therapies for Huntington’s disease: delivery challenges and opportunities. Therapeutic delivery. 2012 Sep;3(9):1061-76.
Sampaio C. Huntington disease–Update on ongoing therapeutic developments and a look toward the future. Parkinsonism & Related Disorders. 2024 Feb 15:106049.


Fig 1: Synthesis of cationic β-cyclodextrin nanoparticles


Fig 2: Particle size of synthesized β-cyclodextrin nanoparticles


Fig 3: Silencing of mHtt expression following treatment with free siRNA, and CD-siRNA