In Vitro Efficacy of PEI-Derived Lipopolymers in Silencing of Toxic Proteins in a Neuronal Model of Huntington’s Disease

<b>Background:</b> Huntington's Disease (HD) is a neurodegenerative disorder caused by an abnormal extension of a CAG repeat stretch located in the exon 1 of the <i>HTT</i> (IT15) gene, leading to production of a mutated and misfolded Huntingtin protein (muHTT) with an abnormally elongated polyglutamine (polyQ) region. This mutation causes muHTT to oligomerize and aggregate in the brain, particularly in the striatum and cortex, causing alterations in intracellular trafficking, caspase activation, and ganglioside metabolism, ultimately leading to neuronal damage and death and causing signs and symptoms such as chorea and cognitive dysfunction. Currently, there is no available cure for HD patients; hence, there is a strong need to look for effective therapies. <b>Methods:</b> This study aims to investigate the efficacy of siRNA-containing nano-engineered lipopolymers in selectively silencing the <i>HTT</i> expression in a neuronal model expressing a chimeric protein formed by the human mutated exon 1 of the <i>HTT</i> gene, tagged with GFP. Toxicity of lipopolymers was assessed using MTT assay, while efficacy of silencing was monitored using qRT-PCR, as well as Western blotting/flow cytometry. Changes in muHTT-GFP aggregation were observed using fluorescence microscopy and image analyses. <b>Results:</b> Here, we show that engineered lipopolymers can be used as delivery vehicles for specific siRNAs, decreasing the transcription of the mutated gene, as well as the muHTT protein production and aggregation, with Leu-Fect C being the most effective candidate amongst the assessed lipopolymers. <b>Conclusions:</b> Our findings have profound implications for genetic disorder therapies, highlighting the potential of nano-engineered materials for silencing mutant genes and facilitating molecular transfection across cellular barriers. This successful in vitro study paves the way for future in vivo investigations with preclinical models, offering hope for previously considered incurable diseases such as HD.