{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T15:36:33Z","timestamp":1787240193158,"version":"build-2736575974"},"reference-count":51,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T00:00:00Z","timestamp":1748563200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Natural Sciences and Engineering Council of Canada (NSERC)"},{"name":"New Frontiers Research Fund (NFRF)"},{"name":"Canadian Institutes of Health Research (CIHR)"},{"name":"Cancer Research Institute of Northern Alberta (CRINA)"},{"name":"RJH Biosciences Inc."},{"name":"Edmonton Civic Employees-Charitable Assistance Fund (ECE-CAF)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: Huntington\u2019s Disease (HD) is a neurodegenerative disorder caused by an abnormal extension of a CAG repeat stretch located in the exon 1 of the HTT (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. Methods: This study aims to investigate the efficacy of siRNA-containing nano-engineered lipopolymers in selectively silencing the HTT expression in a neuronal model expressing a chimeric protein formed by the human mutated exon 1 of the HTT 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. Results: 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. Conclusions: 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.<\/jats:p>","DOI":"10.3390\/pharmaceutics17060726","type":"journal-article","created":{"date-parts":[[2025,6,2]],"date-time":"2025-06-02T12:27:09Z","timestamp":1748867229000},"page":"726","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["In Vitro Efficacy of PEI-Derived Lipopolymers in Silencing of Toxic Proteins in a Neuronal Model of Huntington\u2019s Disease"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4194-2961","authenticated-orcid":false,"given":"Luis C.","family":"Morales","sequence":"first","affiliation":[{"name":"Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada"},{"name":"Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5173-9912","authenticated-orcid":false,"given":"Luv","family":"Modi","sequence":"additional","affiliation":[{"name":"Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4960-5089","authenticated-orcid":false,"given":"Saba","family":"Abbasi Dezfouli","sequence":"additional","affiliation":[{"name":"Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amarnath Praphakar","family":"Rajendran","sequence":"additional","affiliation":[{"name":"Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada"},{"name":"Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3554-7247","authenticated-orcid":false,"given":"Remant","family":"Kc","sequence":"additional","affiliation":[{"name":"Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada"},{"name":"Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vaibhavi","family":"Kadam","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2R3, Canada"},{"name":"Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simonetta","family":"Sipione","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2R3, Canada"},{"name":"Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1118-4170","authenticated-orcid":false,"given":"Hasan","family":"Uluda\u011f","sequence":"additional","affiliation":[{"name":"Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada"},{"name":"Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2R3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,30]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"A review of cognition in Huntington\u2019s disease","volume":"5","author":"Dumas","year":"2013","journal-title":"Front. Biosci. Sch. Ed."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1097\/WCO.0000000000000116","article-title":"An update on Huntington\u2019s disease: From the gene to the clinic","volume":"27","author":"Kim","year":"2014","journal-title":"Curr. Opin. Neurol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1016\/S1474-4422(09)70170-X","article-title":"Biological and clinical manifestations of Huntington\u2019s disease in the longitudinal TRACK-HD study: Cross-sectional analysis of baseline data","volume":"8","author":"Tabrizi","year":"2009","journal-title":"Lancet Neurol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/978-3-642-45106-5_14","article-title":"Huntington\u2019s disease","volume":"Volume 220","author":"Zuccato","year":"2014","journal-title":"Handbook of Experimental Pharmacology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1016\/0092-8674(93)90585-E","article-title":"A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington\u2019s disease chromosomes","volume":"72","author":"MacDonald","year":"1993","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"754","DOI":"10.1038\/aps.2018.11","article-title":"Proteostasis in Huntington\u2019s disease: Disease mechanisms and therapeutic opportunities","volume":"39","author":"Harding","year":"2018","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/S1474-4422(10)70276-3","article-title":"Biological and clinical changes in premanifest and early stage Huntington\u2019s disease in the TRACK-HD study: The 12-month longitudinal analysis","volume":"10","author":"Tabrizi","year":"2011","journal-title":"Lancet Neurol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1953","DOI":"10.1093\/hmg\/11.17.1953","article-title":"Early transcriptional profiles in huntingtin-inducible striatal cells by microarray analyses","volume":"11","author":"Sipione","year":"2002","journal-title":"Hum. Mol. Genet."},{"key":"ref_9","first-page":"245","article-title":"Modeling brain pathologies using neural stem cells","volume":"198","author":"Sipione","year":"2002","journal-title":"Methods Mol. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1016\/S1474-4422(22)00121-1","article-title":"Potential disease-modifying therapies for Huntington\u2019s disease: Lessons learned and future opportunities","volume":"21","author":"Tabrizi","year":"2022","journal-title":"Lancet Neurol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2298","DOI":"10.1093\/brain\/awac458","article-title":"AAV5-miHTT-mediated huntingtin lowering improves brain health in a Huntington\u2019s disease mouse model","volume":"146","author":"Thomson","year":"2023","journal-title":"Brain J. Neurol."},{"key":"ref_12","first-page":"36","article-title":"Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease","volume":"48","author":"Caron","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4043","DOI":"10.1093\/brain\/awae266","article-title":"Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington\u2019s disease models","volume":"147","author":"Landles","year":"2024","journal-title":"Brain"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3421","DOI":"10.1093\/brain\/awab354","article-title":"Therapeutic reversal of Huntington\u2019s disease by in vivo self-assembled siRNAs","volume":"144","author":"Zhang","year":"2021","journal-title":"Brain J. Neurol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1038\/d41573-021-00088-6","article-title":"Double setback for ASO trials in Huntington disease","volume":"20","author":"Kingwell","year":"2021","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Attia, N., Mashal, M., Puras, G., and Pedraz, J.L. (2021). Mesenchymal Stem Cells as a Gene Delivery Tool: Promise, Problems, and Prospects. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13060843"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3059","DOI":"10.1021\/acs.biochem.4c00327","article-title":"Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases","volume":"63","author":"Dezfouli","year":"2024","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1021\/bm034041+","article-title":"Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH","volume":"5","author":"Funhoff","year":"2004","journal-title":"Biomacromolecules"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1002\/mabi.201000402","article-title":"Impact of lipid substitution on assembly and delivery of siRNA by cationic polymers","volume":"11","author":"Aliabadi","year":"2011","journal-title":"Macromol. Biosci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3972","DOI":"10.1039\/C4TB02101K","article-title":"Additive nanocomplexes of cationic lipopolymers for improved non-viral gene delivery to mesenchymal stem cells","volume":"3","author":"Kucharski","year":"2015","journal-title":"J. Mater. Chem. B"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Dezfouli, S.A., Rajendran, A.P., Claerhout, J., and Uludag, H. (2023). Designing Nanomedicines for Breast Cancer Therapy. Biomolecules, 13.","DOI":"10.3390\/biom13101559"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"11404","DOI":"10.1073\/pnas.96.20.11404","article-title":"Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells","volume":"96","author":"Kazantsev","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"17204","DOI":"10.1073\/pnas.0708285104","article-title":"Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits","volume":"104","author":"DiFiglia","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1016\/j.biomaterials.2013.09.068","article-title":"Differential nanotoxicological and neuroinflammatory liabilities of non-viral vectors for RNA interference in the central nervous system","volume":"35","author":"Godinho","year":"2014","journal-title":"Biomaterials"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1651","DOI":"10.1038\/sj.cdd.4401853","article-title":"Interaction of F1L with the BH3 domain of Bak is responsible for inhibiting vaccinia-induced apoptosis","volume":"13","author":"Postigo","year":"2006","journal-title":"Cell Death Differ."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.ejpb.2018.06.018","article-title":"siRNA\/lipopolymer nanoparticles to arrest growth of chronic myeloid leukemia cells in vitro and in vivo","volume":"130","author":"Aliabadi","year":"2018","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wang, H., Liu, X., Ai, X., Remant-Bahadur, K.C., Dick, T.A., Yan, B., Lu, T., Zhou, X., Luo, R., and Liu, M. (2023). Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15020410"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Yotsomnuk, P., Rajendran, A.P., Sundaram, D.N.M., Morales, L.C., Kucharski, C., Nasrullah, M., Skolpap, W., Jiang, X., Gibson, S.B., and Brandwein, J. (2025). Lipopolymers as the Basis of Non-Viral Delivery of Therapeutic siRNA Nanoparticles in a Leukemia (MOLM-13) Model. Biomolecules, 15.","DOI":"10.3390\/biom15010115"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2840","DOI":"10.1021\/acsptsci.4c00336","article-title":"Lipopolymer\/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia","volume":"7","author":"Nasrullah","year":"2024","journal-title":"ACS Pharmacol. Transl. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1436","DOI":"10.1021\/acs.molpharmaceut.3c01077","article-title":"Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems","volume":"21","author":"Morales","year":"2024","journal-title":"Mol. Pharm."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1016\/j.jconrel.2024.02.018","article-title":"Lipopolymer mediated siRNA delivery targeting aberrant oncogenes for effective therapy of myeloid leukemia in preclinical animal models","volume":"367","author":"Ansari","year":"2024","journal-title":"J. Control. Release"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1007\/s10637-022-01282-y","article-title":"Improved delivery of Mcl-1 and survivin siRNA combination in breast cancer cells with additive siRNA complexes","volume":"40","author":"Santadkha","year":"2022","journal-title":"Investig. New Drugs"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"11704","DOI":"10.1128\/JVI.00931-09","article-title":"Nucleosome assembly proteins bind to Epstein-Barr virus nuclear antigen 1 and affect its functions in DNA replication and transcriptional activation","volume":"83","author":"Wang","year":"2009","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.toxlet.2010.12.010","article-title":"Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis","volume":"201","author":"Piao","year":"2011","journal-title":"Toxicol. Lett."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2069","DOI":"10.1093\/hmg\/2.12.2069","article-title":"Characterization and localization of the Huntington disease gene product","volume":"2","author":"Hoogeveen","year":"1993","journal-title":"Hum. Mol. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1002\/mds.870090609","article-title":"Cortical myoclonus in Huntington\u2019s disease","volume":"9","author":"Thompson","year":"1994","journal-title":"Mov. Disord."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1016\/S0092-8674(00)81369-0","article-title":"Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice","volume":"87","author":"Mangiarini","year":"1996","journal-title":"Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1002\/cne.10295","article-title":"Cellular localization and development of neuronal intranuclear inclusions in striatal and cortical neurons in R6\/2 transgenic mice","volume":"449","author":"Meade","year":"2002","journal-title":"J. Comp. Neurol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1046\/j.1471-4159.2003.02009.x","article-title":"Inclusion formation in Huntington\u2019s disease R6\/2 mouse muscle cultures","volume":"87","author":"Orth","year":"2003","journal-title":"J. Neurochem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3248","DOI":"10.1523\/JNEUROSCI.19-08-03248.1999","article-title":"Characterization of progressive motor deficits in mice transgenic for the human Huntington\u2019s disease mutation","volume":"19","author":"Carter","year":"1999","journal-title":"J. Neurosci. Off. J. Soc. Neurosci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.actbio.2022.06.030","article-title":"In Vitro Cytotoxicity and Cytokine Production by Lipid-Substituted Low Molecular Weight Branched PEIs Used for Gene Delivery","volume":"148","author":"Sundaram","year":"2022","journal-title":"Acta Biomater."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"6099","DOI":"10.1093\/nar\/gkae368","article-title":"A programmable dual-targeting siRNA scaffold supports potent two-gene modulation in the central nervous system","volume":"52","author":"Belgrad","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Mathews, E.W., Coffey, S.R., G\u00e4rtner, A., Belgrad, J., Bragg, R.M., O\u2019Reilly, D., Cantle, J.P., McHugh, C., Summers, A., and Fentz, J. (2024). Suppression of Huntington\u2019s Disease Somatic Instability by Transcriptional Repression and Direct CAG Repeat Binding. BioRxiv.","DOI":"10.1101\/2024.11.04.619693"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.omtn.2021.02.007","article-title":"Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS","volume":"24","author":"Hirunagi","year":"2021","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"670543","DOI":"10.3389\/fnano.2021.670543","article-title":"Suppression of Human Coronavirus 229E Infection in Lung Fibroblast Cells via RNA Interference","volume":"3","author":"Aliabadi","year":"2021","journal-title":"Front. Nanotechnol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.4155\/tde.12.80","article-title":"RNAi-based therapies for Huntington\u2019s disease: Delivery challenges and opportunities","volume":"3","author":"Mantha","year":"2012","journal-title":"Ther. Deliv."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1892","DOI":"10.1073\/pnas.0308698100","article-title":"Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells","volume":"101","author":"Scacheri","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1188","DOI":"10.1261\/rna.28106","article-title":"Off-target effects by siRNA can induce toxic phenotype","volume":"12","author":"Fedorov","year":"2006","journal-title":"RNA"},{"key":"ref_49","first-page":"487","article-title":"Mutant huntingtin reduction in astrocytes slows disease progression in the BACHD conditional Huntington\u2019s disease mouse model","volume":"28","author":"Wood","year":"2019","journal-title":"Hum. Mol. Genet."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1016\/j.tibtech.2018.01.006","article-title":"Nanotechnology-Based Strategies for siRNA Brain Delivery for Disease Therapy","volume":"36","author":"Zheng","year":"2018","journal-title":"Trends Biotechnol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1016\/j.jconrel.2012.08.012","article-title":"Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression","volume":"163","author":"Burgess","year":"2012","journal-title":"J. Control. 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