Gene Therapy最新文献

{"title":"Dystrophin/mini-dystrophin expression analysis by immunoaffinity liquid chromatography-tandem mass spectrometry after gene therapy for DMD.","authors":"Jason Walsh, Joe Palandra, Nicole Duriga, David Beidler, Avery McIntosh, Michael Binks, Hendrik Neubert","doi":"10.1038/s41434-025-00554-5","DOIUrl":"https://doi.org/10.1038/s41434-025-00554-5","url":null,"abstract":"<p><p>Adeno-associated virus (AAV)-based gene replacement therapies in Duchenne muscular dystrophy (DMD) aim to restore dystrophin function via the introduction of micro- or mini-dystrophins. We report dystrophin and mini-dystrophin concentrations generated by immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) in skeletal muscle biopsies from ambulatory participants with DMD in a phase 1b study of fordadistrogene movaparvovec, an AAV9-based gene replacement construct. The assay performed robustly for 26 months, as demonstrated by limited variability in calibration standards for peptides LLQV (dystrophin and mini-dystrophin) and LEMP (mini-dystrophin only), quality control samples consisting of spiked mini-dystrophin in DMD skeletal muscle lysate, as well as unspiked, pooled, non-dystrophic skeletal muscle lysate (normal pool). Average values for LLQV in the normal pool tested as part of clinical sample and long-term stability runs were similar to validated values. Biopsy samples showed minor or absent LLQV and absent LEMP signals pre-treatment with fordadistrogene movaparvovec infusion, but signals substantially increased at Days 60 and 360, on average. There was strong concordance in LEMP and LLQV expression change between Days 60 and 360 (R² = 0.91; p < 0.001). IA-LC-MS/MS enables reproducible, stable, and reliable quantification of dystrophin/mini-dystrophin following fordadistrogene movaparvovec infusion. ClinicalTrials.gov identifier: NCT03362502.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.5,"publicationDate":"2025-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768611","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gene editing for Spinocerebellar ataxia type 3 taking advantage of the human ATXN3L paralog as replacement gene.","authors":"Margareta Rybarikova, Maria Rey, Ed Hasanovic, Mélanie Sipion, Lukas Rambousek, Nicole Déglon","doi":"10.1038/s41434-025-00557-2","DOIUrl":"10.1038/s41434-025-00557-2","url":null,"abstract":"<p><p>Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.5,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144729853","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AAV9-mediated transduction of memory circuits following convection-enhanced delivery into the olfactory bulbs.","authors":"Theodore Dimitrov, Vikas Munjal, Allison O'Brien, Matthew T Rocco, Ahmad Karkhah, Kaya E Ceyhan, Daniel Prevedello, Lluis Samaranch","doi":"10.1038/s41434-025-00555-4","DOIUrl":"10.1038/s41434-025-00555-4","url":null,"abstract":"<p><p>This study explores the potential of adeno-associated virus serotype 9 (AAV9) to deliver therapeutic genes directly into the memory circuit throughout the olfactory bulb (OB), a critical memory and sensory processing region. Using convection-enhanced delivery (CED) of AAV9 encoding green fluorescent protein (GFP), we mapped the extensive neural connectivity from the OB to key memory-related brain regions, including the entorhinal cortex (EC) and hippocampus. Our findings reveal significant transduction of neural pathways and underscore the potential of targeting the OB connectome for therapeutic interventions in progressive neurodegenerative disorders such as Alzheimer's disease or mild cognitive impairment. Targeting the OB connectome will pave the way for new therapeutic strategies to preserve neuronal function and slow the progression, offering a promising avenue beyond symptomatic relief to address the underlying mechanisms of the disease.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.5,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144717822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput evaluation of cardiac-specific promoters for adeno-associated virus mediated cardiac gene therapy.","authors":"Dhanya Ravindran, Renuka Rao, Juan Mundisugih, Tracy Titus, Shinya Tsurusaki, Cindy Y Kok, Fairooj N Rashid, Sindhu Igoor, Yasuhito Kotake, Saurabh Kumar, James J H Chong, Ian E Alexander, Leszek Lisowski, Eddy Kizana","doi":"10.1038/s41434-025-00553-6","DOIUrl":"https://doi.org/10.1038/s41434-025-00553-6","url":null,"abstract":"<p><p>The selection of an appropriate promoter is important to the design and optimisation of adeno-associated viral (AAV) vector-based cardiac gene therapies. The expression cassette design can impact efficacy and safety of the vector. This study is the first to use a novel AAV barcode-seq method for the simultaneous evaluation of a panel of cardiac-specific promoters in a high-throughput manner. Functional analyses of our cardiac promoter kit packaged in three different capsids were performed using neonatal rat ventricular myocytes (NRVM), human iPSC-derived cardiomyocytes (hiPSC-CMs), HuH7 hepatocellular carcinoma cells, as well as mouse, rat, sheep and pig models. The cardiac troponin T (cTnT) promoter showed the most promise overall as a cardiac-specific promoter across all cardiac models tested. The results validate the barcode-seq technique as a powerful and versatile approach that enables high-throughput, quantitative analysis of various expression cassettes in commonly used models of cardiac gene therapy.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"^hInGeTox: a human-based in vitro platform to evaluate lentivirus/host interactions that contribute to genotoxicity.","authors":"Saqlain Suleman, Sharmin Alhaque, Andrew Guo, Huairen Zhang, Annette Payne, Marco Zahn, Serena Fawaz, Mohammad S Khalifa, Susan Jobling, David Hay, Matteo Franco, Raffaele Fronza, Wei Wang, Olga Strobel-Freidekind, Annette Deichmann, Yasuhiro Takeuchi, Irene Gil-Farina, Jan Klapwijk, Stefany Perera, Manfred Schmidt, Michael Themis","doi":"10.1038/s41434-025-00550-9","DOIUrl":"https://doi.org/10.1038/s41434-025-00550-9","url":null,"abstract":"<p><p>Lentivirus vectors are effective for treatment of genetic disease. However, safety associated with vector related genotoxicity is of concern and currently available models are not reliably predictive of safety in humans. We have developed ^hInGeTox as the first human in vitro platform that uses induced pluripotent stem cells and their hepatocyte like cell derivatives to better understand vector-host interactions that relate vectors to their potential genotoxicity. Using lentiviral vectors carrying the eGFP expression cassette under SFFV promoter activity, that only differ by their LTR and SIN configuration, we characterised vector host interactions potentially implicated in genotoxicity. To do this, lentiviral infected cells were subjected to an array of assays and data from these was used for multi-omics analyses of vector effects on cells at early and late harvest time points. Data on the integration sites of lentiviral vectors in cancer genes and differential expression levels of these genes, showed that both vector configurations are capable of activating cancer genes. Through IS tracking in bulk infected cell populations, we also saw an increase in the viral sequence count in cancer genes present over time which were differentially regulated. RNASeq also showed each vector had potential to generate fusion transcripts with the human genome suggestive of gene splicing or vector mediated readthrough from the internal SFFV promoter. Initially, after infection, both vector configurations were associated with differential expression of genes associated cytokine production, however, after culturing over time there were differences in differential expression in cells infected by each LV. This was marked in particular by the expression of genes involved in the response to DNA damage in cells transduced by the SIN vector, suggesting effects likely to prevent tumour development, in contrast to the expression of genes involved in methylation, characteristic of tumour development, in cells transduced by the LTR vector. Both sets of lentiviral infected cells were also found associated with differential expression of MECOM and LMO2 genes known to be associated with clonal dominance, supporting their potential genotoxicity. Alignment of transcriptomic signatures from iPSC and HLC infected cultures with known cancer gene signatures showed the LTR vector with a higher cancer score than the SIN vector over time in iPSC and also in HLC, which further suggests higher genotoxic potential by the LTR configuration lentivirus. By application of ^hInGeTox to cells infected with LV at the pre-clinical stage of development, we hope that ^hInGeTox can act as a useful pre-clinical tool to identify lentivirus-host interactions that may be considered contributory to genotoxicity to improve safer lentiviral vector design for gene therapy.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144642457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transabdominal ultrasound guided AAV9-GFP delivery in fetal pigs: a translational and minimally invasive model for in utero fetal gene therapy.","authors":"Alessia Di Donfrancesco, Alessia Adelizzi, Anastasia Giri, Roberto Duchi, Simona Boito, Maria Barandalla, Giulia Massaro, Chiara Santanatoglia, Enrica Cappellozza, Andrea Perota, Ivano Di Meo, Valeria Tiranti, Emanuela Bottani, Cesare Galli, Nicola Persico, Dario Brunetti","doi":"10.1038/s41434-025-00551-8","DOIUrl":"https://doi.org/10.1038/s41434-025-00551-8","url":null,"abstract":"<p><p>In utero fetal gene therapy (IUFGT) has the potential to correct severe monogenic disorders before irreversible damage occurs. Despite promising results in small and large animal models, its translation to clinical practice remains limited by technical challenges, safety concerns, and the lack of standardized protocols in relevant disease models species. We established and validated a minimally invasive, ultrasound-guided approach for systemic gene delivery in fetal pigs using a self-complementary AAV9 vector encoding GFP under a CAG promoter. Injections were performed at different gestational ages (GA 80 and GA 108) via intracardiac or umbilical venous routes. Postnatal outcomes were monitored, and transgene biodistribution and expression were assessed by qPCR, ddPCR, immunofluorescence, and Western blotting. Inflammatory response, toxicity, and maternal safety were evaluated through cytokine profiling and histological analyses. The procedure was well tolerated, with no significant maternal morbidity or adverse obstetric outcomes beyond one preterm delivery. Biodistribution analysis revealed widespread vector presence in peripheral tissues, with robust GFP expression in liver and heart. Importantly, there was no evidence of significant tissue toxicity, necrosis, or fibrosis in any of the organs analyzed. Mild increases in pro-inflammatory cytokines (GM-CSF, GRO-α, IFN-γ) were observed but were not associated with histopathological changes. No anti-AAV9 capsid antibodies were detected in sera from piglets or sows, suggesting a minimal immune response to the vector. These findings demonstrate the safety, feasibility, and efficacy of ultrasound-guided IUFGT in pigs, supporting its potential as a translational platform for therapeutic gene delivery in fetuses affected by severe congenital diseases. This model offers a valuable framework for further preclinical development of prenatal interventions, particularly for disorders with early onset, such as mitochondrial diseases.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144608219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanoparticle hydrogel system delivery of miR-494-3p to improve tendon healing by targeting CXXC4.","authors":"Gu Heng Wang, Lei Wang, Lei Sheng, Hua Jian Shan, Wei Gang Zhu, Ya Lan Chen, Ai Dong Deng, Jun Tan, Xiao Zhong Zhou","doi":"10.1038/s41434-025-00543-8","DOIUrl":"10.1038/s41434-025-00543-8","url":null,"abstract":"<p><p>Due to the poor healing capacity of tendons, the healing process is slow, with a risk of re-rupture post-injury. In this study, we found that miR-494-3p was one of the miRNAs with significant expression differences after tendon injury by sequencing in the rat Achilles tendon injury model. Therefore, we hypothesized that regulating miR-494-3p expression in tendons could improve tendon healing. Considering the long healing process of the tendons and the short half-life of miRNA, we hope to achieve the best efficacy by delivering miR-494-3p using a sustained-release nanoparticle hydrogel system. In the results, with an increase in miR-494-3p, the tendon biomechanics were significantly improved after 2-week repair, and the content of collagen I (Col I) also increased. Through bioinformatics prediction, double luciferase, and immunohistochemistry experiments, we confirmed that miR-494-3p targeting CXXC finger protein 4 (CXXC4) promoted tendon healing. In conclusion, the miR-494-3p/nanoparticles hydrogel delivery system can protect and sustainedly transfer miR-494-3p into tenocytes, block the translation of CXXC4, increase the expression of Col I, and ultimately improve tendon healing. A nanoparticle hydrogel delivery system of miRNA was constructed and applied to injured tendons. Finally, we confirmed that the miR-494-3p/nanoparticles hydrogel delivery system can protect and sustainedly transfer miR-494-3p into tenocytes, block the translation of CXXC4, increase the expression of Col I, and ultimately improve tendon healing.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"hafoe: an interactive tool for the analysis of chimeric AAV libraries after random mutagenesis.","authors":"Tatevik Jalatyan, Erik Aznauryan, Rokib Hasan, Valeri Vardanyan, Stepan Nersisyan, David B Thompson, Noah Davidsohn, Sanya Thomas, Simon van Haren, Jenny Tam, Denitsa Milanova, George M Church, Lilit Nersisyan","doi":"10.1038/s41434-025-00548-3","DOIUrl":"https://doi.org/10.1038/s41434-025-00548-3","url":null,"abstract":"<p><p>Naturally occurring adeno-associated viruses (AAVs) are an integral part of gene therapy, yet engineering novel AAV variants is necessary to expand targetable tissues and treatable diseases. Directed evolution, particularly through DNA shuffling of the capsid genes of wild-type AAV serotypes, is a widely employed strategy to generate novel chimeric variants with desired properties. Yet, the computational analysis of such chimeric sequences presents challenges. We introduce hafoe, a novel computational tool designed for the exploratory analysis of chimeric AAV libraries, which does not require extensive bioinformatics expertise. hafoe accurately deciphers the serotype composition and enrichment patterns of chimeric AAV variants across different tissues. Validation against synthetic datasets demonstrates that hafoe identifies parental serotype compositions with an accuracy of 96.3% to 97.5%. Additionally, we engineered chimeric AAV capsid libraries and screened novel AAV variants for tropism to human dermal fibroblasts and dendritic cells, as well as canine muscle, and liver tissues. Using hafoe we identified and characterized enriched AAV variants in these tissues for potential use in gene therapy and vaccine development. Overall, hafoe can provide valuable insights that may further support the rational design of AAV vectors based on parental serotype and sequence preferences of the capsid genes in target tissues.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systemically delivered lipid nanoparticle-mRNA encoding lysosomal acid β-glucosidase restores the enzyme deficiency in a murine Gaucher disease model.","authors":"Yuanqing Liu, Shasha Wang, Yanni Chen, Zhang Zhang, Xiaojiang Quan, Zhijun Guo, Zihao Wang","doi":"10.1038/s41434-025-00549-2","DOIUrl":"https://doi.org/10.1038/s41434-025-00549-2","url":null,"abstract":"<p><p>Gaucher disease (GD) is a rare genetically inherited illness caused by loss of lysosomal acid β-glucosidase (β-GCase) that leads to progressive accumulation of substrates, sphingolipid glucosylceramide (GL1) and glucosylsphingosine (lyso-GL1). The protein-based enzyme replacement therapy (ERT) requires frequent dosing due to short drug half-life causing challenges in long-term patient compliance. JCXH-301 is a lipid nanoparticle (LNP) encapsulated messenger RNA (mRNA) encoding β-GCase. Intravenous administration of JCXH-301 delivered the target mRNA to various tissues in mice with intracellular expression of β-GCase predominantly in macrophages and dendritic cells in the spleen and bone marrow. In GBA1 D427V homozygous mice treated with JCXH-301, the dose-dependent in vivo production of functional β-GCase resulted in reduction of serum lyso-GL1, a key biomarker of GD. The therapeutic effect of JCXH-301 was sustained for a duration significantly longer than that of protein-based ERT Cerezyme. JCXH-301 administration induced minimal pro-inflammatory cytokines in the liver and spleen. Taken together, these results provide proof-of-concept for using LNP-delivered mRNA as a new drug modality to restore the β-GCase genetic deficiency for GD treatment.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144559972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of an AAV-RNAi strategy to silence the dominant variant GNAO1 c.607G>A linked to encephalopathy.","authors":"Evgenii A Lunev, Natalia V Klementieva, Svetlana G Vassilieva, Egor A Volovikov, David Jappy, Irina M Savchenko, Ekaterina A Svetlova, Anna V Polikarpova, Maria Y Shubina, Danil M Spirin, Ksenia S Anufrieva, Petr R Lebedev, Vladimir M Pokrovsky, Marina V Utkina, Viktoriya G Krut', Mikhail Sintsov, Sergey Popov, Alexey V Deykin, Andrei Rozov, Tatiana V Egorova, Maryana V Bardina","doi":"10.1038/s41434-025-00532-x","DOIUrl":"10.1038/s41434-025-00532-x","url":null,"abstract":"<p><p>Heterozygous mutations in GNAO1 cause an ultra-rare neurodevelopmental disease called GNAO1 encephalopathy, characterized by infantile epilepsy and movement disorder. Here, we provide a functional characterization of the hotspot mutation GNAO1 c.607G>A (p.G203R) and conduct early-phase development of an adeno-associated virus (AAV)-mediated gene therapy approach. The GNAO1 gene encodes the Gαo protein that is involved in neuronal signaling. We showed that the Gαo-G203R lost its ability to enhance forskolin-stimulated cAMP synthesis in HEK293T cells. In primary neuronal culture, Gαo-G203R had a dominant-negative effect on neuronal activity and GABA_B-dependent synaptic release. To ablate the mutant protein, we used selective silencing of the pathogenic variant using effectors of RNA interference (RNAi). We selected the short hairpin RNA (sh1500) that suppressed the c.607G>A transcripts, resulting in a 3.8-fold increase in the ratio of wild-type to mutant GNAO1 transcripts in patient-specific neurons. We also detected off-target effects of sh1500 as well as transcriptome changes associated with AAV transduction and RNAi activation. We improved the AAV construct by using an artificial miRNA (miR1500) and the neuron-specific hSyn promoter. Systemic administration of AAV9-hSyn-miR1500 did not cause pathological changes in Gnao1-GGA mice with a \"humanized\" target sequence. Importantly, AAV9 transduced Gαo-positive neurons in the striatum, thalamus, substantia nigra, and cerebellum, which we defined as primary targets for gene therapy. Our findings pave the road toward the development of AAV-RNAi approaches for dominant-negative GNAO1 variants.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":"430-445"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143976401","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}