Gene Therapy最新文献

{"title":"Nitrous oxide enhances MR-guided focused ultrasound delivery of gene therapy to the murine hippocampus","authors":"Deepshikha Bhardwaj, Ibrahim Youssef, Darren Imphean, Sydni K. Holmes, Venugopal Krishnan, Sandi Jo Estill-Terpack, Marc Diamond, Rajiv Chopra, Rachel M. Bailey, Bhavya R. Shah","doi":"10.1038/s41434-025-00530-z","DOIUrl":"10.1038/s41434-025-00530-z","url":null,"abstract":"Transcranial Magnetic Resonance Guided Focused Ultrasound can oscillate intravenously delivered microbubbles and transiently open the blood brain barrier (BBB) in a targeted brain region. However, high microbubble doses or Focused ultrasound pressures (FUS) leads to injury. So, we administered nitrous oxide (N2O), an anesthetic gas to determine reduced need of FUS pressure and microbubble dose for opening BBB. Swiss Webster mice were treated with N2O or medical air (MA) at varying FUS pressures, while the microbubble dose was kept constant and the vice-versa. Consequently, BBB opening was quantified by acoustic emissions and enhancement rate on T1-weighted MR. To compare the effect of N2O on gene delivery, following BBB opening with either MA or N2O, a viral vector expressing GFP was subsequently delivered. Additionally, Immunohistochemical studies quantified viral transfection efficacy and assessed acute cell injury. We observed that N2O significantly potentiates acoustic emissions and enhancement rate on post-contrast MRI images, compared to MA at all measured pressures (0.39, 0.45, 0.67 MPa). Furthermore, N2O reduces the microbubble dose to 0.02μl/kg and FUS pressures to 0.28 and 0.39 MPa for BBB disruption and enhanced viral gene delivery, respectively. Hence, N2O potentiates microbubble oscillations, allowing reduced microbubble dose and FUS pressures and improved viral gene delivery.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"376-384"},"PeriodicalIF":4.5,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144007622","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":"Unveiling the sex bias: higher preexisting and neutralizing titers against AAV in females and implications for gene therapy","authors":"Stephanee Warrington, Trish T. Hoang, Morten Seirup, Leila Abdelhamid, Hrittal Saha, Sojin Bing, Sima Saleh, Je-Nie Phue, Ronit Mazor","doi":"10.1038/s41434-025-00528-7","DOIUrl":"10.1038/s41434-025-00528-7","url":null,"abstract":"Gene therapy with AAV vectors is a promising approach for treating numerous genetic disorders but is often hindered by preexisting antibodies that neutralize the vectors. Given that females may exhibit stronger immune responses than males, this study hypothesizes that females may have higher preexisting antibody titers against AAV. Serum samples from two U.S. cohorts were analyzed for antibody titers, antibody subtypes, and transduction inhibition activity against AAV serotypes AAV1, AAV2, AAV5, AAV8, and AAV9. We found that among seropositive samples, females had higher preexisting antibody levels and neutralizing activities against AAV9 and other serotypes. Immunoglobulin subclass analysis showed IgG1 dominance in both sexes, but females had higher IgA levels, whereas males had higher levels of IgG2. We further evaluated the cellular level of this differential immune response to AAV by stimulation of male and female human PBMCs. We observed dose-dependent increase in cytokines and chemokines in female PBMCs which suggests a differential inflammatory response. Altogether, our findings suggest that the enhanced immune response in females could lead to neutralization and faster clearance of AAV vectors with potential to impact the efficacy of gene therapy.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"339-348"},"PeriodicalIF":4.5,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144005557","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":"A novel multi-functional chimeric peptide for enhanced safe gene delivery in immunotherapy.","authors":"Mahdiyar Dehshiri, Shokouh Rezaei, Saman Hosseinkhani","doi":"10.1038/s41434-025-00538-5","DOIUrl":"https://doi.org/10.1038/s41434-025-00538-5","url":null,"abstract":"<p><p>Chimeric peptides hold promising potential to be introduced as an ideal gene delivery platform based on their advantages over viral carriers, including but not limited to the safety profile and specific targeting. However, their gene transfer efficiency needs improvement. Here, we designed a new multi-functional chimeric peptide for enhanced gene delivery by adding a cyclic TAT motif to a previously designed MPG2H peptide to enable the targeting of cells with independent/dependent endocytosis cell entry mechanisms. CTATMPG2H was expressed and purified using affinity chromatography; then it was characterized through a gel retardation assay, circular dichroism (CD) spectropolarimetry, transmission electron microscopy (TEM) dynamic light scattering (DLS), and zeta potential analysis. CTATMPG2H was compared with MiRGD as a chimeric peptide control in all steps. After assessing the platform stability in various conditions, its gene transfer efficiency was evaluated in the HEK293T cell line with reporter genes. Additionally, mouse bone marrow-derived dendritic cells (BMDCs) were transfected to test CTATMPG2H potential in immunotherapy. The results illustrated a safe gene transfer profile for CTATMPG2H comparable to MiRGD and Polyethyleneimine (PEI). Flow cytometry results showed up to 48% gene transfer rate for CTATMPG2H to dendritic cells with minimal toxicity (viability rate ~80%). Moreover, the in silico investigation showed that the synergistic effects of electrostatic, hydrogen, and hydrophobic interactions enhance the stability and binding affinity of peptide-pDNA complexes, ensuring robust and specific targeting of nucleic acids. This research sets a foundation for future in vivo studies and potential clinical applications, aiming for safer and more effective gene therapy strategies.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002900","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":"From bench to bedside: the future of stable lentiviral packaging cell lines in gene therapy.","authors":"Kumitaa Theva Das","doi":"10.1038/s41434-025-00537-6","DOIUrl":"https://doi.org/10.1038/s41434-025-00537-6","url":null,"abstract":"","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143975284","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":"Fatal outcomes following onasemnogene abeparvovec in advanced-stage spinal muscular atrophy.","authors":"Peerada Pongsakornkullachart, Pimchanok Kulsirichawaroj, Ratcharin Kongkasuwan, Prakarn Tovichien, Settapong Jitwongwai, Supaluck Kanjanauthai, Nutnicha Preeprem, Sivaporn Limpaninlachat, Nisasri Sermpon, Oranee Sanmaneechai","doi":"10.1038/s41434-025-00535-8","DOIUrl":"https://doi.org/10.1038/s41434-025-00535-8","url":null,"abstract":"<p><p>Supported by encouraging trial outcomes, onasemnogene abeparvovec (OA) was authorized for spinal muscular atrophy (SMA). Nevertheless, efficacy of OA in advanced SMA patients remains underexplored. This investigation assessed clinical effectiveness and adverse effects of OA in a cohort including advanced SMA, and compared to historical survival data for SMA type 1 patients in Thailand. We conducted observational cohort study at Siriraj Hospital, Thailand, from May 2019 to April 2022. The study enrolled eight SMA patients receiving OA therapy. The cohort comprised five SMA type 1 patients treated at 16.7 months (6.5-24.9 months) and three SMA type 2 patients treated at 20.3 months (19-31.5 months). Before receiving OA, all Type 1 patients required 24-hour invasive ventilation and feeding support. Post-treatment, Three of five showed gradual improvement in motor scores, but none achieved new motor milestones. Survival rate was not improved, with all experiencing fatalities. Conversely, Type 2 patients exhibited motor score improvement without serious adverse events. OA did not significantly improve clinical outcomes or survival rates in advanced Type 1 SMA. These findings highlight need for additional caution when administering OA to severe SMA Type 1 and more specific guidelines in selecting subgroups for treatment.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144011513","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":"Ex vivo machine perfusion as a platform for lentiviral gene delivery in rat livers","authors":"Irina Filz von Reiterdank,&nbsp;Mohammadreza Mojoudi,&nbsp;Raphaela Bento,&nbsp;McLean S. Taggart,&nbsp;Antonia T. Dinicu,&nbsp;Gregory Wojtkiewicz,&nbsp;J. H. Coert,&nbsp;Aebele B. Mink van der Molen,&nbsp;Ralph Weissleder,&nbsp;Biju Parekkadan,&nbsp;Korkut Uygun","doi":"10.1038/s41434-025-00536-7","DOIUrl":"10.1038/s41434-025-00536-7","url":null,"abstract":"Developing new strategies for local monitoring and delivery of immunosuppression is critical to making allografts safer and more accessible. Ex vivo genetic modification of grafts using machine perfusion presents a promising approach to improve graft function and modulate immune responses while minimizing risks of off-target effects and systemic immunogenicity in vivo. This proof-of-concept study demonstrates the feasibility of using normothermic machine perfusion (NMP) to mimic in vitro conditions for effective gene delivery. In this study, lentiviral vectors encoding the secreted biomarker Gaussia Luciferase (GLuc) and red fluorescent protein (RFP) were introduced ex vivo to rodent livers during a 72-h machine perfusion protocol. After an initial 24-h exposure to viral vectors, the organs were maintained in perfusion for an additional 48 h to monitor gene expression, aligning with in vitro benchmarks. Control livers were perfused in similar fashion, but without viral injections. Virally perfused livers exhibited nearly a 10-fold increase in luminescence compared to controls (p &lt; 0.0001), indicating successful genetic modification of the organs. These findings validate the use of machine perfusion systems and viral vectors to genetically engineer whole organs ex vivo, laying the groundwork for a broad range of applications in transplantation through genetic manipulation of organ systems. Future studies will focus on refining this technology to enhance precision in gene expression and explore its implications for clinical translation.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"421-429"},"PeriodicalIF":4.5,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143983866","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":"Unveiling molecular secrets: Analysis of stable lentiviral packaging cell lines enables identification of novel viral gene functions","authors":"Jona Röscheise,&nbsp;Maximilian Klimpel,&nbsp;Parameswari Govindarajan,&nbsp;Kerstin Otte,&nbsp;Holger Laux","doi":"10.1038/s41434-025-00533-w","DOIUrl":"10.1038/s41434-025-00533-w","url":null,"abstract":"Lentiviral vectors (LVVs) are widely used in gene therapy due to their ability to infect both dividing and non-dividing cells. For LVV production, the creation of stable packaging cell lines with integrated genes necessary for viral replication offer a more consistent and scalable alternative to transient plasmid transfection approach. Although the development of such stable LVV packaging cell lines has been reported, the molecular changes induced by stable and inducible viral gene expression and the impact of genome integrated viral genes on cellular pathways remain poorly characterized. For better insight, we investigated the molecular characteristics of a stable LVV packaging cell line and its host cell line (HEK293T/17) by comparing differential expressed genes. This pathway analysis revealed significant changes in pathway usage between packaging and host cell lines, influenced by different viral transgenes. Gag-pol expression was found to suppress host translational machinery, while rev and VSV-G expression modulated mitochondrial pathways, including oxidative phosphorylation. HIV-1 tat expression, on the other hand, activated histone-related genes. These regulatory shifts suggest a strategic reprogramming of host cellular states to favor viral replication, curbing protein synthesis and energy production to levels that support viral assembly but impair the host’s immune defense and the production of immune-related proteins. Our findings provide a deeper understanding of the molecular changes associated with stable viral gene expression, which can inform the optimization of LVV production in gene therapy applications.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 3","pages":"266-276"},"PeriodicalIF":4.5,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143981535","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,&nbsp;Natalia V. Klementieva,&nbsp;Svetlana G. Vassilieva,&nbsp;Egor A. Volovikov,&nbsp;David Jappy,&nbsp;Irina M. Savchenko,&nbsp;Ekaterina A. Svetlova,&nbsp;Anna V. Polikarpova,&nbsp;Maria Y. Shubina,&nbsp;Danil M. Spirin,&nbsp;Ksenia S. Anufrieva,&nbsp;Petr R. Lebedev,&nbsp;Vladimir M. Pokrovsky,&nbsp;Marina V. Utkina,&nbsp;Viktoriya G. Krut’,&nbsp;Mikhail Sintsov,&nbsp;Sergey Popov,&nbsp;Alexey V. Deykin,&nbsp;Andrei Rozov,&nbsp;Tatiana V. Egorova,&nbsp;Maryana V. Bardina","doi":"10.1038/s41434-025-00532-x","DOIUrl":"10.1038/s41434-025-00532-x","url":null,"abstract":"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&gt;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 GABAB-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&gt;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.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"430-445"},"PeriodicalIF":4.5,"publicationDate":"2025-04-14","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}

{"title":"Expression of anti-amyloid CARs in microglia promotes efficient and selective phagocytosis of Aβ1‒42","authors":"Christina N. Heiss,&nbsp;Rebecca Riise,&nbsp;Eric Hanse,&nbsp;Stefanie Fruhwürth,&nbsp;Henrik Zetterberg,&nbsp;Andreas Björefeldt","doi":"10.1038/s41434-025-00534-9","DOIUrl":"10.1038/s41434-025-00534-9","url":null,"abstract":"Genetic engineering of microglial cells is a promising therapeutic avenue emerging with advancements in gene delivery techniques. Using a recently developed AAV capsid for efficient in vitro transduction we report the engineering of microglia with CARs (CAR-Mic) targeting phagocytosis of amyloid beta 1‒42 (Aβ42). Functional screening of seven CAR constructs in human iPSC-derived microglia revealed up to 6-fold increases in internalized Aβ relative to viral control. CAR-driven phagocytic enhancement was selective for Aβ, dependent on intracellular domain signaling, and was confirmed in primary mouse microglia. These findings highlight the potential of using this approach to target dysfunctional microglia in Alzheimer’s disease and other CNS disorders.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":"32 4","pages":"333-338"},"PeriodicalIF":4.5,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12310543/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144007430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Is dystrophin immunogenicity a barrier to advancing gene therapy for Duchenne muscular dystrophy?","authors":"Dariusz C Górecki, Pawel Kalinski, Joanna Pomeroy","doi":"10.1038/s41434-025-00531-y","DOIUrl":"10.1038/s41434-025-00531-y","url":null,"abstract":"<p><p>Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that leads to severe disability and premature death in young men. As DMD is caused by the absence of dystrophin, therapeutic development has focused on strategies to restore dystrophin expression. These include readthrough of premature stop codons, exon skipping to restore the reading frame, and gene therapy. The first two methods are mutation-specific, benefiting only subsets of patients, whereas gene therapy could treat all individuals with DMD. Immunogenicity of dystrophin may challenge these efforts. The immune system can recognize dystrophin as a neo-antigen, just as it can recognize newly arising antigens present on mutated cells. An in-depth evaluation of anti-dystrophin immune response as a factor affecting the treatment effectiveness is needed. Key questions include the underlying mechanisms of immunity induction by antigenic epitopes of the re-expressed dystrophin, the impact of such responses on the therapeutic efficacy, and the role of patient-specific risk factors, such as preimmunization due to revertant fibres, chronic muscle inflammation, pre-existing T lymphocytes reactive to dystrophin, which avoided deletion in dystrophic thymus, or antigen cross-reactivity. Patients' immune status assessment before treatment may help mitigating anti-dystrophin responses. Exploring potential therapeutic strategies to enhance treatment outcomes is also essential: Since DMD can be diagnosed at birth, early dystrophin re-expression could prevent damage and also potentially induce neonatal tolerance. In older patients, carefully managed immunosuppression and tolerogenic protocols could pave the way for more successful dystrophin replacement therapies.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.6,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143779746","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}