Human gene therapy最新文献

{"title":"AviNP-Seq: A Blindspot-Free Single-Molecule Framework for Unmasking AAV Genome Heterogeneity and Determining Packaging Limits.","authors":"Guo-Hua Li, Yan Xu, Tong Liu, Xiao-Yu Ma, Meng-Ting Hou, Shan-Chen Pang, Jian-Ping Zhang, Tao Cheng, Xiao-Bing Zhang","doi":"10.1177/10430342261454324","DOIUrl":"10.1177/10430342261454324","url":null,"abstract":"<p><p>Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library preparation biases that obscure genome integrity. We present AviNP-seq, a blindspot-free nanopore sequencing framework utilizing one-end-sufficient ligation and Cas9-ribonucleoprotein (RNP) linearization to minimize terminal selection. Applied to a 1.5-6.5 kb panel, AviNP-seq delineates a sharp packaging cliff at 5.0-5.2 kb and reveals that sequence structure modulates integrity by 2-5× at fixed lengths. It unmasks covalent head-to-tail tandems in sub-3 kb vectors, detecting them with significantly higher sensitivity than PacBio HiFi. The Cas9-RNP step boosts ligation yield ∼7-fold, providing an unbiased assessment of genome integrity (≥95% inverted terminal repeat [ITR]-to-ITR). In addition, the assay quantifies plasmid impurities down to 0.05% with linear response. By integrating integrity mapping, tandem detection, and impurity profiling into a rapid (<36 h), low-input workflow, AviNP-seq provides a robust analytical tool to guide vector design and de-risk early-stage process development.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"887-902"},"PeriodicalIF":4.6,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148224747","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 Dual-Viral Delivery Platform Enables Efficient Site-Specific Integration of Therapeutic-Length Genes in Human Primary Stem Cells.","authors":"Zhu-Ying Gao, Tian-Lai Shen, Chong-Yan Cheng, Yi-Dan Sun, Li-Ming Zhang, Jian-Ping Zhang, Xiao-Bing Zhang","doi":"10.1177/10430342261453040","DOIUrl":"10.1177/10430342261453040","url":null,"abstract":"<p><p>Site-specific integration of large genes in human primary stem cells remains a significant challenge in gene therapy, particularly for treating multiallelic diseases. Gene editing efficiency in primary stem cells is heavily influenced by the delivery strategy, which often faces issues with programmability, efficiency, and specificity. Here, we developed a dual-viral delivery system, targeted integration via virus-like particles and integrase-deficient lentivirus (TIVID). This system combines virus-like Cas9 edit particles for delivering Cas9/sgRNA ribonucleoprotein complexes and integrase-deficient lentiviral vectors for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 65% ± 5% in human induced pluripotent stem cells (iPSCs). In erythroid progenitor HUDEP2 cells, TIVID mediates precise integration of a 7.1 kb HBB-GFP cassette (from cut site to cut site) at the <i>AAVS1</i> locus with 20% efficiency and stable expression. Crucially, we demonstrate that TIVID overcomes stringent packaging constraints to deliver an approximately 6 kb full-length <i>HBB</i> therapeutic cassette into primary human CD34⁺ hematopoietic stem and progenitor cells. This platform achieved 5-10% targeted integration efficiency and preserved robust lineage-specific differentiation capacity, demonstrating its potential for treating β-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (∼50% vs. <10% at <i>AAVS1</i> in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (∼20% vs. ∼1.5%). Compared with traditional electroporation delivery, TIVID offers lower early cytotoxicity, promotes predominantly mono-allelic integration, and exhibits enhanced compatibility with primary stem cells. By decoupling nuclease and donor delivery, TIVID circumvents the payload constraints of single-vector systems and the toxicity of physical transfection, providing a robust <i>ex vivo</i> engineering platform for complex gene replacement therapies.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"858-875"},"PeriodicalIF":4.6,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148042068","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":"Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.","authors":"Jingjing Hu, Jun Zhang, Yu Shao, Huashun Li, Yi Yang, Jinping Zhang","doi":"10.1177/10430342261453858","DOIUrl":"10.1177/10430342261453858","url":null,"abstract":"<p><p>Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and uterine cancer. Our and other laboratories' studies have shown that ROBO1 CAR-NK cells exhibit promising tumor therapeutic effects. However, the results still have some limitations. Cbl-b, an E3 ubiquitin ligase, has been reported to negatively regulate NK cell activation, homeostasis, and antitumor immunity.¹ Therefore, we attempted to further enhance the antitumor activity of ROBO1 CAR-NK92 cells by knocking out <i>Cbl-b</i> using CRISPR/Cas9 gene-editing technology. In this study, we conjugated Cbl-b sgRNA with Cas9 protein to form ribonucleoprotein complexes, which were then delivered into ROBO1 CAR-NK92 and NK-92 cells (control cells) via electroporation. Through fluorescence-activated cell sorting, limiting dilution, and sequencing, we obtained monoclonal Cbl-b-knock-out (KO) cell lines. Both <i>in vitro</i> cytotoxicity assays and <i>in vivo</i> tumor xenograft experiments were conducted to examine whether Cbl-b knockout enhances the target cell killing and tumor suppression capacities of ROBO1 CAR-NK92 cells. In this study, monoclonal cell lines of ROBO1 CAR-NK92-Cbl-b-KO and NK92-Cbl-b-KO were successfully established. <i>In vitro</i>, at an effector-to-target (E:T) ratio of 0.1:1, ROBO1 CAR-NK92-Cbl-b-KO (50.55%) cells exhibited significantly higher cytolytic activity against ROBO1-positive T47D target cells after 3 h of coculture than ROBO1 CAR-NK92 (34.10%), NK92-Cbl-b-KO (22.22%), and parental NK-92 cells (3.28%). <i>In vivo</i>, tumor volume and weight measurements demonstrated that mice treated with ROBO1 CAR-NK92-Cbl-b-KO cells developed significantly smaller tumors than all control groups, achieving a tumor growth inhibition (TGI) rate of 32.45%, indicating enhanced antitumor efficacy conferred by Cbl-b knockout. <i>In vitro</i> and <i>in vivo</i> data confirmed that Cbl-b knockout potentiates the antitumor efficacy of ROBO1 CAR-NK92 cells. The overall cytotoxic capability ranked as follows: ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"849-857"},"PeriodicalIF":4.6,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148042035","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":"<i>Letter:</i> Exon 19 of the <i>GNPTAB</i> Gene is Indispensable for the Catalytic Activity of the Enzyme.","authors":"Balraj Doray, Steven Le, Patricia I Dickson","doi":"10.1177/10430342261468988","DOIUrl":"10.1177/10430342261468988","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"815-818"},"PeriodicalIF":4.6,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148420715","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":"Inflammation-Inducible Expression of Human Indoleamine 2,3-Dioxygenase Driven by the E-Selectin Promoter Ameliorates Collagen-Induced Arthritis.","authors":"Mehmet Karaçay, İzel Yılmaz, Ayşe Özden Barazi Soydaş, Gökçen Güvenç-Bayram, Figen Ersoy, Cansel Güzin Özgüden Akkoç, Arzu Yılmaztepe Oral, Ahmet Akkoç, Murat Yalçın, Haluk Barbaros Oral","doi":"10.1177/10430342261452520","DOIUrl":"10.1177/10430342261452520","url":null,"abstract":"<p><p>Rheumatoid arthritis is a chronic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. Although biological disease-modifying anti-rheumatic drugs (DMARDs) have transformed treatment, their systemic immunosuppression, high cost, and incomplete efficacy in certain joints remain significant challenges. To address these limitations, we developed a localized gene therapy using the human indoleamine 2,3-dioxygenase (<i>hIDO</i>) gene driven by the E-selectin (ELAM-1) promoter, which is responsive to inflammatory cytokines. We assessed ELAM-1 promoter responsiveness to interleukin-1β (IL-1β) stimulation <i>in vitro</i> mainly using the HeLa cell line, measuring <i>hIDO</i> expression and enzymatic activity by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting and kynurenine assays. <i>In vivo</i>, we established a robust collagen-induced arthritis (CIA) model in BALB/c mice, which are normally resistant to CIA, and delivered pELAM-1pro/hIDO plasmids intra-articularly via liposomes. Therapeutic efficacy was evaluated by clinical scoring, ankle circumference, histopathology, immunohistochemical quantification of synovial CD4⁺ T cells and CD68⁺ macrophages, and endpoint immunohistochemical assessment of IDO expression. <i>In vitro</i>, the ELAM-1 promoter allowed rapid, high-level expression of hIDO within 2 h of IL-1β stimulation, without IDO-induced suppression of the promoter. <i>In vivo</i>, pELAM-1pro/hIDO treatment significantly reduced ankle swelling and clinical arthritis scores more rapidly than constitutive CMV-driven hIDO, achieving marked histological improvement on day 7. The treated joints exhibited substantial reductions in pathogenic CD4⁺ T cells and CD68⁺ macrophages, consistent with the IDO immunoregulatory mechanism. This study supports the feasibility of a localized gene therapy approach using an inflammation-responsive promoter and suggests localized immunomodulation at disease sites in a manner consistent with inflammation-associated promoter activity.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"833-848"},"PeriodicalIF":4.6,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148156366","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":"Engineering Human Hematopoietic Stem and Progenitor Cells for Antigen Expression in Antigen-Presenting Cells.","authors":"Clara Heider, Meg Louise Donovan, Carina Walpole, Peter R Murphy, Louisa Alim, Irina Buckle, Liam O'Brien, Maki Nakayama, Simon C Barry, Stuart I Mannering, Kristen J Radford, Raymond J Steptoe","doi":"10.1177/10430342261478743","DOIUrl":"https://doi.org/10.1177/10430342261478743","url":null,"abstract":"<p><p>Gene therapy achieved through genetic manipulation and transfer of hematopoietic stem and progenitor cells (HSPC) is becoming an increasingly attractive option for personalized medicine. In the current clinical scenarios, use is restricted to overcoming genetic disorders, but it is envisioned that more diverse applications will be developed in the future. Immunotherapy of malignant disease already employs non-HSPC-based gene therapies, and there is a body of evidence that HSPC-mediated gene therapy has potential as a powerful immunotherapeutic for some diseases of immune dysregulation such as autoimmune disease and allergies. Administration of HSPC gene-engineered to encode antigen(s) induces antigen-specific immune tolerance in animal models. To translate this technology, we sought to identify whether human dendritic cells (DC) and other antigen-presenting cell populations derived from lentivirally transduced HSPC would express, process, and present antigens of interest. Lentiviral vectors were generated that encoded preproinsulin, a relevant autoantigen implicated in type 1 diabetes, or a control protein carrying a viral epitope along with reporter genes under transcriptional control of ubiquitous or antigen-presenting cell-directed promoters. <i>In vitro</i>, DC were differentiated from transduced HSPC and tested in antigen presentation assays using Jurkat lines carrying relevant type 1 diabetes-associated T cell receptors. <i>In vivo</i>, transduced HSPC were used to generate humanized mice, and the development of a range of antigen-presenting cells and their expression of lentivirus-encoded genes were determined. Antigen-presenting cell progeny of transduced HSPC expressed antigens and reporter genes both <i>in vitro</i> and <i>in vivo</i>, and <i>in vitro</i>-generated DC presented lentivirally encoded antigens. The studies characterize human chimerism and therapeutic gene expression in a new model and provide support for the use of HSPC-based gene therapies for immune dysregulation in humans.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261478743"},"PeriodicalIF":4.6,"publicationDate":"2026-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148812953","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":"Streamlining Genetically Modified Organism Regulatory Classification in UK Gene Therapy Clinical Trials: A UK Pharmacy Perspective.","authors":"Annalisa Del Prete, Beatriz Duran Jimenez, Anne Black","doi":"10.1177/10430342261473434","DOIUrl":"https://doi.org/10.1177/10430342261473434","url":null,"abstract":"<p><p>Accurate classification of Genetically Modified Organism (GMO) gene therapies remains a significant challenge to initiating clinical trials in the United Kingdom, often delaying patient access to innovative treatments. Current regulatory processes require separate GMO risk assessments at each National Health Service (NHS) trial location, leading to inconsistent approaches and duplication of effort. This article presents the development of a decision tree designed to streamline GMO classification for Gene Therapy Investigational Medicinal Products (GTIMPs). The proposed tool aims to address key challenges in trial setup by providing a structured and evidence-based approach to GMO classification, clarifying international misalignment in hazard classification and offering practical guidance for the <i>in vivo</i> and <i>ex vivo</i> GMO GTIMPs most encountered in clinical practice. By harmonizing decision-making and clarifying containment requirements, the adoption of the decision tree across NHS organizations could reduce variability, accelerate trial setup, and strengthen the United Kingdom's position as a competitive destination for GTIMP clinical trials.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261473434"},"PeriodicalIF":4.6,"publicationDate":"2026-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148712244","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":"Physiologically Regulated Frataxin Gene Replacement Restores Neurological Function in a Mouse Model of Friedreich Ataxia.","authors":"Federica Pilotto, Laure Dall'Agnol, Laurence Reutenauer, Marie Paschaki, Hélène Puccio","doi":"10.1177/10430342261474315","DOIUrl":"https://doi.org/10.1177/10430342261474315","url":null,"abstract":"<p><p>Friedreich ataxia (FA) is a progressive neurodegenerative disorder caused by reduced expression of frataxin (FXN), a mitochondrial protein essential for iron-sulfur (Fe-S) cluster biogenesis. Although gene therapy strategies aimed at restoring FXN have shown promise, excessive expression can lead to mitochondrial dysfunction, emphasizing the importance of maintaining FXN within a physiological range. Here, we evaluated a gene therapy approach based on a human mini-frataxin construct (miniFXN7) incorporating an endogenous regulatory element to enable controlled FXN expression. The construct was delivered systemically using an AAV-PHP.eB vector in the <i>Pvalb</i>-cKO mouse model of FA. MiniFXN7 treatment resulted in widespread neuronal transduction and restoration of FXN expression toward a near-physiological range in the neuronal populations examined. Treated mice exhibited sustained improvements in motor coordination and proprioceptive function, including normalization of H-reflex responses. At the cellular level, miniFXN7 restored succinate dehydrogenase activity, a mitochondrial Fe-S enzyme, and was associated with partial normalization of mitochondrial morphology. In parallel, neuronal integrity was preserved and astrogliosis reduced across the cerebellum. These findings demonstrate that physiologically regulated FXN replacement is sufficient to achieve substantial functional rescue in FA, supporting a gene therapy strategy based on a transgene expression driven by endogenous regulatory elements.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261474315"},"PeriodicalIF":4.6,"publicationDate":"2026-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148700881","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":"Safety, Tolerability, and Efficacy of a Prophylactic Sirolimus Protocol for Patients Receiving Delandistrogene Moxeparvovec-Rokl Gene Therapy.","authors":"Zahraa Siddiqui, Serena Neumann, Daniel Guillen, Ruchee Patel, Rachel Cox, W Bryan Burnette, Rachel Harris, Saeed Mohammad, Jonathan H Soslow","doi":"10.1177/10430342261472609","DOIUrl":"https://doi.org/10.1177/10430342261472609","url":null,"abstract":"<p><p>Delandistrogene moxeparvovec-rokl (DMR) is the only U.S. Food and Drug Administration-approved gene therapy for patients with Duchenne muscular dystrophy (DMD). After reports of two deaths due to acute liver injury (ALI)/acute liver failure, our center began prophylaxis with sirolimus to help prevent ALI. The objective of this study is to report the initial safety, tolerability, and efficacy of sirolimus prophylaxis. The clinical course of patients receiving sirolimus prophylaxis with a target trough of 4-6 ng/mL was retrospectively evaluated and compared with those who did not receive sirolimus. Laboratory testing, particularly gamma glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, and total bilirubin, was collected. A Fisher's exact test was used to assess the difference between groups. Twelve patients were started on sirolimus prophylaxis, 10 prior to DMR infusion and 2 within 2 weeks of DMR. The median age at DMR dosing was 11 years old (interquartile range = 8.5-14); 11 patients were ambulatory. No patients experienced a serious adverse event related to sirolimus administration. Seven patients developed elevated triglyceride or cholesterol levels and began omega-3 supplementation. None of the patients receiving sirolimus prophylaxis experienced ALI compared with 3 of the 14 patients (21%) infused prior to implementation of the sirolimus protocol. Sirolimus prophylaxis appears to be safe and well-tolerated in DMD patients receiving DMR. While not statistically significant, these results suggest that further study of sirolimus prophylaxis is warranted.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261472609"},"PeriodicalIF":4.6,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148673597","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":"Adeno-Associated Virus-Mediated Central Nervous System Gene Transfer to Suppress Alzheimer's Disease High-Risk APOE4 Variant and Replace with Protective APOE2.","authors":"Kalpita R Karan, Neil R Hackett, Ronald G Crystal","doi":"10.1177/10430342261473427","DOIUrl":"https://doi.org/10.1177/10430342261473427","url":null,"abstract":"<p><p>Common genetic variants of APOE are major risk factors for sporadic late-onset Alzheimer's disease (AD). APOE has three common variants: APOE3, APOE4, and APOE2. Epidemiological, clinical, and experimental evidence demonstrate that APOE3 is associated with an average risk for AD, APOE4 is pathogenic and conveys a high risk, and APOE2 is protective and reduces risk. In prior mouse studies, we have demonstrated that (1) adeno-associated virus (AAV)-mediated central nervous system (CNS) gene transfer of APOE2 is highly protective against the toxic effects of APOE4 and (2) AAV-mediated CNS transfer of anti-APOE4 microRNA (miRNA) significantly suppresses the expression of APOE4. The ideal therapy for APOE4 homozygotes would both reduce CNS levels of APOE4 and substitute APOE4 with the addition of APOE2. We have developed a \"silence-and-replace\" therapy, where suppression of CNS APOE4 is achieved through AAV-mediated expression of miRNAs designed to silence the endogenous human APOE4 messenger RNA (mRNA), together with simultaneous replacement by an APOE2 coding sequence [APOE2(R)] resistant to those miRNAs. AAV vectors expressing APOE2(R) with and without the miRNAs were administered to the hippocampus of human APOE4 mice, and APOE levels at the mRNA and protein levels were the same for both vectors. Dideoxy Sanger sequencing and allele-specific real-time quantitative PCR were used to assess the CNS APOE2 to APOE4 ratio. As expected, both vectors with APOE2(R) increased the E2/E4 ratio relative to controls by 3.5 ± 0.6-fold for APOE2(R) and 6.3 ± 0.4-fold for APOE2(R) + miRNAs. We conclude that a single AAV vector can deliver a cassette with dual functionality: suppression of the toxic APOE4 variant and simultaneous delivery of the protective APOE2. This dual approach may provide a more potent gene therapy for APOE4-dependent AD than either strategy.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261473427"},"PeriodicalIF":4.6,"publicationDate":"2026-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148663736","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}