Human gene therapy最新文献

{"title":"Halting Recombinant Adeno-Associated Virus Transgene Expression Using mRNA-Lipid Nanoparticle-Delivered Meganucleases.","authors":"Rubens Tavora, Lizhou Zhang, Mai H Tran, Hao Li, Dan O'Hagan, Andi Pan, Lorenzo Barrett, Joseph A Jablonski, Sonia Mediouni, Alexander Lopez, Zachary Comella, Charles Bailey, Hyeryun Choe, Michael Farzan, Susana T Valente","doi":"10.1089/hum.2025.011","DOIUrl":"10.1089/hum.2025.011","url":null,"abstract":"<p><p>Recombinant adeno-associated virus (rAAV) vectors are increasingly preferred for <i>in vivo</i> gene therapy due to their broad tropism, low immunogenicity, and sustained transgene expression. Nevertheless, in cases of adverse reactions to these expressions, a method to suppress or permanently halt rAAV transgene activity could significantly enhance the safety of these vectors. To address this need, we employed meganucleases-highly specific DNA endonucleases with long recognition sequences. By placing meganuclease target sites within rAAV transgenes, we created a system in which targeted cleavage leads to controlled disruption of transgene expression. Utilizing a luciferase assay, we screened various meganucleases and identified I-AniI-Y2, I-BmoI, and I-PpoI as prime candidates due to their high cleavage efficiencies. By strategically placing multiple meganuclease target sequences within introns, as well as in the 5' and 3' untranslated regions (UTRs) of transgenes, we significantly enhanced the cleavage efficiency of these meganucleases, ensuring robust and targeted suppression of transgene expression. Finally, we employed an mRNA-loaded lipid nanoparticledelivery system to demonstrate the ability of meganucleases to robustly inhibit rAAV-mediated transgene expression <i>in vitro</i>. Our findings underscore the potential of meganucleases as a viable safety mechanism in rAAV gene therapies, marking a significant advance toward safer long-term gene therapy approaches.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"870-883"},"PeriodicalIF":3.9,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12171709/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144011669","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":"Progress, Applications and Prospects of CRISPR-Based Genome Editing Technology in Gene Therapy for Cancer and Sickle Cell Disease.","authors":"Sha-Sha Zang, Ruirui Zhang, Jia-Run Zhang, Xi Zhang, Jun Li","doi":"10.1089/hum.2024.262","DOIUrl":"10.1089/hum.2024.262","url":null,"abstract":"<p><p>The advent of genome-editing technologies, particularly the RNA-guided the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) 9, which originates from prokaryotic CRISPR/Cas adaptive immune mechanisms, has revolutionized molecular biology. Renowned for its simplicity, cost-effectiveness, and capacity for multiplexed gene editing, CRISPR/Cas9 has emerged as the most versatile and widely adopted genome-editing platform. Its applications span fundamental research, biotechnology, medicine, and therapeutics. This review highlights recent advancements in CRISPR-based technologies, focusing on CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas12f. It emphasizes precision editing methods like base editing and prime editing, which enable targeted nucleotide changes without double-strand breaks. The specificity of these tools, including on-target accuracy and off-target risks, is critically evaluated. Additionally, recent preclinical and clinical efforts to treat diseases such as cancer and sickle cell disease using CRISPR are summarized. Finally, the challenges and future directions of CRISPR-mediated gene therapy are discussed, emphasizing its potential to integrate with other molecular approaches to address unmet medical needs.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"858-869"},"PeriodicalIF":3.9,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143965538","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":"BCMA-CAR Therapy for Multiple Myeloma in NOG Mice Prevents the Progression of Anemia and Bone Lesions.","authors":"Ryosuke Uchibori, Ken Ohmine, Takeshi Teruya, Junichi Mineno, Keiya Ozawa","doi":"10.1089/hum.2024.263","DOIUrl":"10.1089/hum.2024.263","url":null,"abstract":"<p><p>Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. Myeloma cells interfere with hematopoietic activities of the bone marrow, often leading to anemia, and can cause the bones to develop osteoporotic and lytic lesions. Clinical experience with chimeric antigen receptor T-cell (CAR-T) therapy targeting B-cell maturation antigen (BCMA) has been promising, with good response rates, favorable safety profiles, and low incidences of severe cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. However, CAR-T therapy in MM is accompanied by several new challenges, including therapeutic failure and relapse, and much attention has been paid to the further development of B-cell maturation antigen-chimeric antigen receptor (BCMA-CAR). Although most of the reported benefits of BCMA-CAR have been discussed, whether cancer can be eliminated, as well as the efficacy of CAR-T therapy for anemia and bone lesions, both myeloma-defining events, have not yet been reported in any animal model. In this study, we designed and verified a novel BCMA-specific chimeric antigen receptor (CAR). Our BCMA-CAR demonstrated the fundamental properties of CAR-T cells, including target-specific cytotoxic activity, cytokine production, and <i>in vivo</i> antitumor effects. In addition, we evaluated the therapeutic effect of BCMA-CAR in mice by imaging bone lesions and conducting blood examinations. Tumor mouse models showed systemic progression of MM in the bone marrow, and mice treated with saline or nongene modified T cells showed continued tumor progression, progressive bone lesions, and prolonged anemia. In contrast, all mice treated with gene modified T cells achieved a complete response, improved anemia to the level observed in normal mice, and suppressed progression of bone lesions. We concluded that anemia was improved with BCMA-CAR-T cell therapy. However, novel strategies to support the recovery of bone lesions by enhancing CAR-T cell function must be developed.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"902-913"},"PeriodicalIF":3.9,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144077480","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":"European Society of Gene & Cell Therapy Spring School 2025.","authors":"Thomas Gallagher","doi":"10.1089/hum.2025.088","DOIUrl":"10.1089/hum.2025.088","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"856-857"},"PeriodicalIF":3.9,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144101787","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":"Safe and Efficacious Permanent Removal of Large COL7A1 Exons for Gene Reframing as a Reliable Therapeutic Strategy for Recessive Dystrophic Epidermolysis Bullosa.","authors":"Sergio López-Manzaneda, Ángeles Mencía, José Bonafont, Alex Bassons-Bascuñana, Marta García, Alexander Nyström, Blanca Duarte, Sara Llames, Rodolfo Murillas, Silvia Modamio-Hoybjor, Matías Morín, Lucía Soletto, María J Escamez, Miguel A Moreno-Pelayo, Marcela Del Rio, Fernando Larcher","doi":"10.1089/hum.2024.238","DOIUrl":"https://doi.org/10.1089/hum.2024.238","url":null,"abstract":"<p><p>Mutations leading to premature termination codons in <i>COL7A1</i> are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing mutated <i>COL7A1</i> exons along with the consequent reframing of <i>COL7A1</i> may not pose noticeable impact on protein function, offering a potential therapeutic strategy. However, investigations into the long-term <i>in vivo</i> effects of genome editing-mediated removal of mutant exons have only focused on the small exon 80 thus far. Hence, this study focuses on exons 73 and 105 of <i>COL7A1</i> to explore whether targeted exon removal, through a CRISPR/Cas9-assisted, Non-homologous end joining (NHEJ)-mediated approach, could be extended to other larger exons. Introducing ribonucleoprotein complexes carrying Cas9 and optimized sgRNA guide pairs for each exon (73 and 105) through electroporation efficiently led to their removal, consequently restoring type VII collagen (C7) synthesis in RDEB primary patient cells carrying frameshift mutations in these exons. <i>In vitro</i> tests indicated the normal stability of the resulting C7 variants expressed at physiological levels, while <i>in vivo</i> analyses of regenerated skin grafted onto immunodeficient mice using E73 or E105 RDEB edited cells demonstrated the proper deposition of C7 at the basement membrane zone, thereby restoring normal dermo-epidermal adherence. This study enhances the broader potential of the exon deletion approach in the treatment of RDEB.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144158326","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":"Stromal Gene Therapy Mediates Prolonged Protection Against Corneal Neovascularization Induced by an Aggressive Angiogenic Insult.","authors":"Mark Basche, Scott Robbie, D Frank P Larkin, Alexander J Smith, Rachael A Pearson, Robin R Ali","doi":"10.1089/hum.2024.248","DOIUrl":"https://doi.org/10.1089/hum.2024.248","url":null,"abstract":"<p><p>Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral vector (AAV)-based gene therapy targeting both hematic and lymphatic neovascularization in a murine model of severe CoNV. We first assessed the profile of transgene expression mediated by intrastromal injection of AAV2/8[Y733F] via longitudinal visualization of an enhanced Green Fluorescent Protein (eGFP) transgene and found that this serotype mediates a temporary (∼18 day) transduction of the corneal epithelium and sustained (≥148 day) transduction within the stroma. Constitutively expressed <i>sFlt1</i> or <i>sFlt4</i> were prophylactically delivered via intrastromal injection of AAV2/8[Y733F] vector at various intervals prior to aggressive induction of CoNV in a murine model. The extent of CoNV induced was quantified by fluorescein angiography and immunohistochemistry 17 days after induction. AAV2/8[Y733F]-CMV-sFlt1 was highly effective in the prevention of hemangiogenesis (HA) induced at 3, 28, and 210 days after intrastromal injection, but ineffective in the prevention of lymphangiogenesis. Two variants of AAV2/8[Y733F]-CMV-sFlt4 were ineffective in the prevention of angiogenesis when delivered alone, but combined delivery of AAV2/8[Y733F]-CMV-sFlt1 and AAV2/8[Y733F]-CMV-sFlt4 suggested a synergistic effect. Our results show that a single intrastromal injection of AAV2/8[Y733F]-CMV-sFlt1 is sufficient to protect against a robust stimulus for corneal HA over the long term. This technique could also be applied <i>ex vivo</i> to reduce the risk of failure in cases of \"high-risk\" corneal transplantation.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144158328","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":"Timely Intervention: Navigating Ethical Challenges in <i>OTOF</i>-Gene Therapy Trials.","authors":"Ellen Reisinger, Hans-Jörg Ehni, Oliver Feeney, Urban Wiesing","doi":"10.1089/hum.2025.042","DOIUrl":"https://doi.org/10.1089/hum.2025.042","url":null,"abstract":"<p><p><i>OTOF</i>-gene therapy for profound deafness in children has entered clinical trials. Given that there is an approved alternative therapy with cochlear implants, it is imperative to scrutinize the risks, while also highlighting the novel benefits, of this experimental gene therapy. Since the untreated inner ear subsequently degenerates in this form of inherited deafness, the <i>OTOF</i>-gene therapy will be most effective in young children. Moreover, the best outcome in terms of hearing and speech comprehension is expected when the gene therapy is applied before the age of 3 years. Given such \"earlier the better\" considerations, the optimal time for these clinical trials and this particular therapy is at an age when children are too young to give informed consent. Enrolling children, which are a vulnerable category of persons, in clinical trials where the balance of benefits and risks is uncertain, raises a series of ethical considerations. In this article, we outline how this research can be pursued in an ethically responsible manner.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144158331","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":"Recombinant Adeno-Associated Virus Vector Mediated Gene Editing in Proliferating and Polarized Cultures of Human Airway Epithelial Cells.","authors":"Soo Yeun Park, Zehua Feng, Soon H Choi, Xiujuan Zhang, Yinghua Tang, Grace N Gasser, Donovan Richart, Feng Yuan, Jianming Qiu, John F Engelhardt, Ziying Yan","doi":"10.1089/hum.2024.260","DOIUrl":"https://doi.org/10.1089/hum.2024.260","url":null,"abstract":"<p><p>Cystic fibrosis (CF) is caused by mutations in the <i>cystic fibrosis transmembrane conductance regulator</i> (<i>CFTR</i>) gene. While CRISPR-based <i>CFTR</i> editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi^{Cas9(Y66S)eGFP} reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of <i>CFTR</i> functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del <i>CFTR</i> and Y66S <i>eGFP</i> was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP⁺ (Y66S-corrected) cells and eGFP^- (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP^- population exhibited 25.2% of the CFTR-specific transepithelial Cl^- transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP⁺ cells than eGFP^- cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009310","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":"Encapsulation of a Single-Stranded Form of DNA Impurities into the Capsid of a Recombinant Adeno-Associated Virus.","authors":"Kazuhisa Uchida, Emi Ito-Kudo, Kiyoko Higashiyama, Kyoko Masumi-Koizumi, Keisuke Yusa, Yuzhe Yuan","doi":"10.1089/hum.2024.264","DOIUrl":"10.1089/hum.2024.264","url":null,"abstract":"<p><p>Recombinant adeno-associated viruses (rAAVs) are widely used viral vectors in human gene therapy. However, DNA impurities, such as plasmid DNA and host cell DNA, remain a significant quality control concern for final products. Our study examined purified rAAV1-ZsGreen1, rAAV2-ZsGreen1, rAAV5-ZsGreen1, and rAAV6-ZsGreen1 samples and found that they contained 0.69-3.27% DNA impurities derived from three plasmids, as detected by droplet digital PCR. These plasmid-derived impurities primarily consisted of those derived from the pAAV plasmid (≥98.88%), with small amounts of pRC1, pRC2mi342, pRC5, or pRC6 (≤0.91%), and pHelper (≤0.21%) plasmids. To determine the DNA strand form of these impurities within the capsids, we used two different DNases with distinct substrate specificities. The extracted DNA impurities from the rAAV samples exhibited high sensitivity to nuclease P1 but not to lambda exonuclease. Similarly, host cell DNA encapsulated within the capsids revealed similar sensitivities to the nucleases. These findings indicate that DNA impurities derived from the plasmids and host cell DNA are encapsulated into rAAV capsids as single-stranded DNA, likely through a mechanism similar to that of the rAAV genome.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"814-822"},"PeriodicalIF":3.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657031","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":"Bioinformatic Analysis of the Genetic Basis of Differential Adeno-Associated Virus Production Capability of 293 Variants.","authors":"Christopher R Herzog, Junping Zhang, Xiaomin Feng, Thao Thi Dang, Xiangping Yu, Jie Huang, Fang Fang, Hongyu Gao, Xuhong Yu, Yue Wang, Renzhi Han, Yulong Liu, Kenneth Cornetta, Weidong Xiao, Weihong Xu","doi":"10.1089/hum.2025.002","DOIUrl":"10.1089/hum.2025.002","url":null,"abstract":"<p><p>Human embryonic kidney 293 (HEK 293) cells are the main producer cell line for recombinant adeno-associated virus (rAAV) production. However, AAV vector yields among 293 clones vary considerably. To elucidate the biological basis for these differences, whole genomes of an adherent and a suspension 293 cell clone with high-yield rAAV were sequenced using nanopore technology. All 293 cell derivative lines showed a twofold copy number gain at the adenoviral integration site across, suggesting a genome duplication event. To our surprise, the two high-producer clones, despite having been separately developed, are biologically closely grouped together as compared to other commonly used 293 clones. Their genomes contain a similar adenoviral gene integration region, which likely leads to high expression of proteins that facilitate AAV replication and packaging. Thus, genome duplication in the adenovirus integration locus may be a key factor affecting AAV production yield.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":"36 9-10","pages":"801-813"},"PeriodicalIF":3.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12171708/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143968149","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}