Human gene therapy最新文献

{"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}

{"title":"Advancements in Gene-Based Therapeutic Angiogenesis for Chronic Limb-Threatening Ischemia.","authors":"Peng Wang, Xiao Di, Fengshi Li, Zhihua Rong, Wenzhuo Lian, Guoqiang Sun, ChangWei Liu, Leng Ni","doi":"10.1089/hum.2024.245","DOIUrl":"https://doi.org/10.1089/hum.2024.245","url":null,"abstract":"<p><p>The objective of this article is to summarize the research progress and discuss the current difficulties of gene-based therapeutic angiogenesis in lower limb ischemic diseases, so as to provide new research directions for the non-invasive treatment of lower limb ischemia. The basic and clinical trials of gene-based therapeutic angiogenesis in lower limb ischemia in recent years were read and reviewed. Growth factors such as vascular endothelial growth factor, hepatocyte growth factor, and fibroblast growth factor have been extensively studied for their application in lower limb ischemic diseases. However, clinical studies across various phases have shown inconsistent efficacy endpoints. The efficacy of gene therapy remains questionable. Before exploring efficient methods of delivering pro-angiogenic genes to ischemic tissues, clarification is needed regarding whether the goal of gene therapy is to simply promote collateral circulation or create a conducive tissue microenvironment for angiogenesis. In conclusion, pre-clinical and clinical studies have demonstrated the potential of therapeutic angiogenesis, but more systematic and comprehensive research is needed to explore safer, more effective, and cost-effective treatment methods.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":"36 9-10","pages":"787-800"},"PeriodicalIF":3.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144013423","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":"Prediction of Adeno-Associated Virus Fitness with a Protein Language-Based Machine Learning Model.","authors":"Jason Wu, Yu Qiu, Eugenia Lyashenko, Tess Torregrosa, Edith L Pfister, Michael J Ryan, Christian Mueller, Sourav R Choudhury","doi":"10.1089/hum.2024.227","DOIUrl":"https://doi.org/10.1089/hum.2024.227","url":null,"abstract":"<p><p>Adeno-associated virus (AAV)-based therapeutics have the potential to transform the lives of patients by delivering one-time treatments for a variety of diseases. However, a critical challenge to their widespread adoption and distribution is the high cost of goods. Reducing manufacturing costs by developing AAV capsids with improved yield, or fitness, is key to making gene therapies more affordable. AAV fitness is largely determined by the amino acid sequence of the capsid, however, engineered AAVs are rarely optimized for manufacturability. Here, we report a state-of-the art machine learning (ML) model that predicts the fitness of AAV2 capsid mutants based on the amino acid sequence of the capsid monomer. By combining a protein language model (PLM) and classical ML techniques, our model achieved a significantly high prediction accuracy (Pearson correlation = 0.818) for capsid fitness. Importantly, tests on completely independent datasets showed robustness and generalizability of our model, even for multimutant AAV capsids. Our accurate ML-based model can be used as a surrogate for laborious <i>in vitro</i> experiments, thus saving time and resources, and can be deployed to increase the fitness of clinical AAV capsids to make gene therapies economically viable for patients.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":"36 9-10","pages":"823-829"},"PeriodicalIF":3.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008316","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":"Trojan Horse-Like Vehicles for CRISPR-Cas Delivery: Engineering Extracellular Vesicles and Virus-Like Particles for Precision Gene Editing in Cystic Fibrosis.","authors":"Laudonia Lidia Dipalo, Jacob Giehm Mikkelsen, Rik Gijsbers, Marianne S Carlon","doi":"10.1089/hum.2024.258","DOIUrl":"https://doi.org/10.1089/hum.2024.258","url":null,"abstract":"<p><p>The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced \"living drugs.\" We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in <i>in vivo</i> gene editing for this disease.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144003132","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":"Deciphering Key Adenoviral Elements in the Production of Recombinant Adeno-Associated Virus Vectors.","authors":"Sofia Fernandes, Júlia Guerra, Mariana V Ferreira, Ana Sofia Coroadinha","doi":"10.1089/hum.2024.265","DOIUrl":"https://doi.org/10.1089/hum.2024.265","url":null,"abstract":"<p><p>Over the last two decades, adeno-associated viruses (AAVs) have been widely used as viral vectors in gene therapy due to their ability to infect both dividing and nondividing cells, broad tissue specificity, and favorable safety profile. Recombinant AAV (rAAV) production requires a helper virus, typically adenovirus (AdV), which provides essential genes for AAV replication. However, the increasing demand for safer and more efficient rAAV production methods led to the need to develop helper plasmids with minimal AdV components. In this study, we evaluate the impact of AdV E2 and E4 in the productivity and genome packaging of rAAV serotypes 2, 5, 8, and 9, produced by transient transfection. We designed and tested eight novel helper plasmids with different deletions in E2 and E4 genes. Results indicated that deletions in these genes significantly affected rAAV productivity and packaging, particularly for serotypes 8 and 9. Helper plasmids containing minimal essential genes-E2-DBP, E4orf6, and VA RNA-showed near to 10-fold reduction in viral genome packaging compared to the control. However, including E2 L4-22/33K and E4orf3 regions significantly improved viral production, particularly for serotypes 8, and 9. In this study, we also demonstrated that the full E4 gene is crucial to achieving high full-empty ratios, minimizing the production of empty capsids, and enhancing viral release into the culture medium of rAAV8. Accordingly, we created a smaller plasmid, without adenoviral structural proteins that allows a similar rAAV production across all tested serotypes. Overall, these findings provide insights into the genetic requirements for efficient rAAV production and highlight the importance of the E2 and E4 regions for optimizing viral yield and quality. This approach could lead to the development of improved strategies for large-scale rAAV vector production by enabling safer and more cost-effective systems.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143981324","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":"Deep Intronic SVA_E Retrotransposition as a Novel Factor in Canavan Disease Pathogenesis.","authors":"Melina Weiß, Mareike Selig, Johannes Friedrich, Anna Wierczeiko, Stefan Diederich, Helen Sigel, Janna Bredow, Florian S Eichler, Amanda Nagy, Denise Seyler, Laura Holthöfer, Susanne Gerber, Susann Schweiger, Matthias Linke, Annette Bley","doi":"10.1089/hum.2025.006","DOIUrl":"https://doi.org/10.1089/hum.2025.006","url":null,"abstract":"<p><p>Canavan disease (CD) is a rare autosomal recessive leukodystrophy caused by biallelic pathogenic variants in the <i>ASPA</i> gene. CD is characterized by developmental delay, macrocephaly, and abnormal muscle tone. The biochemical diagnosis is confirmed by increased <i>N</i>-acetylaspartic acid levels. The phenotypic presentation varies, with 85-90% of individuals exhibiting the severe, typical form, while 10-15% present with a milder, atypical form. Here we report on five patients with a clinical and biochemically proven diagnosis in whom a second pathogenic variant had not yet been identified. Targeted long-read sequencing of the entire <i>ASPA</i> gene revealed an SVA_E retrotransposable element located in intron 4 that had been missed by standard short-read-based diagnostic procedures. Haplotype analysis of all patients showed linkage of the SVA_E element with a noncoding variant in intron 1. Functional characterization of the SVA_E element suggests that transcripts of the affected allele are prone to highly efficient mRNA degradation processes. These findings enhance the precision of genetic diagnostics and enable improved guidance for families as well as facilitating potential access to targeted therapies.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008840","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":"Advancing Potency Assay Development for Advanced Therapy Medicinal Products: A Comprehensive Approach and Regulatory Insights.","authors":"Alaa Abdellatif, Melissa Bou Jaoudeh, Alex Zwiers, Gabrièle Breda","doi":"10.1089/hum.2024.249","DOIUrl":"https://doi.org/10.1089/hum.2024.249","url":null,"abstract":"<p><p>The development of potency assays for Advanced Therapy Medicinal Products (ATMPs) presents significant challenges due to the variability of starting materials and the complex mechanisms of action involved. This article aims to address the following key question: <i>How can we design robust and reliable potency assays for ATMPs that accommodate product-specific challenges and align with evolving regulatory standards?</i> To answer this, we employed a mixed-methods approach, synthesizing data from scientific literature, industry reports, and regulatory guidelines to identify current limitations and innovative solutions for potency assay development. Our methodology integrates a systematic review of academic publications (2018-2024) to capture recent advancements in biotechnology and their applicability to potency testing. We complemented this with an analysis of industry perspectives, drawn from webinars and white papers, as well as a detailed comparison of global regulatory frameworks, including the FDA's new guidance on potency assurance for Cellular and Gene Therapy Products (CGTs/ATMPs). Additionally, we developed a comprehensive database to analyze potency assays used in approved, rejected, and withdrawn CGT/ATMP products, focusing on technical and regulatory challenges. Based on this multilevel analysis, we propose a product-specific framework for designing, developing, and validating potency assays for different ATMP categories, taking into account their unique technical and regulatory constraints. We also highlight emerging technologies, such as droplet digital polymerase chain reaction and reporter gene assays, as innovative tools for improving the precision and reliability of potency testing. Our findings underscore the need for flexible, risk-based strategies in potency assay development that evolve throughout product development and clinical trial phases. Future recommendations emphasize assay standardization, the definition of acceptable variability, and stronger correlations between in vitro potency data and clinical outcomes.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143997803","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":"Meta-Analysis and Optimization of the <i>In Vitro</i> Immortalization Assay for Safety Assessment of Retroviral Vectors in Gene Therapy.","authors":"Antonella L Bastone, Philipp John-Neek, Violetta Dziadek, Friederike Mansel, Maike Hagedorn, Jenni Fleischauer, Bettina Weigel, Gabi Paul, Axel Schambach, Michael Rothe","doi":"10.1089/hum.2024.221","DOIUrl":"https://doi.org/10.1089/hum.2024.221","url":null,"abstract":"<p><p>The underlying risk of retroviral vector-induced insertional oncogenesis in gene therapies requires a reliable preclinical safety assessment. Dysregulation of genes neighboring the vector's integration sites has triggered hematopoietic malignancies in patients treated with different vector genera and designs. With ca. 18 years in practical use, the <i>in vitro</i> immortalization (IVIM) assay can quantify this mutagenic potential and is actively requested by regulatory authorities during preclinical stages. Here, we present a thorough meta-analysis of IVIM data alongside a step-by-step cell culture protocol. On this basis, we propose clonal outgrowth as the single indicator of mutagenicity, simplifying the IVIM assay cost- and time-wise.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811322","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}