Human gene therapy最新文献

{"title":"FDA Issues Plausible Mechanism Pathway Draft Guidance.","authors":"Alex Philippidis","doi":"10.1177/10430342261437961","DOIUrl":"10.1177/10430342261437961","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"387-391"},"PeriodicalIF":4.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147580277","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":"Prime Editing of Alzheimer's Disease High-Risk APOE4 Allele by Brain-Directed Adeno-Associated Virus Vectors.","authors":"Caner Günaydin, Neil R Hackett, Victor Wakim, Dolan Sondhi, Stephen M Kaminsky, Ronald G Crystal","doi":"10.1177/10430342251401888","DOIUrl":"10.1177/10430342251401888","url":null,"abstract":"<p><p>Common variants of the apolipoprotein E (APOE) gene have a major impact on the risk of developing Alzheimer's disease (AD). Relative to homozygotes with the common E3 allele, the APOE4 variant (C112R) increases risk by 3.5-fold in E3/E4 heterozygotes and 15-fold in E4 homozygotes. Since the E3 and E4 alleles differ only by a single nucleotide, gene editing of E4 to E3 is a potential strategy to reduce AD risk in E4 homozygotes. Because the APOE pool in the brain is separate from systemic APOE, editing to treat AD would ideally be directed to the brain. Following <i>in vitro</i> optimization of prime editing guide RNAs, efficient prime editing expression cassettes were inserted into the adeno-associated virus (AAV) split-intein system and packaged into pairs of AAV vectors for <i>in vivo</i> editing. The AAV vectors were administered to human homozygous APOE4-targeted replacement mice (TRE4), and APOE4 to APOE3 editing efficiency was assessed after 4 weeks. The prime editing construct designated APOE3/4-3_10 was the most efficient at APOE4 to APOE3 conversion, both in liver following intravenous delivery and in brain following intrahippocampal delivery. To assess brain-wide editing, two AAV capsids were compared, including AAVrh.10 with administration either directly to the hippocampus or to the cerebrospinal fluid via the cisterna magna and AAV-CAP.B10 administered intravenously. Other than minor differences in APOE4/3-3_10 mediated E4 to E3 editing in the cerebellum, the different capsids and routes yielded similar editing efficacy throughout the brain. This may represent a candidate treatment to reduce the risk of AD.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"403-411"},"PeriodicalIF":4.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145833815","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":"AAV-Mediated Base Editing for Correction of <i>RSPH4A</i> Mutations in Primary Ciliary Dyskinesia: A Proof-of-Concept Study.","authors":"Alessandro De Carli, Sara Pastore, Debora Maj, Fabio Filippini, Matteo Matteucci, Maria Elisa Di Cicco, Diego Peroni, Gabriele Donzelli, Stefania Crucitta, Lorena Zentilin, Angela Michelucci, Chiara Gabellini, Giulia Freer, Michele Lai, Massimo Pifferi, Mauro Pistello","doi":"10.1177/10430342261442511","DOIUrl":"https://doi.org/10.1177/10430342261442511","url":null,"abstract":"<p><p>Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder, with abnormal ciliary motility, usually due to an ultrastructural defect, with chronic airway infections. Currently, no curative therapy exists for PCD. Given the prevalence of single nucleotide variants (SNVs) among causative mutations, we evaluated a novel base-editing approach. Specifically, we used a nickase Cas9 fused to adenosine deaminase to correct mutations in the radial spoke head component 4 A (<i>RSPH4A</i>) gene, causing PCD. We selected two PCD patients sharing the same SNV in <i>RSPH4A</i>, one with compound heterozygosity (child, patient 1) and one with homozygosity (adult, patient 2). After designing gRNAs, HEK293T cells with or without a DNA fragment containing the SNV in <i>RSPH4A</i>, were co-transfected with base editor plasmids. Complex formation and editing efficiency were validated by Western blot and digital PCR. We then treated patient cells with AAV containing the base editors and assessed ciliary beat frequency and motion pattern using high-speed video and confocal microscopy to evaluate delivery.Base editor complexes formed efficiently <i>in vitro</i>. AAV-mediated delivery in patient 1 cells led to an approximately 30.4% increase in normal motion pattern, with a corresponding reduction in circular motions (<i>p</i> < 0.001) compared with pre-treatment, and a 20% of editing efficiency detected by dPCR in transduced cells. Our data indicate that this limited editing efficiency is due to reduced AAV penetration in the lower layers of cells.This proof-of-concept study demonstrates the therapeutic potential of base editing for PCD, though current limitations include low editing efficiency and restricted delivery to inner cell layers in our experimental model. Future work should focus on optimizing base editors and testing novel delivery strategies to target progenitor cells, thereby enhancing the prospects for personalized gene therapy in PCD.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261442511"},"PeriodicalIF":4.0,"publicationDate":"2026-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147769862","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":"Strategies and Advances in Site-Specific Integration of Exogenous Large Genes.","authors":"Zhencheng Wu, Jia Chen, Manqi Huang, Wenqi Hu, Yiyu Liu, Hongwei Shao, Wenfeng Zhang","doi":"10.1177/10430342261445050","DOIUrl":"https://doi.org/10.1177/10430342261445050","url":null,"abstract":"<p><p>Large genomic deletions (≥1 kb) are a recurrent class of disease-causing lesions in monogenic disorders, frequently leading to complete gene inactivation or the loss of critical cis-regulatory elements. Addressing these defects in a therapeutically relevant manner requires integration modalities capable of delivering and stably installing large exogenous DNA sequences at predefined genomic loci with an improved safety profile. By contrast, legacy approaches-including viral-vector delivery, recombinase-based strategies, and transposon-mediated insertion-typically achieve integration through random or semi-random mechanisms, which, despite their practicality and often favorable efficiencies, limit control over insertion site and copy number and may increase the risk of insertional mutagenesis and position-dependent variability in transgene expression. The past few years have witnessed rapid methodological diversification driven by genome editing, resulting in a growing repertoire of locus-specific strategies for large-fragment DNA insertion that are reshaping both disease-model construction and genetic therapeutics. In this Review, we synthesize the main classes of targeted large-fragment integration technologies reported to date. We begin with homology-directed repair (HDR)-dependent CRISPR-Cas9 knock-in strategies and discuss how donor architecture and local donor recruitment can be leveraged to improve integration outcomes for kilobase-scale payloads. We then examine approaches centered on prime editing, particularly those that couple prime editing with engineered serine/tyrosine recombinases to support programmable insertion of large DNA cargos. We close by surveying emerging HDR-independent systems based on CRISPR-guided transposition and retrotransposition, and we provide a comparative perspective on their performance envelopes, constraints, and trajectories toward broader biomedical applications.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261445050"},"PeriodicalIF":4.0,"publicationDate":"2026-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147769900","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":"Harvest Process and Affinity Resin Selection Impacts on Adeno-Associated Virus Residual Host Cell Protein Retention.","authors":"Thomas M Leibiger, Lie Min, Kelvin H Lee","doi":"10.1177/10430342261443799","DOIUrl":"https://doi.org/10.1177/10430342261443799","url":null,"abstract":"<p><p>Scalable purification platforms have been developed for adeno-associated virus (AAV) processing to support large-scale vector manufacturing. The ability of column chromatography to recover packaged vectors and remove empty capsids has been well-established, but knowledge gaps remain for understanding process parameter impacts on impurity retention. In this work, we examine the impacts of two key process parameters-the harvest method and affinity resin selection-on residual host cell protein (HCP) retention. Sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) proteomics is applied to comprehensively profile residual HCPs in affinity chromatography (AC) elution pools from four AAV serotypes (AAV2, -5, -8, and -9) produced by suspension HEK293 cells. Vectors were purified from cell culture lysates and from supernatants using POROS^™ CaptureSelect^™ AAVX (AAVX) and one additional serotype-specific affinity resin-Capto^™ AVB (AVB), POROS CaptureSelect AAV8 (PAAV8), or POROS CaptureSelect AAV9 (PAAV9). Significant divergence in residual HCP profiles was observed with the use of different affinity resins, with AVB and PAAV9 showing reduced residual HCP content in elution pools compared with AAVX and PAAV8. Processing of null culture lysates with fresh resins and resins with digested single-domain antibody fragments (sdAbs) shows that differences in resin performance are driven by variable nonspecific sdAb association with cellular impurities. Proteomic analysis of vector preparations from lysates compared with supernatants demonstrates product quality advantages of designing a media-only harvest process, specifically for AAV8, which was measured to contain an average of 66% of total vector genome content in the cell culture media. This work highlights the importance of serotype-specific tailoring of AAV downstream process design for improved product quality attributes to support clinical manufacture and small-scale analytics workflows.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261443799"},"PeriodicalIF":4.0,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147698688","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":"Monitoring Fetal Somatic Cell Genome Editing <i>In Vivo</i> with Total-Body Positron Emission Tomography: Studies in Rhesus Macaques (<i>Macaca mulatta</i>).","authors":"Alice F Tarantal, Michele L Martinez, Lionel Sanz, Charles I Lee, He Yang, Henriette O'Geen, Dennis J Hartigan-O'Connor, David J Segal","doi":"10.1177/10430342261438941","DOIUrl":"https://doi.org/10.1177/10430342261438941","url":null,"abstract":"<p><p>Translational development of somatic cell genome editing requires monitoring the extent of editing in the body at a given time, the specificity of editing, durability, and the potential for adverse events. A noninvasive approach that can identify edited cells <i>in vivo</i> is beneficial for addressing these and related questions. These studies used total-body positron emission tomography (PET) to identify somatic cell gene editing <i>in vivo</i> in fetal rhesus macaques. Rhesus dams were screened to confirm they were seronegative for AAV serotype 9 and <i>Sa</i>Cas9 antibodies, then selected for the study. Fetuses were administered a dual imaging vector (AAV9/<i>Sa</i>Cas9 and AAV9/<i>PCSK9</i> gRNA/HSV-sr39TK) <i>in utero</i> using an ultrasound-guided fetal intrahepatic approach in the second trimester (10¹² vector genomes/fetus). After maternal intravenous administration of 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) (∼3 mCi/kg), PET imaging was performed in the second and third trimesters. PET imaging provided evidence of editing in the fetal liver, which was sustained. Appropriate insertion of the promoterless HSV-sr39TK reporter in frame with the <i>PCSK9</i> gene was confirmed in the fetal liver near term using RNA sequencing, and correctly targeted insertions were observed. These studies have shown that total-body PET can provide insights into gene-edited somatic cells <i>in utero</i> and without evidence of adverse effects.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261438941"},"PeriodicalIF":4.0,"publicationDate":"2026-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147689433","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>Corrigendum to:</i> Leveraging CRISPR-Cas9 for Accurate Detection of AAV-Neutralizing Antibodies: The AAV-HDR Method.","authors":"","doi":"10.1177/10430342261436594","DOIUrl":"https://doi.org/10.1177/10430342261436594","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"10430342261436594"},"PeriodicalIF":4.0,"publicationDate":"2026-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147638621","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":"Preferential Encapsulation of DNA Impurities Derived from pAAV Plasmid Backbone into Recombinant Adeno-Associated Virus Capsids.","authors":"Yuzhe Yuan, Kiyoko Higashiyama, Emi Ito-Kudo, Kyoko Masumi-Koizumi, Keisuke Yusa, Kazuhisa Uchida","doi":"10.1177/10430342251415385","DOIUrl":"10.1177/10430342251415385","url":null,"abstract":"<p><p>Recombinant adeno-associated viruses (rAAVs) are useful vectors for clinical gene therapy. It is crucial to examine DNA impurities, such as plasmid DNA, for quality control of rAAV products. In this study, we examined highly purified rAAV1, rAAV2, rAAV5, and rAAV6 samples produced on a three-plasmid platform, using a high-throughput sequencer. These samples contained 0.49-3.80% detectable DNA impurities derived from the three plasmids, as estimated by the ampicillin resistance gene (<i>amp</i>^R). The plasmid impurities consisted of 90.62-95.84% pAAV, 3.21-6.83% pRC, and 0.95-2.55% pHelper DNA. These trends were consistent with those of DNA impurities determined by droplet digital PCR (ddPCR), indicating that cleaved pAAV backbone DNA was the primary source of DNA impurities encapsulated into the capsids. To examine the preferential encapsulation of pAAV backbone DNA into capsids, short sequencing reads were mapped to the entire pAAV backbone sequence, and we found that the reads were relatively evenly distributed across the backbone sequence, with occasional sharp drops. Furthermore, the read length distribution containing the pAAV backbone sequence showed a main peak at 3.2 kb in Oxford Nanopore Technologies sequencing. This length was consistent with that of backbone DNA nicked at two terminal resolution sites by Rep78/68. Analysis of the terminal sequences of the long reads containing backbone sequences revealed that 92.7-98.4% of them contained Rep-binding elements. These results indicate that the pAAV backbone in linear DNA form was cleaved from pAAV-ZsGreen1 by Rep78/68 nicking at <i>trs</i> in the nucleus, and that the single-stranded DNA was efficiently translocated into the capsids through the encapsulating machinery, similar to the rAAV genome.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"349-364"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146142213","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":"Comparative Analysis of Induced Pancreatic Stem Cells Generated with Different Factors.","authors":"Hirofumi Noguchi, Chika Miyagi-Shiohira, Takuya Sadahira, Masami Watanabe, Issei Saitoh","doi":"10.1177/10430342251414882","DOIUrl":"10.1177/10430342251414882","url":null,"abstract":"<p><p>Recently, our research group generated induced tissue-specific stem/progenitor (iTS/iTP) cells. Compared with induced pluripotent stem (iPS) cells, iTS/iTP cells offer several advantages, that is, easy generation, higher differentiation efficiency, and no teratoma formation. In this study, iTS cells were generated from mouse pancreatic tissues (iTS-P cells) using two different methods. Plasmid vectors were used for expressing <i>Oct3/4</i>, <i>Sox2</i>, <i>Klf4</i>, <i>c-Myc</i> (<i>OSKM</i>), or <i>Yap1</i> (<i>YAP</i>) to evaluate the efficiency and differentiation potential of the resulting cells. No significant difference in reprogramming efficiency between the <i>OSKM</i>- and <i>YAP</i>-based methods was observed. Among the established clones, iTS-P OSKM2 and iTS-P YAP9 cells, which demonstrated high efficiency in differentiating to insulin-producing cells (IPCs), were selected for further comparison. Both iTS-P OSKM2 and iTS-P YAP9 cells expressed genetic markers of endoderm and pancreatic progenitors and differentiated into IPCs more efficiently than the embryonic stem (ES) cells. Genomic bisulfite sequencing revealed that the pluripotency factors <i>Oct3/4</i> and <i>Nanog</i> were partially methylated in both iTS-P OSKM2 and iTS-P YAP9 cells. Unsupervised hierarchical clustering of gene expression profiles showed that iTS-P YAP9 cells clustered more closely with the ES cells than with the iTS-P OSKM2 cells. However, the expression levels of <i>Oct3/4</i> and <i>Nanog</i> were significantly lower in both iTS-P OSKM2 and YAP9 cells than in the ES cells. These results conclude that no substantial difference is present in the characteristics between iTS-P cells induced by <i>OSKM</i> or <i>YAP</i>, and that their higher differentiation efficiency than the ES cells indicates promising potential for clinical applications.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"329-340"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146112853","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":"Lilly, Seamless Ink Up-to-$1.12B Hearing Loss Collaboration.","authors":"Alex Philippidis","doi":"10.1177/10430342261426584","DOIUrl":"10.1177/10430342261426584","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":"295-299"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147325297","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}