Molecular Therapy-Methods & Clinical Development最新文献

Highly efficient in vivo hematopoietic stem cell transduction using an optimized self-complementary adeno-associated virus.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-21 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101438

Carsten T Charlesworth, Shota Homma, Anais K Amaya, Carla Dib, Sriram Vaidyanathan, Tze-Kai Tan, Masashi Miyauchi, Yusuke Nakauchi, Fabian P Suchy, Sicong Wang, Kyomi J Igarashi, M Kyle Cromer, Amanda M Dudek, Alvaro Amorin, Agnieszka Czechowicz, Adam C Wilkinson, Hiromitsu Nakauchi

{"title":"Highly efficient in vivo hematopoietic stem cell transduction using an optimized self-complementary adeno-associated virus.","authors":"Carsten T Charlesworth, Shota Homma, Anais K Amaya, Carla Dib, Sriram Vaidyanathan, Tze-Kai Tan, Masashi Miyauchi, Yusuke Nakauchi, Fabian P Suchy, Sicong Wang, Kyomi J Igarashi, M Kyle Cromer, Amanda M Dudek, Alvaro Amorin, Agnieszka Czechowicz, Adam C Wilkinson, Hiromitsu Nakauchi","doi":"10.1016/j.omtm.2025.101438","DOIUrl":"10.1016/j.omtm.2025.101438","url":null,"abstract":"In vivo gene therapy targeting hematopoietic stem cells (HSCs) holds significant therapeutic potential for treating hematological diseases. This study uses adeno-associated virus serotype 6 (AAV6) vectors and Cre recombination to systematically optimize the parameters for effective in vivo HSC transduction. We evaluated various genetic architectures and delivery methods of AAV6, establishing an optimized protocol that achieved functional recombination in more than two-thirds of immunophenotypic HSCs. Our findings highlight that second-strand synthesis is a critical limiting factor for transgene expression in HSCs, leading to significant under-detection of HSC transduction with single-stranded AAV6 vectors. We also demonstrate that HSCs in the bone marrow (BM) are readily accessible to transduction, with neither localized injection nor mobilization of HSCs into the bloodstream, enhancing transduction efficacy. Additionally, we observed a surprising preference for HSC transduction over other BM cells, regardless of the AAV6 delivery route. Together, these findings not only underscore the potential of AAV vectors for in vivo HSC gene therapy but also lay a foundation that can inform the development of both in vivo AAV-based HSC gene therapies and potentially in vivo HSC gene therapies that employ alternative delivery modalities.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101438"},"PeriodicalIF":4.6,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11930595/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143702325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Efficient nonviral integration of large transgenes into human T cells using Cas9-CLIPT.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-18 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101437

Anna Tommasi, Dan Cappabianca, Madison Bugel, Kirstan Gimse, Karl Lund-Peterson, Hum Shrestha, Denis Arutyunov, James A Williams, Seshidhar Reddy Police, Venkata Indurthi, Sage Z Davis, Muhammed Murtaza, Christian M Capitini, Krishanu Saha

{"title":"Efficient nonviral integration of large transgenes into human T cells using Cas9-CLIPT.","authors":"Anna Tommasi, Dan Cappabianca, Madison Bugel, Kirstan Gimse, Karl Lund-Peterson, Hum Shrestha, Denis Arutyunov, James A Williams, Seshidhar Reddy Police, Venkata Indurthi, Sage Z Davis, Muhammed Murtaza, Christian M Capitini, Krishanu Saha","doi":"10.1016/j.omtm.2025.101437","DOIUrl":"10.1016/j.omtm.2025.101437","url":null,"abstract":"CRISPR-Cas9 ribonucleoproteins (RNPs) combined with a nucleic acid template encoding a chimeric antigen receptor (CAR) transgene can edit human cells to produce CAR T cells with precise CAR insertion at a single locus. However, many human cells have adverse innate immune responses to foreign nucleic acids, particularly circular double-stranded DNA (dsDNA). Here, we introduce Cleaved, LInearized with Protein Template (Cas9-CLIPT), a circular plasmid containing a single target sequence for the Cas9 RNP, such that during manufacturing, Cas9-RNP binds and cleaves the plasmid to linearize the dsDNA in vitro. Cas9-RNP remains bound to the linearized template and is delivered to cells to promote precise knock-in via homology-directed repair with Cas9-CLIPT. Cas9-CLIPT Nanoplasmids generate up to 1.7-fold higher rates of precise knock-in relative to linearized dsDNA, reaching efficiencies up to 60% with non-homologous end joining inhibition. Cas9-CLIPT-manufactured GD2 TRAC-CAR T cells are potent against GD2+ neuroblastoma cells and exhibit an enriched stem cell memory phenotype. On several electroporation instruments and approaching clinically relevant yields, we successfully manufactured TRAC-CAR T cells using Cas9-CLIPT plasmids containing large (2-6 kb) transgenes. Cas9-CLIPT strategies have the potential to simplify donor template production and integrate large transgenes, allowing for more efficient nonviral manufacturing of multifunctional, genome-edited immune cell therapies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101437"},"PeriodicalIF":4.6,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11930092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143694426","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Why do lipid nanoparticles target the liver? Understanding of biodistribution and liver-specific tropism.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-15 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101436

Mahboubeh Hosseini-Kharat, Kristen E Bremmell, Clive A Prestidge

{"title":"Why do lipid nanoparticles target the liver? Understanding of biodistribution and liver-specific tropism.","authors":"Mahboubeh Hosseini-Kharat, Kristen E Bremmell, Clive A Prestidge","doi":"10.1016/j.omtm.2025.101436","DOIUrl":"10.1016/j.omtm.2025.101436","url":null,"abstract":"Lipid nanoparticles (LNPs) are now highly effective transporters of nucleic acids to the liver. This liver-specificity is largely due to their association with certain serum proteins, most notably apolipoprotein E (ApoE), which directs them to liver cells by binding to the low-density lipoprotein (LDL) receptors on hepatocytes. The liver's distinct anatomy, with its various specialized cell types, also influences how LNPs are taken up from the circulation, cleared, and how effective they are in delivering treatments. In this review, we consider factors that facilitate LNP's effective liver targeting and explore the latest advances in liver-targeted LNP technologies. Understanding how LNPs are targeted to the liver can help for effective design and optimization of nanoparticle-based therapies. Comprehension of the cellular interaction and biodistribution of LNPs not only leads to better treatments for liver diseases but also delivers insight for directing nanoparticles to other tissues, potentially broadening their range of therapeutic applications.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101436"},"PeriodicalIF":4.6,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11919328/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143659688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of efficacy and safety of AAV8-ΔC4ATP7B gene therapy in a mutant mouse model of Wilson's disease.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-13 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101435

Chunhua Zeng, Yunting Lin, Xinshuo Lu, Shehong Chen, Yan Xia, Kangdi Zhang, Yongxian Shao, Zhihong Guan, Rong Du, Zongcai Liu, Mingqi Zhao, Xiaoling Jiang, Yanna Cai, Taolin Li, Xueying Su, Yaoyong Chen, Xiaoyan Dong, Wen Zhang, Li Liu, Wenhao Zhou

{"title":"Evaluation of efficacy and safety of AAV8-ΔC4ATP7B gene therapy in a mutant mouse model of Wilson's disease.","authors":"Chunhua Zeng, Yunting Lin, Xinshuo Lu, Shehong Chen, Yan Xia, Kangdi Zhang, Yongxian Shao, Zhihong Guan, Rong Du, Zongcai Liu, Mingqi Zhao, Xiaoling Jiang, Yanna Cai, Taolin Li, Xueying Su, Yaoyong Chen, Xiaoyan Dong, Wen Zhang, Li Liu, Wenhao Zhou","doi":"10.1016/j.omtm.2025.101435","DOIUrl":"10.1016/j.omtm.2025.101435","url":null,"abstract":"Wilson's disease (WD) is an autosomal recessive disorder caused by pathogenic variants in the ATP7B gene, resulting in the toxic accumulation of copper (Cu). Impaired Cu homeostasis in WD is characterized by low serum ceruloplasmin, excess hepatic Cu, and elevated urinary Cu. WD often presents with hepatic and/or neurological diseases and is fatal if untreated. Adeno-associated virus (AAV)-mediated gene therapy holds promise for WD, but challenges remain in efficacy and safety. Here, we established an Atp7b R780L knockin (KI) mouse model corresponding to the human ATP7B R778L variant and investigated the therapeutic efficacy and safety of liver-targeted AAV8-mediated ATP7B (AAV8-ΔC4ATP7B) gene therapy in this model. The results demonstrated the Atp7b KI/KI mice recapitulated key features of impaired Cu metabolism in WD but had mild liver disease. Ten-week-old Atp7b KI/KI mice received a single-dose of AAV8-ΔC4ATP7B and were sacrificed at 8 or 30 weeks after treatment. Treated Atp7b KI/KI mice showed normalization of serum ceruloplasmin, reduced hepatic Cu, decreased urinary Cu, and reversed liver histopathology. Serum transaminases had a transient increase at 8 weeks after treatment but returned to normal at 30 weeks after treatment. These data provide evidence for the efficacy and safety of AAV8-ΔC4ATP7B in animals, supporting clinical translation to patients with WD.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101435"},"PeriodicalIF":4.6,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11919453/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143659677","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A comprehensive study of AAV tropism across C57BL/6 mice, BALB/c mice, and crab-eating macaques.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-13 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101434

Kailun Fang, Xiali Yang, Yuanhua Liu, Junhui Xia, Ruoxi Wu, Fan Yang, Canbin Feng, Xinyu Liu, Linyu Shi, Guannan Geng, Hui Yang

{"title":"A comprehensive study of AAV tropism across C57BL/6 mice, BALB/c mice, and crab-eating macaques.","authors":"Kailun Fang, Xiali Yang, Yuanhua Liu, Junhui Xia, Ruoxi Wu, Fan Yang, Canbin Feng, Xinyu Liu, Linyu Shi, Guannan Geng, Hui Yang","doi":"10.1016/j.omtm.2025.101434","DOIUrl":"10.1016/j.omtm.2025.101434","url":null,"abstract":"Recombinant adeno-associated viruses (AAVs) have been widely used for gene delivery and gene therapy. However, certain AAV serotypes exhibited distinct transduction patterns among different mouse strains or between mice and non-human primates (NHPs). These variations prompted us to investigate the AAV tropism of 21 capsid variants using barcoded AAV libraries among different tissues in C57BL/6 and BALB/c mice, as well as in crab-eating macaques. Our study unveiled that AAV tropisms varied significantly among different mouse strains and species, particularly in capsid variants such as AAV4, AAV9, PHP.B, and CAP-B10. Notably, AAV4 exhibited liver-detargeting properties in both mice and NHPs, and was remarkably efficient in transducing the lung, glomerulus, and pancreatic islet. These findings furnish crucial insights into the variations of AAV tropism among different mouse strains and species and facilitate the selection of appropriate AAV capsids for target tissues among different mouse strains and in NHPs.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101434"},"PeriodicalIF":4.6,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11919325/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143659675","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Selective RNAi silencing of Schwann cell Piezo1 alleviates mechanical hypersensitization following peripheral nerve injury.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-12 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101433

Brandon Itson-Zoske, Uarda Gani, Alexander Mikesell, Chensheng Qiu, Fan Fan, Cheryl L Stucky, Quinn H Hogan, Seung Min Shin, Hongwei Yu

{"title":"Selective RNAi silencing of Schwann cell Piezo1 alleviates mechanical hypersensitization following peripheral nerve injury.","authors":"Brandon Itson-Zoske, Uarda Gani, Alexander Mikesell, Chensheng Qiu, Fan Fan, Cheryl L Stucky, Quinn H Hogan, Seung Min Shin, Hongwei Yu","doi":"10.1016/j.omtm.2025.101433","DOIUrl":"10.1016/j.omtm.2025.101433","url":null,"abstract":"The present study was designed to investigate the role of Schwann cell (SC) Piezo1 in peripheral nociception. We first developed an AAV vector that has primary SC tropism after delivery into the sciatic (or tibial) nerve. This was achieved by packing AAV-GFP transcribed by a CBA promoter using a capsid AAVolig001 to generate AAVolig001-CBA-GFP. Six weeks after intraneural injection of AAVolig001-CBA-GFP in naive rats, GFP expression was detected selectively in both myelinating SCs (mSCs) and non-myelinating SCs (nmSCs). A dual promoter and bidirectional AAV encoding a U6-driven short hairpin RNA against rat Piezo1 (PZ1shRNA) and CBA-transcribed GFP was packed with capsid olig001 (AAVolig001-PZ1shRNA), and AAV was injected into unilateral sciatic (or tibial) nerve immediately after induction of common peroneal nerve injury (CPNI). Results showed that the development of mechanical hypersensitivity in the CPNI rats injected with AAVolig001-PZ1shRNA was mitigated compared to rats subjected to AAVolig001-scramble. Selective in vivo SC transduction and functional block of Piezo1 channel activity of primary cultured SCs was confirmed. These data demonstrate that (1) AAVolig001 has unique and selective primary tropism to SCs via intraneural delivery, and (2) SC Piezo1 contributes to mechanical hypersensitivity following nerve injury.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101433"},"PeriodicalIF":4.6,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11910156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143651916","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cathepsin B inhibition blocks amyloidogenesis in the mouse models of neurological lysosomal diseases MPS IIIC and sialidosis.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-11 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101432

Gustavo M Viana, Xuefang Pan, Shuxian Fan, TianMeng Xu, Alexandra Wyatt, Alexey V Pshezhetsky

{"title":"Cathepsin B inhibition blocks amyloidogenesis in the mouse models of neurological lysosomal diseases MPS IIIC and sialidosis.","authors":"Gustavo M Viana, Xuefang Pan, Shuxian Fan, TianMeng Xu, Alexandra Wyatt, Alexey V Pshezhetsky","doi":"10.1016/j.omtm.2025.101432","DOIUrl":"https://doi.org/10.1016/j.omtm.2025.101432","url":null,"abstract":"Neuronal accumulation of amyloid aggregates is a hallmark of brain pathology in neurological lysosomal storage diseases (LSDs), including mucopolysaccharidoses (MPS); however, the molecular mechanism underlying this pathology has not been understood. We demonstrate that elevated lysosomal cathepsin B (CTSB) levels and CTSB leakage to the cytoplasm triggers amyloidogenesis in two neurological LSDs. CTSB levels were elevated 3- to 5-fold in the cortices of mouse models of MPS IIIC (Hgsnat-Geo and Hgsnat P304L ) and sialidosis (Neu1 ΔEx3 ), as well as in cortical samples of MPS I, IIIA, IIIC, and IIID patients. CTSB was found in the cytoplasm of pyramidal layer IV-V cortical neurons containing thioflavin-S+, β-amyloid+ aggregates consistent with a pro-senile phenotype. In contrast, CTSB-deficient MPS IIIC (Hgsnat P304L /Ctsb -/- ) mice as well as Hgsnat P304L and Neu1 ΔEx3 mice chronically treated with irreversible brain-penetrable CTSB inhibitor E64 showed a drastic reduction in neuronal thioflavin-S+/APP+ deposits. Neurons of Hgsnat P304L /Ctsb -/- mice and E64-treated Hgsnat P304L mice also showed reduced levels of P62+, LC3+ puncta, GM2 ganglioside, and misfolded subunit C of mitochondrial ATP synthase, consistent with restored autophagy. E64 treatment also rescued hyperactivity and reduced anxiety in Hgsnat P304L mice, implying that CTSB may become a novel pharmacological target for MPS III and similar LSDs.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101432"},"PeriodicalIF":4.6,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11910108/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143651892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Nonclinical study of ixo-vec gene therapy for nAMD supports efficacy for a human dose of 6E10 vg/eye and staggered dosing of fellow eyes.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-10 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101430

Kris Poulsen, Kelly Hanna, Julio Nieves, Ngoc Nguyen, Pallavi Sharma, Ruslan Grishanin, Romu Corbau, Szilárd Kiss

{"title":"Nonclinical study of ixo-vec gene therapy for nAMD supports efficacy for a human dose of 6E10 vg/eye and staggered dosing of fellow eyes.","authors":"Kris Poulsen, Kelly Hanna, Julio Nieves, Ngoc Nguyen, Pallavi Sharma, Ruslan Grishanin, Romu Corbau, Szilárd Kiss","doi":"10.1016/j.omtm.2025.101430","DOIUrl":"https://doi.org/10.1016/j.omtm.2025.101430","url":null,"abstract":"Ixoberogene soroparvovec (ixo-vec), formerly ADVM-022, is an adeno-associated virus (AAV) gene therapy using the AAV.7m8 capsid for intravitreal delivery (IVT) to transduce retinal tissue and produce sustained intraocular aflibercept for treating neovascular age-related macular degeneration (nAMD). Non-clinical studies show that aflibercept production by ixo-vec is less than dose proportional, while intraocular inflammation (IOI) increases with dose, suggesting that lower doses could yield effective aflibercept levels with reduced IOI risk. Our evaluation confirmed that doses as low as 3E10 vg (vector genome)/eye (6E10 vg/eye human equivalent) maintained effective aflibercept production. The concept behind ADVM-022 is supported by clinical studies OPTIC (NCT03748784) and LUNA (NCT05536973), where a single IVT administration eliminated or significantly reduced the need for additional anti-VEGF injections in patients. Moreover, LUNA confirmed the clinical efficacy of a 6E10-vg/eye dose, demonstrating robust and sustained aflibercept levels. Additionally, we evaluated staggered dosing in contralateral eyes to treat asynchronous disease development. Staggered dosing, administered 2 months apart, did not exacerbate IOI, and both eyes maintained therapeutic aflibercept levels. These findings support the tolerability and efficacy of staggered dosing, indicating the potential for bilaterally relevant aflibercept levels with ixo-vec, due to immune response confinement to the dosed eye.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101430"},"PeriodicalIF":4.6,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11910100/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143651910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Deciphering key parameters enhancing lentiviral vector producer cells yields: Vector components copy number and expression. 解密提高慢病毒载体生产细胞产量的关键参数：载体成分的拷贝数和表达量

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-10 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101431

Ana S Formas-Oliveira, Mariana Valentim Ferreira, Ana Sofia Coroadinha

{"title":"Deciphering key parameters enhancing lentiviral vector producer cells yields: Vector components copy number and expression.","authors":"Ana S Formas-Oliveira, Mariana Valentim Ferreira, Ana Sofia Coroadinha","doi":"10.1016/j.omtm.2025.101431","DOIUrl":"10.1016/j.omtm.2025.101431","url":null,"abstract":"The use of lentiviral vectors (LVs) in gene therapy is expanding, demanding high-quality viral preparations. Producer cell lines for LV production offer robust manufacturing platforms. However, their development is still progressing and more knowledge on the impact of vector components expression levels on vector yields and quality is essential. This work studies the impact of vector cassette expression and stability on vector titer and quality, identifying key parameters in cell line development. Ten heterogeneous LV stable producer clones established through random cassette integration were characterized. The gag-pol and rev cassettes, expressed under the control of constitutive promoters, showed robust expression generating titers of 109 physical particles (P.P.s)/mL. However, Pol and reverse transcriptase expressions were shown to be better indicators of potential functional titers. Envelope and transfer vector expression levels were key to attaining high functional particles yields. The stability analysis of two top clones and their trans-complementation with each genetic cassette further supported this conclusion. The producer LV clones expressed constitutively the 4070A envelope, but the overexpression of the VSV-G envelope increased 30-fold the titer supporting the envelope as key determinant in LV quality. This work further elucidates bottlenecks in LV producer cell line development providing insights for their optimization.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101431"},"PeriodicalIF":4.6,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11925171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143671857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterization of VSV-GP morphology by cryo-EM imaging and SEC-MALS. 通过低温电子显微镜成像和 SEC-MALS 分析 VSV-GP 的形态特征。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-06 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101429

Dongyue Xin, Leela Kurien, Katherine Briggs, Adrian Schimek, Richard Dambra, Daniel Hochdorfer, Tanja A Arnouk, Marija Brgles, Saurabh Gautam, Dominik Hotter, Johannes Solzin, Thomas Kriehuber, Joseph Ashour, Adam Vigil, Michael Hawley, Xiaorong He

{"title":"Characterization of VSV-GP morphology by cryo-EM imaging and SEC-MALS.","authors":"Dongyue Xin, Leela Kurien, Katherine Briggs, Adrian Schimek, Richard Dambra, Daniel Hochdorfer, Tanja A Arnouk, Marija Brgles, Saurabh Gautam, Dominik Hotter, Johannes Solzin, Thomas Kriehuber, Joseph Ashour, Adam Vigil, Michael Hawley, Xiaorong He","doi":"10.1016/j.omtm.2025.101429","DOIUrl":"10.1016/j.omtm.2025.101429","url":null,"abstract":"Vesicular stomatitis virus expressing the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) is a promising platform for oncolytic viruses and cancer vaccines. In this work, cryoelectron microscopy (cryo-EM) imaging was employed to directly visualize VSV-GP particles. Several different subpopulations of virus particle morphology were observed. Definition and fraction counting of subpopulations enabled quantitative comparison of subpopulation profiles between several VSV-GP samples. In developing an orthogonal method with higher throughput, we showed that the morphological profile of the VSV-GP particles can be characterized by size exclusion chromatography coupled with a multi-angle light scattering detector (SEC-MALS) based on a novel shape-based separation mechanism. Together, the two complementary techniques enable the analysis of morphological profile for VSV-GP and potentially other non-spherical viruses or nanoparticles.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101429"},"PeriodicalIF":4.6,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11904549/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143625974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0