Molecular Therapy-Methods & Clinical Development最新文献_第10页

Expanded specific T cells to hypomutated regions of the SARS-CoV-2 using mRNA electroporated antigen presenting cells. 利用 mRNA 电穿孔抗原递呈细胞，将特异性 T 细胞扩增到 SARS-CoV-2 的低突变区。

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-22 DOI: 10.1016/j.omtm.2024.101192

Elizabeth Ogando-Rivas, Paul Castillo, Changlin Yang, Vrunda Trivedi, Dingpeng Zhang, Fernanda Pohl-Guimarães, Ruixuan Liu, Arnav Barpujari, Kate M. Candelario, Hector Mendez-Gomez, Elias J. Sayour, Duane A. Mitchell

{"title":"Expanded specific T cells to hypomutated regions of the SARS-CoV-2 using mRNA electroporated antigen presenting cells.","authors":"Elizabeth Ogando-Rivas, Paul Castillo, Changlin Yang, Vrunda Trivedi, Dingpeng Zhang, Fernanda Pohl-Guimarães, Ruixuan Liu, Arnav Barpujari, Kate M. Candelario, Hector Mendez-Gomez, Elias J. Sayour, Duane A. Mitchell","doi":"10.1016/j.omtm.2024.101192","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101192","url":null,"abstract":"The COVID-19 pandemic has caused about seven million deaths worldwide. Preventative vaccines have been developed including Spike gp mRNA-based vaccines that provide protection to immunocompetent patients. However, patients with primary immunodeficiencies, patients with cancer, or hematopoietic stem cell transplant recipients are not able to mount robust immune responses against current vaccine approaches. We propose to target structural SARS-CoV-2 antigens (i.e., Spike gp, Membrane, Nucleocapsid, and Envelope) using circulating human antigen presenting cells electroporated with full length SARS-CoV-2 structural protein-encoding mRNAs to activate and expand specific T cells. Based on the Th1-type cytokine and cytolytic enzyme secretion upon antigen rechallenge, we were able to generate SARS-CoV-2 specific T cells in up to 70% of unexposed unvaccinated healthy donors (HDs) after 3 subsequent stimulations and in 100% of recovered patients (RPs) after 2 stimulations. By means of SARS-CoV-2 specific TCRβ repertoire analysis, T cells specific to Spike gp-derived hypomutated regions were identified in HDs and RPs despite viral genomic evolution. Hence, we demonstrated that SARS-CoV-2 mRNA-loaded antigen presenting cells are effective activating and expanding COVID19-specific T cells. This approach represents an alternative to patients who are not able to mount adaptive immune responses to current COVID19 vaccines with potential protection across new variants that have conserved genetic regions.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139579341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

AAV8 Gene Therapy Reverses Cardiac Pathology and Prevents Early Mortality in a Mouse Model of Friedreich’s Ataxia AAV8 基因疗法可逆转弗里德里希共济失调小鼠模型的心脏病理变化并防止早期死亡

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-22 DOI: 10.1016/j.omtm.2024.101193

Joshua C. Chang, Molly R. Ryan, Marie C. Stark, Su Liu, Pravinkumar Purushothaman, Fria Bolan, Caitlin A. Johnson, Mark Champe, Hui Meng, Michael W. Lawlor, Sarah Halawani, Lucie V. Ngaba, David R. Lynch, Crystal Davis, Elena Gonzalo-Gil, Cathleen Lutz, Fabrizia Urbinati, Bala Medicherla, Carlos Fonck

{"title":"AAV8 Gene Therapy Reverses Cardiac Pathology and Prevents Early Mortality in a Mouse Model of Friedreich’s Ataxia","authors":"Joshua C. Chang, Molly R. Ryan, Marie C. Stark, Su Liu, Pravinkumar Purushothaman, Fria Bolan, Caitlin A. Johnson, Mark Champe, Hui Meng, Michael W. Lawlor, Sarah Halawani, Lucie V. Ngaba, David R. Lynch, Crystal Davis, Elena Gonzalo-Gil, Cathleen Lutz, Fabrizia Urbinati, Bala Medicherla, Carlos Fonck","doi":"10.1016/j.omtm.2024.101193","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101193","url":null,"abstract":"Friedreich’s ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein, frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of FRDA’s cardiac phenotype was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxnflox/null::MCK-Cre conditional knockout mouse (FXN-MCK) has a FXN gene ablation that prevents frataxin expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss and morbidity. FXN-MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN gene under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN-MCK control mice, AAV-treated FXN-MCK mice displayed increases in body weight, reversal of cardiac deficits and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks post dose. Frataxin protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild-type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139552992","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High-titer manufacturing of SARS-CoV-2 spike-pseudotyped VSV in stirred-tank bioreactors 在搅拌罐生物反应器中制造高滴度 SARS-CoV-2 尖峰伪型 VSV

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-17 DOI: 10.1016/j.omtm.2024.101189

Hayley M. Todesco, Chris Gafuik, Cini M. John, Erin L. Roberts, Breanna S. Borys, Alexis Pawluk, Michael S. Kallos, Kyle G. Potts, Douglas J. Mahoney

{"title":"High-titer manufacturing of SARS-CoV-2 spike-pseudotyped VSV in stirred-tank bioreactors","authors":"Hayley M. Todesco, Chris Gafuik, Cini M. John, Erin L. Roberts, Breanna S. Borys, Alexis Pawluk, Michael S. Kallos, Kyle G. Potts, Douglas J. Mahoney","doi":"10.1016/j.omtm.2024.101189","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101189","url":null,"abstract":"The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms were rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped Vesicular Stomatitis Virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 + 0.42 ×106 cells/mL in 1 L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 + 0.58 ×108 plaque-forming units (pfu)/mL at 3 days post-infection. When compared to growth in plate-based cultures this was a 29-fold increase in virus production, meaning a 1 L bioreactor produces the same amount of virus as 1284 15cm plates. Additionally, the omicron BA.1 S-VSV reached a peak titer of 5.58 + 0.35 ×106 pfu/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-pfu ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139506246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Mannose-coupled AAV2: a second generation AAV vector for increased retinal gene therapy efficiency 甘露糖偶联 AAV2：提高视网膜基因治疗效率的第二代 AAV 载体

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-17 DOI: 10.1016/j.omtm.2024.101187

Mathieu Mével, Virginie Pichard, Mohammed Bouzelha, Dimitri Alvarez-Dorta, Pierre-Alban Lalys, Nathalie Provost, Marine Allais, Alexandra Mendes, Elodie Landagaray, Jean-Baptiste Ducloyer, Estelle Toublanc, Anne Galy, Nicole Brument, Gaëlle M. Lefevre, Sébastien G. Gouin, Carolina Isiegas, Guylène Le Meur, Thérèse Cronin, Caroline Le Guiner, Michel Weber, Oumeya Adjali

{"title":"Mannose-coupled AAV2: a second generation AAV vector for increased retinal gene therapy efficiency","authors":"Mathieu Mével, Virginie Pichard, Mohammed Bouzelha, Dimitri Alvarez-Dorta, Pierre-Alban Lalys, Nathalie Provost, Marine Allais, Alexandra Mendes, Elodie Landagaray, Jean-Baptiste Ducloyer, Estelle Toublanc, Anne Galy, Nicole Brument, Gaëlle M. Lefevre, Sébastien G. Gouin, Carolina Isiegas, Guylène Le Meur, Thérèse Cronin, Caroline Le Guiner, Michel Weber, Oumeya Adjali","doi":"10.1016/j.omtm.2024.101187","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101187","url":null,"abstract":"Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139506345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia 在半乳糖血症小鼠模型中，将全身半乳糖氧化作为一种稳健的功能检测方法来评估基于基因的疗法的疗效

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-17 DOI: 10.1016/j.omtm.2024.101191

Bijina Balakrishnan, Xinhua Yan, Marshall D. McCue, Olivia Bellagamba, Aaron Guo, Felicity Winkler, Jason Thall, Lisa Crawford, Rain Dimen, Sara Chen, Sean McEnaney, Yiman Wu, Mike Zimmer, Joe Sarkis, Paolo GV. Martini, Patrick F. Finn, Kent Lai

{"title":"Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia","authors":"Bijina Balakrishnan, Xinhua Yan, Marshall D. McCue, Olivia Bellagamba, Aaron Guo, Felicity Winkler, Jason Thall, Lisa Crawford, Rain Dimen, Sara Chen, Sean McEnaney, Yiman Wu, Mike Zimmer, Joe Sarkis, Paolo GV. Martini, Patrick F. Finn, Kent Lai","doi":"10.1016/j.omtm.2024.101191","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101191","url":null,"abstract":"Despite the implementation of life-saving newborn screening programs and a galactose-restricted diet, many patients with Classic Galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which not only decreased galactose-1 phosphate (gal-1P) in the liver, but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, non-invasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of Galactosemia. While our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that target the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139510387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection. 在 HEK293 细胞培养转染过程中，细胞外囊泡耗竭和 UGCG 过表达可减轻细胞密度效应。

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-17 DOI: 10.1016/j.omtm.2024.101190

Pol Pérez-Rubio, Jesús Lavado-García, Laia Bosch-Molist, Elianet Lorenzo Romero, Laura Cervera, Francesc Gòdia

{"title":"Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection.","authors":"Pol Pérez-Rubio, Jesús Lavado-García, Laia Bosch-Molist, Elianet Lorenzo Romero, Laura Cervera, Francesc Gòdia","doi":"10.1016/j.omtm.2024.101190","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101190","url":null,"abstract":"The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compounds in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV-depletion from the extracellular medium restored transfection efficiency in conditions suffering from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UGCG enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV-depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12x106 cells/mL. These results suggest that the interaction between polyplexes and extracelluar and intracellular vesicles plays a crutial role in the CDE, providing insights for the development of strategies to mitigate its impact.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139506342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Recombinant AAV Genome Size Effect on Viral Vector Production, Purification, and Thermostability 重组 AAV 基因组大小对病毒载体生产、纯化和耐热性的影响

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-17 DOI: 10.1016/j.omtm.2024.101188

Nermin Ibreljic, Benjamin E. Draper, Carl W. Lawton

{"title":"Recombinant AAV Genome Size Effect on Viral Vector Production, Purification, and Thermostability","authors":"Nermin Ibreljic, Benjamin E. Draper, Carl W. Lawton","doi":"10.1016/j.omtm.2024.101188","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101188","url":null,"abstract":"Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. This work studied the effect of the genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension adapted HEK293 cells via triple transfection using AAV plasmids containing the same green fluorescent protein (GFP) transgene with DNA stuffers for variable size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography and the results suggest that the triple transfection condition significantly impacts the AEX retention time and resolution between the full and empty capsid peaks. Charge detection mass spectrometry (CD-MS) was performed on all AEX full capsid peak samples showing a wide distribution of empty, partial, full length, and co-packaged DNA in the capsids. The AEX purified samples were then analyzed by differential scanning fluorimetry (DSF) and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139510188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

New perspectives for gene therapy of the X-linked form of Charcot-Marie-Tooth disease X连锁型夏科-玛丽-牙病基因疗法的新前景

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-12 DOI: 10.1016/j.omtm.2023.101184

Rafael Balada Caballé, Mario Bortolozzi

引用次数: 0

Can mitochondria brown the lower-limb adipocytes? 线粒体能使下肢脂肪细胞变褐吗？

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-05 DOI: 10.1016/j.omtm.2023.101181

Ilias P. Doulamis, Aspasia Tzani

引用次数: 0

Genetic surgery for a cystic fibrosis-causing splicing mutation 针对导致囊性纤维化的剪接突变的基因手术

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-02 DOI: 10.1016/j.omtm.2023.101177

Mattijs Bulcaen, Marianne S. Carlon

引用次数: 0