Pub Date : 2024-06-24DOI: 10.1016/j.omtm.2024.101286
Robert D.E. Clark, Felix Rabito, Ferris T. Munyonho, T. Parks Remcho, Jay K. Kolls
Although the last decade has seen tremendous progress in drugs that treat cystic fibrosis (CF) due to mutations that lead to protein misfolding, there are approximately 8%–10% of subjects with mutations that result in no significant CFTR protein expression demonstrating the need for gene editing or gene replacement with inhaled mRNA or vector-based approaches. A limitation for vector-based approaches is the formation of neutralizing humoral responses. Given that αCD20 has been used to manage post-transplant lymphoproliferative disease in CF subjects with lung transplants, we studied the ability of αCD20 to module both T and B cell responses in the lung to one of the most immunogenic vectors, E1-deleted adenovirus serotype 5. We found that αCD20 significantly blocked luminal antibody responses and efficiently permitted re-dosing. αCD20 had more limited impact on the T cell compartment, but reduced tissue resident memory T cell responses in bronchoalveolar lavage fluid. Taken together, these pre-clinical studies suggest that αCD20 could be re-purposed for lung gene therapy protocols to permit re-dosing.
尽管在过去十年中，治疗囊性纤维化（CF）的药物取得了巨大进步，但仍有约 8%-10%的受试者因基因突变而导致 CFTR 蛋白无明显表达，这表明有必要通过吸入 mRNA 或基于载体的方法进行基因编辑或基因置换。基于载体的方法的一个局限性是会形成中和体液反应。鉴于αCD20已被用于控制CF受试者肺移植后的淋巴组织增生性疾病，我们研究了αCD20在肺部形成T细胞和B细胞对最具免疫原性的载体之一--E1删减腺病毒血清型5--的反应模块的能力。我们发现，αCD20 能显著阻断管腔抗体反应，并有效允许再给药。αCD20 对 T 细胞区的影响较为有限，但能减少支气管肺泡灌洗液中的组织常驻记忆 T 细胞反应。总之，这些临床前研究表明，αCD20 可以重新用于肺基因治疗方案，以允许再次给药。
{"title":"Evaluation of anti-vector immune responses to adenovirus-mediated lung gene therapy and modulation by αCD20","authors":"Robert D.E. Clark, Felix Rabito, Ferris T. Munyonho, T. Parks Remcho, Jay K. Kolls","doi":"10.1016/j.omtm.2024.101286","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101286","url":null,"abstract":"Although the last decade has seen tremendous progress in drugs that treat cystic fibrosis (CF) due to mutations that lead to protein misfolding, there are approximately 8%–10% of subjects with mutations that result in no significant CFTR protein expression demonstrating the need for gene editing or gene replacement with inhaled mRNA or vector-based approaches. A limitation for vector-based approaches is the formation of neutralizing humoral responses. Given that αCD20 has been used to manage post-transplant lymphoproliferative disease in CF subjects with lung transplants, we studied the ability of αCD20 to module both T and B cell responses in the lung to one of the most immunogenic vectors, E1-deleted adenovirus serotype 5. We found that αCD20 significantly blocked luminal antibody responses and efficiently permitted re-dosing. αCD20 had more limited impact on the T cell compartment, but reduced tissue resident memory T cell responses in bronchoalveolar lavage fluid. Taken together, these pre-clinical studies suggest that αCD20 could be re-purposed for lung gene therapy protocols to permit re-dosing.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141553041","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}
Pub Date : 2024-06-22DOI: 10.1016/j.omtm.2024.101287
Daima Bukini, Julie Makani, Joseph McCune, Dennis Lee, Cathy Bansbach, Serena De Vita, Dominic Kemps, Elianna Amin, Jonathan Spector, John Tisdale
Therapeutic innovation to address sickle cell disease (SCD) is at an historical apex, characterized by a drug discovery, development, and commercialization landscape that includes potentially curative gene therapies. Given the wide geographic distribution of SCD, with a major presence in Africa, it is imperative that new medicines are designed to meet the specific needs of persons with SCD everywhere. Target product profiles (TPPs) detail the desired attributes of new medicines and serve as a guide for drug developers. To support research efforts for curative treatments for SCD, we mobilized a large multi-disciplinary expert group to generate consensus-driven TPPs for and SCD gene therapies, utilizing a modified Delphi methodology supplemented with virtual workshops. The main findings are TPPs that describe 20 minimal and optimal criteria for novel gene therapy products in categories of scope (3 criteria), performance/safety (11 criteria), manufacturing (4 criteria), and administration (2 criteria). TPPs for and products differed in some performance/safety criteria and all criteria pertaining to manufacturing and administration. These outputs will ideally support development of durable treatments that are safe, efficacious, and practical for persons with SCD in global settings.
{"title":"Consensus-driven target product profiles for curative sickle cell disease gene therapies","authors":"Daima Bukini, Julie Makani, Joseph McCune, Dennis Lee, Cathy Bansbach, Serena De Vita, Dominic Kemps, Elianna Amin, Jonathan Spector, John Tisdale","doi":"10.1016/j.omtm.2024.101287","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101287","url":null,"abstract":"Therapeutic innovation to address sickle cell disease (SCD) is at an historical apex, characterized by a drug discovery, development, and commercialization landscape that includes potentially curative gene therapies. Given the wide geographic distribution of SCD, with a major presence in Africa, it is imperative that new medicines are designed to meet the specific needs of persons with SCD everywhere. Target product profiles (TPPs) detail the desired attributes of new medicines and serve as a guide for drug developers. To support research efforts for curative treatments for SCD, we mobilized a large multi-disciplinary expert group to generate consensus-driven TPPs for and SCD gene therapies, utilizing a modified Delphi methodology supplemented with virtual workshops. The main findings are TPPs that describe 20 minimal and optimal criteria for novel gene therapy products in categories of scope (3 criteria), performance/safety (11 criteria), manufacturing (4 criteria), and administration (2 criteria). TPPs for and products differed in some performance/safety criteria and all criteria pertaining to manufacturing and administration. These outputs will ideally support development of durable treatments that are safe, efficacious, and practical for persons with SCD in global settings.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141551962","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}
Pub Date : 2024-06-08DOI: 10.1016/j.omtm.2024.101278
Alok Tanala Patra, Evan Tan, Yee Jiun Kok, Say Kong Ng, Xuezhi Bi
The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.
{"title":"Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells","authors":"Alok Tanala Patra, Evan Tan, Yee Jiun Kok, Say Kong Ng, Xuezhi Bi","doi":"10.1016/j.omtm.2024.101278","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101278","url":null,"abstract":"The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141529693","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}
Pub Date : 2024-05-29DOI: 10.1016/j.omtm.2024.101273
Amit Chhabra, George Bashirians, Christos J. Petropoulos, Terri Wrin, Yuvika Paliwal, Peter V. Henstock, Suryanarayan Somanathan, Candida da Fonseca Pereira, Ian Winburn, John E.J. Rasko
Adeno-associated virus (AAV) vectors are promising gene therapy candidates, but pre-existing anti-AAV neutralizing antibodies (NAbs) pose a significant challenge to successful gene delivery. Knowledge of NAb seroprevalence remains limited and inconsistent. We measured activity of NAbs against six clinically relevant AAV serotypes across 10 countries in adults ( = 502) and children ( = 50) using a highly sensitive transduction inhibition assay. NAb prevalence was generally highest for AAV1 and lowest for AAV5. There was considerable variability across countries and geographical regions. NAb prevalence increased with age and was higher in females, participants of Asian ethnicity, and participants in cancer trials. Co-prevalence was most frequently observed between AAV1 and AAV6 and less frequently between AAV5 and other AAVs. Machine learning analyses revealed a unique clustering of AAVs that differed from previous phylogenetic classifications. These results offer insights into the biological relationships between the immunogenicity of AAVs in humans beyond that observed previously using standard clades, which are based on linear capsid sequences. Our findings may inform improved vector design and facilitate the development of AAV vector-mediated clinical gene therapies.
{"title":"Global seroprevalence of neutralizing antibodies against adeno-associated virus serotypes used for human gene therapies","authors":"Amit Chhabra, George Bashirians, Christos J. Petropoulos, Terri Wrin, Yuvika Paliwal, Peter V. Henstock, Suryanarayan Somanathan, Candida da Fonseca Pereira, Ian Winburn, John E.J. Rasko","doi":"10.1016/j.omtm.2024.101273","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101273","url":null,"abstract":"Adeno-associated virus (AAV) vectors are promising gene therapy candidates, but pre-existing anti-AAV neutralizing antibodies (NAbs) pose a significant challenge to successful gene delivery. Knowledge of NAb seroprevalence remains limited and inconsistent. We measured activity of NAbs against six clinically relevant AAV serotypes across 10 countries in adults ( = 502) and children ( = 50) using a highly sensitive transduction inhibition assay. NAb prevalence was generally highest for AAV1 and lowest for AAV5. There was considerable variability across countries and geographical regions. NAb prevalence increased with age and was higher in females, participants of Asian ethnicity, and participants in cancer trials. Co-prevalence was most frequently observed between AAV1 and AAV6 and less frequently between AAV5 and other AAVs. Machine learning analyses revealed a unique clustering of AAVs that differed from previous phylogenetic classifications. These results offer insights into the biological relationships between the immunogenicity of AAVs in humans beyond that observed previously using standard clades, which are based on linear capsid sequences. Our findings may inform improved vector design and facilitate the development of AAV vector-mediated clinical gene therapies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141516443","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}
Pub Date : 2024-04-27DOI: 10.1016/j.omtm.2024.101258
Massimo F. Cau, Francesca Ferraresso, Monica Seadler, Katherine Badior, Youjie Zhang, Laura M. Ketelboeter, Geoffrey Rodriguez, Taylor Chen, Matteo Ferraresso, Amanda Wietrzny, Madelaine Robertson, Amber Haugen, Pieter R. Cullis, Marc de Moya, Mitchell Dyer, Christian J. Kastrup
Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.
{"title":"siRNA-mediated reduction of a circulating protein in swine using lipid nanoparticles","authors":"Massimo F. Cau, Francesca Ferraresso, Monica Seadler, Katherine Badior, Youjie Zhang, Laura M. Ketelboeter, Geoffrey Rodriguez, Taylor Chen, Matteo Ferraresso, Amanda Wietrzny, Madelaine Robertson, Amber Haugen, Pieter R. Cullis, Marc de Moya, Mitchell Dyer, Christian J. Kastrup","doi":"10.1016/j.omtm.2024.101258","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101258","url":null,"abstract":"Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140883094","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}
Pub Date : 2024-04-26DOI: 10.1016/j.omtm.2024.101260
Thomas Williams-Fegredo, Lee Davies, Carol Knevelman, Kyriacos Mitrophanous, James Miskin, Qasim A. Rafiq
Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.
{"title":"Development of novel lipoplex formulation methodologies to improve large-scale transient transfection for lentiviral vector manufacture","authors":"Thomas Williams-Fegredo, Lee Davies, Carol Knevelman, Kyriacos Mitrophanous, James Miskin, Qasim A. Rafiq","doi":"10.1016/j.omtm.2024.101260","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101260","url":null,"abstract":"Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140883138","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}
Pub Date : 2024-04-26DOI: 10.1016/j.omtm.2024.101259
Charlotte A. René, Robin J. Parks
Extracellular vesicles (EVs) have the innate ability to carry proteins, lipids, and nucleic acids between cells, and thus these vesicles have gained much attention as potential therapeutic delivery vehicles. Many strategies have been explored to enhance the loading of specific cargoes of interest into EVs, which could result in the delivery of more therapeutic to recipient cells, thus enhancing therapeutic efficacy. In this review, we discuss the natural biogenesis of EVs, the mechanism by which proteins and nucleic acids are selected for inclusion in EVs, and novel methods that have been employed to enhance loading of specific cargoes into EVs. As well, we discuss biodistribution of administered EVs and summarize clinical trials that have attempted to harness the therapeutic potential of EVs.
{"title":"Bioengineering extracellular vesicle cargo for optimal therapeutic efficiency","authors":"Charlotte A. René, Robin J. Parks","doi":"10.1016/j.omtm.2024.101259","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101259","url":null,"abstract":"Extracellular vesicles (EVs) have the innate ability to carry proteins, lipids, and nucleic acids between cells, and thus these vesicles have gained much attention as potential therapeutic delivery vehicles. Many strategies have been explored to enhance the loading of specific cargoes of interest into EVs, which could result in the delivery of more therapeutic to recipient cells, thus enhancing therapeutic efficacy. In this review, we discuss the natural biogenesis of EVs, the mechanism by which proteins and nucleic acids are selected for inclusion in EVs, and novel methods that have been employed to enhance loading of specific cargoes into EVs. As well, we discuss biodistribution of administered EVs and summarize clinical trials that have attempted to harness the therapeutic potential of EVs.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140883186","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}
Pub Date : 2024-04-26DOI: 10.1016/j.omtm.2024.101257
Joaquin Muriel, Valeriy Lukyanenko, Thomas A. Kwiatkowski, Yi Li, Sayak Bhattacharya, Kassidy K. Banford, Daniel Garman, Hannah R. Bulgart, Roger B. Sutton, Noah Weisleder, Robert J. Bloch
Mutations in the gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca signaling after membrane disruption. The gene encodes 7–8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amendable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed “nanodysferlins,” designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca signaling and that membrane repair does not require these C2 domains.
编码蛋白质 dysferlin 的基因突变会导致多种形式的肌肉萎缩症。在健康的骨骼肌中，dysferlin 集中在横纹肌小管中，参与修复肌浆和稳定膜破坏后的钙离子信号传导。该基因编码 7-8 个 C2 结构域、几个 Fer 和 Dysf 结构域以及一个 C 端跨膜序列。由于其编码序列过于庞大，无法封装在腺相关病毒中，因此全长序列无法采用目前的基因传递方法。因此，我们研究了较小版本的dysferlin，称为 "纳米dysferlin"，旨在消除几个C2结构域，特别是C2结构域D、E和F；B、D和E；以及B、D、E和F。我们发现，虽然这些纳米铁蛋白未能集中在横向小管中，但其中三种支持激光损伤后的膜修复，而所有四种都与膜修复蛋白 TRIM72/MG53 结合，与 WT dysferlin 相似。相反，在肌纤维受到轻度低渗冲击损伤后，它们未能抑制钙波。我们的研究结果表明，正常的微管定位和钙信号转导需要dysferlin的内部C2结构域，而膜修复不需要这些C2结构域。
{"title":"Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling","authors":"Joaquin Muriel, Valeriy Lukyanenko, Thomas A. Kwiatkowski, Yi Li, Sayak Bhattacharya, Kassidy K. Banford, Daniel Garman, Hannah R. Bulgart, Roger B. Sutton, Noah Weisleder, Robert J. Bloch","doi":"10.1016/j.omtm.2024.101257","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101257","url":null,"abstract":"Mutations in the gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca signaling after membrane disruption. The gene encodes 7–8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amendable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed “nanodysferlins,” designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca signaling and that membrane repair does not require these C2 domains.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140889716","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}
Pub Date : 2024-04-24DOI: 10.1016/j.omtm.2024.101255
Davide Monteferrario, Marion David, Satish K. Tadi, Yuanyue Zhou, Irène Marchetti, Caroline Jeanneau, Gaëlle Saviane, Coralie F. Dupont, Angélique E. Martelli, Lynn N. Truong, Jason A. Eshleman, Colman C. Ng, Marshall W. Huston, Gregory D. Davis, Jason D. Fontenot, Andreas Reik, Maurus de la Rosa, David Fenard
Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient and during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.
无需基因编辑的基因沉默技术在开发安全的治疗应用方面具有巨大潜力。在这里，我们描述了一种利用融合了克鲁珀尔相关盒抑制结构域（ZF-Rs）的锌指蛋白同时抑制多个基因的新策略。这是通过优化慢病毒系统实现的，该系统专为在造血细胞中传递 ZF-Rs 而定制。我们的研究表明，慢病毒骨架的优化设计对于复用多达三种 ZF-R 或两种 ZF-R 和一种嵌合抗原受体至关重要。ZF-R 的表达对转导细胞的完整性和功能性没有影响。此外，ZF-R 表达的 T 细胞中的基因抑制是高效的，而且在整个监测期间（长达 10 周）都是如此，同时还伴随着表观遗传重塑事件。最后，我们介绍了一种提高 ZF-R 特异性的方法，以说明产生具有安全临床特征的 ZF-R 的途径。总之，我们成功开发了一种基于表观遗传学的细胞工程方法，可同时调节多个基因的表达，避免了 DNA 编辑带来的风险。
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Pub Date : 2024-04-10DOI: 10.1016/j.omtm.2024.101249
Dan Cappabianca, Dan Pham, Matthew H. Forsberg, Madison Bugel, Anna Tommasi, Anthony Lauer, Jolanta Vidugiriene, Brookelyn Hrdlicka, Alexandria McHale, Quaovi Sodji, Melissa C. Skala, Christian M. Capitini, Krishanu Saha
Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant () gene resulting in -CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 -CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays , and potency against human GD2+ xenograft neuroblastoma models . Compared with standard -CAR T cells, MP -CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1β, IL-17, and TGF-β production at the end of manufacturing , with increased central memory CAR T cells and better persistence observed . MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes , which could lead to better responses against solid tumors .
制造嵌合抗原受体（CAR）T细胞疗法非常复杂，人们对培养基成分如何影响T细胞表型的了解有限。CRISPR-Cas9核糖核蛋白可精确插入CAR序列，同时破坏内源性T细胞受体α常量（）基因，从而产生具有丰富干细胞记忆T细胞群的-CAR T细胞，这一过程可通过改变培养基成分进一步优化。在这项研究中，我们利用 "代谢引物"（MP）生成了抗 GD2 -CAR T 细胞，即在葡萄糖/谷氨酰胺低培养基中激活细胞，然后在葡萄糖/谷氨酰胺高培养基中扩增。通过光谱流式细胞仪、代谢测定、细胞因子产生、细胞毒性测定以及对人类 GD2+ 异种移植神经母细胞瘤模型的效力，对 T 细胞产品进行了评估。与标准 -CAR T 细胞相比，MP -CAR T 细胞的糖酵解较少，CCR7/CD62L 表达较高，结合 NAD(P)H 活性较高，在制造末期 IFN-γ、IL-2、IP-10、IL-1β、IL-17 和 TGF-β 的产生减少，观察到中心记忆 CAR T 细胞增加，持久性更好。在 CAR T 细胞生物制造过程中使用培养基 MP 可以最大限度地减少糖酵解并丰富记忆表型，从而提高对实体瘤的反应。
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