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":"14 1","pages":""},"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":"24 1","pages":""},"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":"25 1","pages":""},"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。
{"title":"Epigenetic control of multiple genes with a single lentiviral vector encoding transcriptional repressors fused to compact zinc finger arrays","authors":"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","doi":"10.1016/j.omtm.2024.101255","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101255","url":null,"abstract":"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.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"17 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841572","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 : 2024-04-10DOI: 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。
{"title":"Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence","authors":"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","doi":"10.1016/j.omtm.2024.101249","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101249","url":null,"abstract":"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 .","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"140 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140636895","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 : 2024-04-06DOI: 10.1016/j.omtm.2024.101248
Emilie Audouard, Nicolas Khefif, Charlotte Mansat, Océane Nelcha, Elena-Gaia Banchi, Camille Lupiet, Dominique Farabos, Antonin Lamaziere, Caroline Sevin, Françoise Piguet
Metachromatic leukodystrophy (MLD) is a rare, autosomal recessive neurodegenerative disease caused by deficient activity of the lysosomal enzyme arylsulfatase A (ARSA), resulting in sulfatide accumulation and subsequent demyelination and neuronal damage within the central and peripheral nervous systems. Three clinical forms of MLD have been described, based on age at symptom onset. The most frequent and severe forms have an early onset, with the disease progressing rapidly toward severe motor and cognitive regression and ultimately premature death. There are currently no approved therapies for most of these early-onset patients once symptoms are present. Thus, it is crucial to develop new approaches to treat symptomatic patients. Here, we proposed a gene therapy approach based on the intravenous delivery of AAVPHP.eB encoding ARSA. MLD mice were treated at 6 months for a dose-response study and at 9 months to assess late-treatment efficacy. Therapeutic efficacy was evaluated 3 or 6 months after injection. We demonstrated a broad transduction in the central nervous system, a complete correction of sulfatide storage, and a significant improvement in neuroinflammation at low dose and late treatment. Taken together, this work establishes a strong rationale for proposing a phase I/II clinical trial in MLD patients.
{"title":"Dose-response evaluation of intravenous gene therapy in a symptomatic mouse model of metachromatic leukodystrophy","authors":"Emilie Audouard, Nicolas Khefif, Charlotte Mansat, Océane Nelcha, Elena-Gaia Banchi, Camille Lupiet, Dominique Farabos, Antonin Lamaziere, Caroline Sevin, Françoise Piguet","doi":"10.1016/j.omtm.2024.101248","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101248","url":null,"abstract":"Metachromatic leukodystrophy (MLD) is a rare, autosomal recessive neurodegenerative disease caused by deficient activity of the lysosomal enzyme arylsulfatase A (ARSA), resulting in sulfatide accumulation and subsequent demyelination and neuronal damage within the central and peripheral nervous systems. Three clinical forms of MLD have been described, based on age at symptom onset. The most frequent and severe forms have an early onset, with the disease progressing rapidly toward severe motor and cognitive regression and ultimately premature death. There are currently no approved therapies for most of these early-onset patients once symptoms are present. Thus, it is crucial to develop new approaches to treat symptomatic patients. Here, we proposed a gene therapy approach based on the intravenous delivery of AAVPHP.eB encoding ARSA. MLD mice were treated at 6 months for a dose-response study and at 9 months to assess late-treatment efficacy. Therapeutic efficacy was evaluated 3 or 6 months after injection. We demonstrated a broad transduction in the central nervous system, a complete correction of sulfatide storage, and a significant improvement in neuroinflammation at low dose and late treatment. Taken together, this work establishes a strong rationale for proposing a phase I/II clinical trial in MLD patients.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"79 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140569379","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}
Adeno-associated virus (AAV) is an optimal gene vector for monogenic disorders. However, neutralizing antibodies (Nabs) against AAV hinder its widespread application in gene therapy. In this study, we biosynthesized peptides recognized by the binding antibodies (Babs) from the sera containing high Nab titers against AAV2. We established four immunological methods to detect immune epitopes of the AAV2-derived peptides, including a Bab assay, Nab assay, B cell receptor (BCR) detecting assay, and immunoglobin-producing B cell enzyme-linked immunosorbent spot (B cell ELISpot) assay. Correlations among the epitopes determined by these four methods were analyzed using the serum samples and peripheral blood mononuclear cells from 89 patients with hemophilia A/B. As decoys, the peptides’ ability to block the Nab of AAV2 particles was assessed using AAV transduction models both and . Overall, we provide insights into AAV2-capsid-derived peptide immune epitopes, involving the Nab, Bab, BCR, and B cell ELISpot assays, offering alternative immunological evaluation approaches and strategies to overcome Nab barriers in AAV-mediated gene therapy.
{"title":"An investigation of the immune epitope properties of adeno-associated virus capsid-derived peptides among hemophilia patients","authors":"Li Liu, Bingqi Xu, Lingling Chen, Jia Liu, Wei Liu, Feng Xue, Sizhou Feng, Erlie Jiang, Mingzhe Han, Wenwei Shao, Lei Zhang, Xiaolei Pei","doi":"10.1016/j.omtm.2024.101245","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101245","url":null,"abstract":"Adeno-associated virus (AAV) is an optimal gene vector for monogenic disorders. However, neutralizing antibodies (Nabs) against AAV hinder its widespread application in gene therapy. In this study, we biosynthesized peptides recognized by the binding antibodies (Babs) from the sera containing high Nab titers against AAV2. We established four immunological methods to detect immune epitopes of the AAV2-derived peptides, including a Bab assay, Nab assay, B cell receptor (BCR) detecting assay, and immunoglobin-producing B cell enzyme-linked immunosorbent spot (B cell ELISpot) assay. Correlations among the epitopes determined by these four methods were analyzed using the serum samples and peripheral blood mononuclear cells from 89 patients with hemophilia A/B. As decoys, the peptides’ ability to block the Nab of AAV2 particles was assessed using AAV transduction models both and . Overall, we provide insights into AAV2-capsid-derived peptide immune epitopes, involving the Nab, Bab, BCR, and B cell ELISpot assays, offering alternative immunological evaluation approaches and strategies to overcome Nab barriers in AAV-mediated gene therapy.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"13 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140569378","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 : 2024-03-22DOI: 10.1016/j.omtm.2024.101242
Silja Hansen Haldrup, Bjørn K. Fabian-Jessing, Thomas Stax Jakobsen, Anna Bøgh Lindholm, Rikke L. Adsersen, Lars Aagaard, Toke Bek, Anne Louise Askou, Thomas J. Corydon
Neovascular age-related macular degeneration (nAMD) is a frequent cause of vision loss among the elderly in the Western world. Current disease management with repeated injections of anti-VEGF agents accumulates the risk for adverse events and constitutes a burden for society and the individual patient. Sustained suppression of VEGF using gene therapy is an attractive alternative, which we explored using adeno-associated virus (AAV)-based delivery of novel RNA interference (RNAi) effectors in a porcine model of choroidal neovascularization (CNV). The potency of -targeting, Ago2-dependent short hairpin RNAs placed in pri-microRNA scaffolds (miR-agshRNA) was established and in mice. Subsequently, AAV serotype 8 (AAV2.8) vectors encoding -targeting or irrelevant miR-agshRNAs under the control of a tissue-specific promotor were delivered to the porcine retina via subretinal injection before CNV induction by laser. Notably, -targeting miR-agshRNAs resulted in a significant and sizable reduction of CNV compared with the non-targeting control. We also demonstrated that single-stranded and self-complementary AAV2.8 vectors efficiently transduce porcine retinal pigment epithelium cells but differ in their transduction characteristics and retinal safety. Collectively, our data demonstrated a robust anti-angiogenic effect of -targeting miR-aghsRNAs in a large translational animal model, thereby suggesting AAV-based delivery of anti-VEGFA RNAi therapeutics as a valuable tool for the management of nAMD.
{"title":"Subretinal AAV delivery of RNAi-therapeutics targeting VEGFA reduces choroidal neovascularization in a large animal model","authors":"Silja Hansen Haldrup, Bjørn K. Fabian-Jessing, Thomas Stax Jakobsen, Anna Bøgh Lindholm, Rikke L. Adsersen, Lars Aagaard, Toke Bek, Anne Louise Askou, Thomas J. Corydon","doi":"10.1016/j.omtm.2024.101242","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101242","url":null,"abstract":"Neovascular age-related macular degeneration (nAMD) is a frequent cause of vision loss among the elderly in the Western world. Current disease management with repeated injections of anti-VEGF agents accumulates the risk for adverse events and constitutes a burden for society and the individual patient. Sustained suppression of VEGF using gene therapy is an attractive alternative, which we explored using adeno-associated virus (AAV)-based delivery of novel RNA interference (RNAi) effectors in a porcine model of choroidal neovascularization (CNV). The potency of -targeting, Ago2-dependent short hairpin RNAs placed in pri-microRNA scaffolds (miR-agshRNA) was established and in mice. Subsequently, AAV serotype 8 (AAV2.8) vectors encoding -targeting or irrelevant miR-agshRNAs under the control of a tissue-specific promotor were delivered to the porcine retina via subretinal injection before CNV induction by laser. Notably, -targeting miR-agshRNAs resulted in a significant and sizable reduction of CNV compared with the non-targeting control. We also demonstrated that single-stranded and self-complementary AAV2.8 vectors efficiently transduce porcine retinal pigment epithelium cells but differ in their transduction characteristics and retinal safety. Collectively, our data demonstrated a robust anti-angiogenic effect of -targeting miR-aghsRNAs in a large translational animal model, thereby suggesting AAV-based delivery of anti-VEGFA RNAi therapeutics as a valuable tool for the management of nAMD.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"115 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140569733","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 : 2024-03-18DOI: 10.1016/j.omtm.2024.101241
Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics
While recombinant Adenoviruses are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated terminal resolution aiding high-efficiency rescue of recombinant Adenoviruses from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based recombinant Adenoviruses libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement allows the introduction of >10ˆ6 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5x10ˆ4 unique recombinant Adenoviruses per cmˆ2 of transfected cell culture.
虽然重组腺病毒被广泛应用于实验室和医学基因转移，但使用这种载体平台的基于文库的应用并不容易获得。最近，我们开发了一种新方法--CRISPR/Cas9 介导的末端解析，有助于从重组 DNA 中高效拯救重组腺病毒。在此，我们报告了一种基因工作流程，它允许利用高效的末端解析构建基于 BAC 的重组腺病毒文库。我们利用频繁出现的、预先存在的基因组序列来插入选择标记，将两个选定的目标位点补充到新型内切酶识别位点中。第二步，通过 Gibson 组装，用转基因或感兴趣的突变取代选择标记。我们的方法不会造成不必要的基因组脱靶突变，同时为基因修饰的位点和性质提供了极大的灵活性。我们将这种新的基因工作流程称为半位定向片段置换，它可以利用实验室规模的方法在 rAd 编码 BAC 中引入大于 10ˆ6 的独特修饰。为了证明 HFR 的威力，我们拯救了条形码病毒载体文库，在每厘米 2 的转染细胞培养物中产生了 2.5x10ˆ4 种独特的重组腺病毒。
{"title":"Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries","authors":"Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics","doi":"10.1016/j.omtm.2024.101241","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101241","url":null,"abstract":"While recombinant Adenoviruses are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated terminal resolution aiding high-efficiency rescue of recombinant Adenoviruses from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based recombinant Adenoviruses libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement allows the introduction of >10ˆ6 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5x10ˆ4 unique recombinant Adenoviruses per cmˆ2 of transfected cell culture.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"126 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140150515","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 : 2024-03-13DOI: 10.1016/j.omtm.2024.101220
Sonal Bhatia, Yann Le Cam, Juan Carrion, Lauren Diamond, Paul Fennessy, Safiyya Gassman, Felix Gutzwiller, Stephen Kagan, Diana Pankevich, Jennifer Young Maloney, Nitin Mahadev, Martin Schulz, Durhane Wong-Rieger, Paolo Morgese
{"title":"Strengthening health systems for access to gene therapy in rare genetic disorders","authors":"Sonal Bhatia, Yann Le Cam, Juan Carrion, Lauren Diamond, Paul Fennessy, Safiyya Gassman, Felix Gutzwiller, Stephen Kagan, Diana Pankevich, Jennifer Young Maloney, Nitin Mahadev, Martin Schulz, Durhane Wong-Rieger, Paolo Morgese","doi":"10.1016/j.omtm.2024.101220","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101220","url":null,"abstract":"","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"2013 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140156559","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}
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