Pub Date : 2024-03-18DOI: 10.1016/j.omtn.2024.102161
Taiki Matsubayashi, Kotaro Yoshioka, Su Su Lei Mon, Maho Katsuyama, Chunyan Jia, Takao Yamaguchi, Rintaro Iwata Hara, Tetsuya Nagata, Osamu Nakagawa, Satoshi Obika, Takanori Yokota
An increasing number of antisense oligonucleotides (ASOs) have been approved for clinical use. However, improvements of both efficacy and safety in the central nervous system (CNS) are crucial for the treatment with CNS diseases. We aimed to overcome the crucial issues by our development of various gapmer ASOs with a novel nucleoside derivative including a 2′,4′-BNA/LNA with 9-(aminoethoxy)phenoxazine (BNAP-AEO). The various gapmer ASOs with BNAP-AEO were evaluated for thermal stability, and efficacy, and acute CNS toxicity. Thermal stability analysis of the duplexes with their complementary RNAs showed that ASOs with BNAP-AEO had a higher binding affinity than those without BNAP-AEO. assays, when transfected into neuroblastoma cell lines, demonstrated that ASOs with BNAP-AEO, had a more efficient gene silencing effect than those without BNAP-AEO. assays, involving intracerebroventricular injections into mice, revealed ASOs with BNAP-AEO potently suppressed gene expression in the brain. Surprisingly, the acute CNS toxicity in mice, as assessed through open field tests and scoring systems, was significantly lower for ASOs with BNAP-AEO than for those without BNAP-AEO. This study underscores the efficient gene-silencing effect and low acute CNS toxicity of ASOs incorporating BNAP-AEO, indicating the potential for future therapeutic applications.
越来越多的反义寡核苷酸（ASO）已被批准用于临床。然而，提高在中枢神经系统（CNS）中的疗效和安全性对于中枢神经系统疾病的治疗至关重要。为了解决这些关键问题，我们开发了各种间隙聚合 ASO，其中包括一种新型核苷衍生物，包括含有 9-（氨基乙氧基）吩噁嗪的 2′,4′-BNA/LNA（BNAP-AEO）。对含有 BNAP-AEO 的各种间隙聚合物 ASO 的热稳定性、药效和急性中枢神经系统毒性进行了评估。转染到神经母细胞瘤细胞系的实验表明，与不含 BNAP-AEO 的 ASOs 相比，含 BNAP-AEO 的 ASOs 具有更有效的基因沉默效应。令人惊讶的是，通过开放性现场试验和评分系统评估，含有 BNAP-AEO 的 ASOs 对小鼠急性中枢神经系统的毒性明显低于不含 BNAP-AEO 的 ASOs。这项研究强调了含有 BNAP-AEO 的 ASOs 具有高效的基因沉默效应和较低的急性中枢神经系统毒性，显示了其在未来治疗应用中的潜力。
{"title":"Favorable efficacy and reduced acute neurotoxicity by antisense oligonucleotides with 2′,4′-BNA/LNA with 9-(aminoethoxy)phenoxazine","authors":"Taiki Matsubayashi, Kotaro Yoshioka, Su Su Lei Mon, Maho Katsuyama, Chunyan Jia, Takao Yamaguchi, Rintaro Iwata Hara, Tetsuya Nagata, Osamu Nakagawa, Satoshi Obika, Takanori Yokota","doi":"10.1016/j.omtn.2024.102161","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102161","url":null,"abstract":"An increasing number of antisense oligonucleotides (ASOs) have been approved for clinical use. However, improvements of both efficacy and safety in the central nervous system (CNS) are crucial for the treatment with CNS diseases. We aimed to overcome the crucial issues by our development of various gapmer ASOs with a novel nucleoside derivative including a 2′,4′-BNA/LNA with 9-(aminoethoxy)phenoxazine (BNAP-AEO). The various gapmer ASOs with BNAP-AEO were evaluated for thermal stability, and efficacy, and acute CNS toxicity. Thermal stability analysis of the duplexes with their complementary RNAs showed that ASOs with BNAP-AEO had a higher binding affinity than those without BNAP-AEO. assays, when transfected into neuroblastoma cell lines, demonstrated that ASOs with BNAP-AEO, had a more efficient gene silencing effect than those without BNAP-AEO. assays, involving intracerebroventricular injections into mice, revealed ASOs with BNAP-AEO potently suppressed gene expression in the brain. Surprisingly, the acute CNS toxicity in mice, as assessed through open field tests and scoring systems, was significantly lower for ASOs with BNAP-AEO than for those without BNAP-AEO. This study underscores the efficient gene-silencing effect and low acute CNS toxicity of ASOs incorporating BNAP-AEO, indicating the potential for future therapeutic applications.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"48 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140147265","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.omtn.2024.102175
Hamideh Parhiz, Vladimir V. Shuvaev, Qin Li, Tyler E. Papp, Awurama A. Akyianu, Ruiqi Shi, Amir Yadegari, Hamna Shahnawaz, Sean C. Semple, Barbara L. Mui, Drew Weissman, Vladimir R. Muzykantov, Patrick M. Glassman
RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically-based pharmacokinetic/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue pharmacokinetics of lipid nanoparticles, as well as the kinetics of tissue luciferase expression measured by activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the behavior of RNA-containing lipid nanoparticles developed for an array of conditions and across species.
{"title":"Physiologically-based modeling of LNP-mediated delivery of mRNA in the vascular system","authors":"Hamideh Parhiz, Vladimir V. Shuvaev, Qin Li, Tyler E. Papp, Awurama A. Akyianu, Ruiqi Shi, Amir Yadegari, Hamna Shahnawaz, Sean C. Semple, Barbara L. Mui, Drew Weissman, Vladimir R. Muzykantov, Patrick M. Glassman","doi":"10.1016/j.omtn.2024.102175","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102175","url":null,"abstract":"RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically-based pharmacokinetic/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue pharmacokinetics of lipid nanoparticles, as well as the kinetics of tissue luciferase expression measured by activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the behavior of RNA-containing lipid nanoparticles developed for an array of conditions and across species.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"1 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140147255","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 : 2024-03-17DOI: 10.1016/j.omtn.2024.102174
Sacha V. Kepreotis, Jae Gyun Oh, Mina Park, Jimeen Yoo, Cholong Lee, Mark Mercola, Roger J. Hajjar, Dongtak Jeong
Dystrophic cardiomyopathy is a significant feature of Duchenne Muscular Dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both Sarcoplasmic Reticulum Calcium ATPase 2a (SERCA2a) and Mothers Against Decapentaplegic Homolog-7 (Smad7) proteins. In this study, we created a gene transfer using a miR-25 Tough Decoy (TuD) RNAs inhibitor delivered via recombinant Adeno-Associated Virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice (), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca homeostasis and abrogate tissue fibrosis in DMD.
萎缩性心肌病是杜氏肌肉萎缩症（DMD）的一个重要特征。心肌细胞细胞质钙（Ca）增加和间质纤维化是主要的病理生理学特征，最终导致心脏功能障碍。MicroRNA-25（miR-25）已被确定为肉质网钙ATP酶2a（SERCA2a）和母亲抗截瘫同源物-7（Smad7）蛋白的抑制因子。在这项研究中，我们利用通过重组腺相关病毒血清型9（AAV9）递送的miR-25 Tough Decoy (TuD) RNAs抑制剂创建了一种基因转移方法，以评估miR-25抑制剂对老龄肌营养不良症/中营养不良症单倍体不足小鼠（）的心脏和骨骼肌功能的影响，老龄肌营养不良症/中营养不良症单倍体不足小鼠是一种有效的DMD转基因小鼠模型。我们发现，静脉注射 AAV9 miR-25 TuD 能强烈而稳定地抑制心脏 miR-25 水平，同时恢复 SERCA2a 和 Smad7 的表达。这与心肌细胞间质纤维化的改善和心脏功能的恢复有关。此外，直接向小鼠四肢肌肉注射 AAV9 miR-25 TuD 能显著恢复骨骼肌 Smad7 的表达，减少组织纤维化，并提高（+/-）小鼠骨骼肌的性能。这些结果表明，miR-25 TuD 基因转移可能是恢复 DMD 心肌细胞钙稳态和减轻组织纤维化的一种新型治疗方法。
{"title":"Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice","authors":"Sacha V. Kepreotis, Jae Gyun Oh, Mina Park, Jimeen Yoo, Cholong Lee, Mark Mercola, Roger J. Hajjar, Dongtak Jeong","doi":"10.1016/j.omtn.2024.102174","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102174","url":null,"abstract":"Dystrophic cardiomyopathy is a significant feature of Duchenne Muscular Dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both Sarcoplasmic Reticulum Calcium ATPase 2a (SERCA2a) and Mothers Against Decapentaplegic Homolog-7 (Smad7) proteins. In this study, we created a gene transfer using a miR-25 Tough Decoy (TuD) RNAs inhibitor delivered via recombinant Adeno-Associated Virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice (), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca homeostasis and abrogate tissue fibrosis in DMD.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"128 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140147553","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 : 2024-03-17DOI: 10.1016/j.omtn.2024.102173
Hywel Dunn-Davies, Tatiana Dudnakova, Antonella Nogara, Julie Rodor, Anita C. Thomas, Elisa Parish, Philippe Gautier, Alison Meynert, Igor Ulitsky, Paolo Madeddu, Andrea Caporali, Andrew Baker, David Tollervey, Tijana Mitić
Epigenetic processes involving long non-coding RNAs (lncRNAs) regulate endothelial genes expression. However, the underlying regulatory mechanisms causing endothelial dysfunction yet remain to be elucidated. nhancer of este omologue 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischaemic angiogenesis. We used ormaldehyde/UV assisted cross-linking igation nd equencing of ybrids (FLASH) and identified new role for maternally xpressed ene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 () promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted expression and improved EC migration and adhesion to fibronectin, A-395 inhibitor re-directed -assisted chromatin remodelling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show context specific role for in guiding EZH2 to repress . Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies.
{"title":"Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression","authors":"Hywel Dunn-Davies, Tatiana Dudnakova, Antonella Nogara, Julie Rodor, Anita C. Thomas, Elisa Parish, Philippe Gautier, Alison Meynert, Igor Ulitsky, Paolo Madeddu, Andrea Caporali, Andrew Baker, David Tollervey, Tijana Mitić","doi":"10.1016/j.omtn.2024.102173","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102173","url":null,"abstract":"Epigenetic processes involving long non-coding RNAs (lncRNAs) regulate endothelial genes expression. However, the underlying regulatory mechanisms causing endothelial dysfunction yet remain to be elucidated. nhancer of este omologue 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischaemic angiogenesis. We used ormaldehyde/UV assisted cross-linking igation nd equencing of ybrids (FLASH) and identified new role for maternally xpressed ene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 () promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted expression and improved EC migration and adhesion to fibronectin, A-395 inhibitor re-directed -assisted chromatin remodelling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show context specific role for in guiding EZH2 to repress . Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"17 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140147250","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.omtn.2024.102153
John J. Rossi
{"title":"Accessory oligos for neuronal delivery of therapeutic siRNAs for ALS","authors":"John J. Rossi","doi":"10.1016/j.omtn.2024.102153","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102153","url":null,"abstract":"","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"390 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140147183","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 : 2024-03-11DOI: 10.1016/j.omtn.2024.102171
Esteban Finol, Sarah E. Krul, Sean J. Hoehn, Xudong Lyu, Carlos E. Crespo-Hernández
Nucleoside-modified mRNA technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260 nm wavelength (A). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260 nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5-15% error, in an mRNA sequence dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260 nm wavelength, the RNA composition of the mRNA, and the A of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs. The webserver is freely available at .
{"title":"The mRNACalc webserver accounts for the hypochromicity of modified nucleosides and enables the accurate quantification of nucleoside-modified mRNA.","authors":"Esteban Finol, Sarah E. Krul, Sean J. Hoehn, Xudong Lyu, Carlos E. Crespo-Hernández","doi":"10.1016/j.omtn.2024.102171","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102171","url":null,"abstract":"Nucleoside-modified mRNA technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260 nm wavelength (A). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260 nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5-15% error, in an mRNA sequence dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260 nm wavelength, the RNA composition of the mRNA, and the A of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs. The webserver is freely available at .","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"11 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140097383","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 : 2024-03-06DOI: 10.1016/j.omtn.2024.102165
Ming Jin, Jiajia Lin, Haisen Li, Zhifang Li, Dong Yang, Yin Wang, Yuyang Yu, Zhurui Shao, Long Chen, Zhiqiang Wang, Yu Zhang, Xiumei Zhang, Ning Wang, Chunlong Xu, Hui Yang, Wan-Jin Chen, Guoling Li
Duchenne muscular dystrophy (DMD) is the most prevalent herediatry disease in men, characterized by dystrophin deficiency, progressive muscle wasting, cardiac insufficiency, and premature mortality, with no effective therapeutic options. Here, we investigated whether adenine base editing can correct pathological non-sense point mutations leading to premature stop codons in the dystrophin gene. We identified 27 causative nonsense mutations in our DMD patient cohort. Treatment with adenine base editor (ABE) could restore dystrophin expression by direct A-to-G editing of pathological nonsense mutations in cardiomyocytes generated from DMD patient-derived induced pluripotent stem cells. We also generated two humanized mouse models of DMD expressing mutation-bearing exons 23 or 30 of human dystrophin gene. Intramuscular administration of ABE, driven by ubiquitous or muscle-specific promoters could correct these non-sense mutations , albeit with higher efficiency in exon 30, restoring dystrophin expression in skeletal fibers of humanized DMD mice. Moreover, a single systemic delivery of ABE with human sgRNA could induce body-wide dystrophin expression and improve muscle function in rotarod tests of humanized DMD mice. These findings demonstrate that ABE with human sgRNAs can confer therapeutic alleviation of DMD in mice, providing a basis for development of adenine base editing therapies in monogenic diseases.
{"title":"Correction of human non-sense mutation via adenine base editing for Duchenne muscular dystrophy treatment in mouse","authors":"Ming Jin, Jiajia Lin, Haisen Li, Zhifang Li, Dong Yang, Yin Wang, Yuyang Yu, Zhurui Shao, Long Chen, Zhiqiang Wang, Yu Zhang, Xiumei Zhang, Ning Wang, Chunlong Xu, Hui Yang, Wan-Jin Chen, Guoling Li","doi":"10.1016/j.omtn.2024.102165","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102165","url":null,"abstract":"Duchenne muscular dystrophy (DMD) is the most prevalent herediatry disease in men, characterized by dystrophin deficiency, progressive muscle wasting, cardiac insufficiency, and premature mortality, with no effective therapeutic options. Here, we investigated whether adenine base editing can correct pathological non-sense point mutations leading to premature stop codons in the dystrophin gene. We identified 27 causative nonsense mutations in our DMD patient cohort. Treatment with adenine base editor (ABE) could restore dystrophin expression by direct A-to-G editing of pathological nonsense mutations in cardiomyocytes generated from DMD patient-derived induced pluripotent stem cells. We also generated two humanized mouse models of DMD expressing mutation-bearing exons 23 or 30 of human dystrophin gene. Intramuscular administration of ABE, driven by ubiquitous or muscle-specific promoters could correct these non-sense mutations , albeit with higher efficiency in exon 30, restoring dystrophin expression in skeletal fibers of humanized DMD mice. Moreover, a single systemic delivery of ABE with human sgRNA could induce body-wide dystrophin expression and improve muscle function in rotarod tests of humanized DMD mice. These findings demonstrate that ABE with human sgRNAs can confer therapeutic alleviation of DMD in mice, providing a basis for development of adenine base editing therapies in monogenic diseases.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"15 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140097467","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 : 2024-03-05DOI: 10.1016/j.omtn.2024.102162
Ayşegül Yıldız, Aida Hasani, Tina Hempel, Nina Köhl, Aline Beicht, René Becker, Stefanie Hubich-Rau, Martin Suchan, Marco A. Poleganov, Ugur Sahin, Tim Beissert
The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and -amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in . To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes.
{"title":"Trans-amplifying RNA expressing functional miRNA mediates target-specific gene suppression and simultaneous transgene expression","authors":"Ayşegül Yıldız, Aida Hasani, Tina Hempel, Nina Köhl, Aline Beicht, René Becker, Stefanie Hubich-Rau, Martin Suchan, Marco A. Poleganov, Ugur Sahin, Tim Beissert","doi":"10.1016/j.omtn.2024.102162","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102162","url":null,"abstract":"The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and -amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in . To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"67 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140097652","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 : 2024-02-28DOI: 10.1016/j.omtn.2024.102160
Xinghua Wang, Syeda S. Baksh, Richard E. Pratt, Victor J. Dzau, Conrad P. Hodgkinson
Reprogramming scar fibroblasts into cardiomyocytes has been proposed to reverse the damage associated with myocardial infarction. However, the limited improvement in cardiac function calls for enhanced strategies. We reported enhanced efficacy of our miR reprogramming cocktail miR combo (miR-1, miR-133a, miR-208a, and miR-499) via RNA-sensing receptor stimulation. We hypothesized that we could combine RNA-sensing receptor activation with fibroblast reprogramming by chemically modifying miR combo. To test the hypothesis, miR combo was modified to enhance interaction with the RNA-sensing receptor Rig1 via the addition of a 5′-triphosphate (5′ppp) group. Importantly, when compared with unmodified miR combo, 5′ppp-modified miR combo markedly improved reprogramming efficacy . Enhanced reprogramming efficacy correlated with a type-I interferon immune response with strong and selective secretion of interferon β (IFNβ). Antibody blocking studies and media replacement experiments indicated that 5′ppp-miR combo utilized IFNβ to enhance fibroblast reprogramming efficacy. In conclusion, miRs can acquire powerful additional roles through chemical modification that potentially increases their clinical applications.
有人提出将瘢痕成纤维细胞重新编程为心肌细胞，以逆转与心肌梗死相关的损伤。然而，对心脏功能的改善有限，因此需要加强策略。我们的 miR 重编程鸡尾酒 miR 组合（miR-1、miR-133a、miR-208a 和 miR-499）通过 RNA 感受体刺激增强了疗效。我们假设，可以通过化学修饰 miR 组合，将 RNA 感知受体激活与成纤维细胞重编程结合起来。为了验证这一假设，我们对 miR combo 进行了修饰，通过添加 5′-三磷酸（5′ppp）基团来增强其与 RNA 感知受体 Rig1 的相互作用。重要的是，与未修饰的 miR 组合相比，5′ppp 修饰的 miR 组合明显提高了重编程功效。重编程功效的提高与Ⅰ型干扰素免疫反应有关，Ⅰ型干扰素免疫反应具有很强的选择性干扰素β（IFNβ）分泌。抗体阻断研究和培养基置换实验表明，5′ppp-miR 组合利用 IFNβ 提高了成纤维细胞重编程的功效。总之，miRs 可以通过化学修饰获得强大的附加作用，从而有可能增加其临床应用。
{"title":"Modifying miRs for effective reprogramming of fibroblasts to cardiomyocytes","authors":"Xinghua Wang, Syeda S. Baksh, Richard E. Pratt, Victor J. Dzau, Conrad P. Hodgkinson","doi":"10.1016/j.omtn.2024.102160","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102160","url":null,"abstract":"Reprogramming scar fibroblasts into cardiomyocytes has been proposed to reverse the damage associated with myocardial infarction. However, the limited improvement in cardiac function calls for enhanced strategies. We reported enhanced efficacy of our miR reprogramming cocktail miR combo (miR-1, miR-133a, miR-208a, and miR-499) via RNA-sensing receptor stimulation. We hypothesized that we could combine RNA-sensing receptor activation with fibroblast reprogramming by chemically modifying miR combo. To test the hypothesis, miR combo was modified to enhance interaction with the RNA-sensing receptor Rig1 via the addition of a 5′-triphosphate (5′ppp) group. Importantly, when compared with unmodified miR combo, 5′ppp-modified miR combo markedly improved reprogramming efficacy . Enhanced reprogramming efficacy correlated with a type-I interferon immune response with strong and selective secretion of interferon β (IFNβ). Antibody blocking studies and media replacement experiments indicated that 5′ppp-miR combo utilized IFNβ to enhance fibroblast reprogramming efficacy. In conclusion, miRs can acquire powerful additional roles through chemical modification that potentially increases their clinical applications.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"33 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140097379","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}
{"title":"Immunostimulatory short non-coding RNAs in the circulation of patients with tuberculosis infection","authors":"Justin Gumas, Takuya Kawamura, Megumi Shigematsu, Yohei Kirino","doi":"10.1016/j.omtn.2024.102156","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102156","url":null,"abstract":"","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"15 2","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139947540","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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