Molecular Therapy. Nucleic Acids最新文献

Engineered AAV capsid transport mutants overcome transduction deficiencies in the aged CNS 工程化 AAV 胶囊运输突变体克服了老年中枢神经系统的转导缺陷

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-12 DOI: 10.1016/j.omtn.2024.102332

Ivette M. Sandoval, Christy M. Kelley, Luis Daniel Bernal-Conde, Kathy Steece-Collier, David J. Marmion, Marcus Davidsson, Sean M. Crosson, Sanford L. Boye, Shannon E. Boye, Fredric P. Manfredsson

{"title":"Engineered AAV capsid transport mutants overcome transduction deficiencies in the aged CNS","authors":"Ivette M. Sandoval, Christy M. Kelley, Luis Daniel Bernal-Conde, Kathy Steece-Collier, David J. Marmion, Marcus Davidsson, Sean M. Crosson, Sanford L. Boye, Shannon E. Boye, Fredric P. Manfredsson","doi":"10.1016/j.omtn.2024.102332","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102332","url":null,"abstract":"Adeno-associated virus (AAV)-based gene therapy has enjoyed great successes over the past decade, with Food and Drug Administration-approved therapeutics and a robust clinical pipeline. Nonetheless, barriers to successful translation remain. For example, advanced age is associated with impaired brain transduction, with the diminution of infectivity depending on anatomical region and capsid. Given that CNS gene transfer is often associated with neurodegenerative diseases where age is the chief risk factor, we sought to better understand the causes of this impediment. We assessed two AAV variants hypothesized to overcome factors negatively impacting transduction in the aged brain; specifically, changes in extracellular and cell-surface glycans, and intracellular transport. We evaluated a heparin sulfate proteoglycan null variant with or without mutations enhancing intracellular transport. Vectors were injected into the striatum of young adult or aged rats to address whether improving extracellular diffusion, removing glycan receptor dependence, or improving intracellular transport are important factors in transducing the aged brain. We found that, regardless of the viral capsid, there was a reduction in many of our metrics of transduction in the aged brain. However, the transport mutant was less sensitive to age, suggesting that changes in the cellular transport of AAV capsids are a key factor in age-related transduction deficiency.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142247942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quality by design for mRNA platform purification based on continuous oligo-dT chromatography 基于连续寡聚-dT 色谱的 mRNA 平台纯化质量设计

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-11 DOI: 10.1016/j.omtn.2024.102333

Jixin Qu, Adithya Nair, George W. Muir, Kate A. Loveday, Zidi Yang, Ehsan Nourafkan, Emma N. Welbourne, Mabrouka Maamra, Mark J. Dickman, Zoltán Kis

{"title":"Quality by design for mRNA platform purification based on continuous oligo-dT chromatography","authors":"Jixin Qu, Adithya Nair, George W. Muir, Kate A. Loveday, Zidi Yang, Ehsan Nourafkan, Emma N. Welbourne, Mabrouka Maamra, Mark J. Dickman, Zoltán Kis","doi":"10.1016/j.omtn.2024.102333","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102333","url":null,"abstract":"Oligo-deoxythymidine (oligo-dT) ligand-based affinity chromatography is a robust method for purifying mRNA drug substances within the manufacturing process of mRNA-based products, including vaccines and therapeutics. However, the conventional batch mode of operation for oligo-dT chromatography has certain drawbacks that reduce this process’s productivity. Here, we report a new continuous oligo-dT chromatography process for the purification of <ce:italic>in vitro</ce:italic> transcribed mRNA, which reduces losses, improves the efficiency of oligo-dT resin use, and intensifies the chromatography process. Furthermore, the quality by design (QbD) framework was used to establish a design space for the newly developed method. The optimization of process parameters (PPs), including salt type, salt concentration, load flow rate and mRNA load concentration both in batch and the continuous mode, achieved a greater than 90% yield (mRNA recovery) along with greater than 95% mRNA integrity and greater than 99% purity. The productivity of continuous chromatography was estimated to be 5.75-fold higher, and the operating cost was estimated 15% lower, when compared with batch chromatography. Furthermore, the QbD framework was further used to map the relationship between critical quality attributes and key performance indicators as a function of critical process parameters and critical material attributes.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142247943","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

ELX-02 Suppresses nonsense mutations and restores type VII collagen and laminin 332 function in recessive dystrophic and junctional epidermolysis bullosa ELX-02 抑制无义突变，恢复隐性萎缩性和交界性表皮松解症中 VII 型胶原蛋白和层粘连蛋白 332 的功能

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-10 DOI: 10.1016/j.omtn.2024.102334

Brandon Levian, Yingping Hou, Xin Tang, Liat Bainvoll, Kate Zheng, Vasu Badarinarayana, Soheil Aghamohammadzadeh, Mei Chen

{"title":"ELX-02 Suppresses nonsense mutations and restores type VII collagen and laminin 332 function in recessive dystrophic and junctional epidermolysis bullosa","authors":"Brandon Levian, Yingping Hou, Xin Tang, Liat Bainvoll, Kate Zheng, Vasu Badarinarayana, Soheil Aghamohammadzadeh, Mei Chen","doi":"10.1016/j.omtn.2024.102334","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102334","url":null,"abstract":"Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are lethal blistering skin disorders resulting from mutations in genes coding for type VII collagen (<ce:italic>COL7A1</ce:italic>) and laminin 332 (<ce:italic>LAMA3</ce:italic>, <ce:italic>LAMB3</ce:italic>, or <ce:italic>LAMC2</ce:italic>), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in <ce:italic>LAMB3</ce:italic> are nonsense mutations (80%). ELX-02, an aminoglycoside analog, has demonstrated superior PTC readthrough activity and lower toxicity compared to gentamicin in various genetic disorders. This study investigated the ability of ELX-02 to suppress PTCs and promote the expression of C7 and laminin 332 in primary RDEB keratinocytes/fibroblasts and primary JEB keratinocytes harboring nonsense mutations. ELX-02 induced a dose-dependent production of C7 or laminin β3 that surpassed the results achieved with gentamicin. ELX-02 reversed RDEB and JEB cellular hypermotility and improved poor cell-substratum adhesion in JEB cells. Importantly, ELX-02-induced C7 and laminin 332 localized to the dermal-epidermal junction. This is the first study demonstrating that ELX-02 can induce PTC readthrough and restore functional C7 and laminin 332 in RDEB and JEB caused by nonsense mutations. Therefore, ELX-02 may offer a novel and safe therapy for RDEB, JEB, and other inherited skin diseases caused by nonsense mutations.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142247945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The beauty of symmetry: siRNA phosphorodithioate modifications reduce stereocomplexity, ease analysis, and can improve in vivo potency 对称之美：siRNA 二硫代磷酸酯修饰可降低立体复杂性，便于分析，并能提高体内效力

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-10 DOI: 10.1016/j.omtn.2024.102336

Sophie Schöllkopf, Stefan Rathjen, Micaela Graglia, Nina Was, Eliot Morrison, Adrien Weingärtner, Lucas Bethge, Judith Hauptmann, Marie Wikström Lindholm

{"title":"The beauty of symmetry: siRNA phosphorodithioate modifications reduce stereocomplexity, ease analysis, and can improve in vivo potency","authors":"Sophie Schöllkopf, Stefan Rathjen, Micaela Graglia, Nina Was, Eliot Morrison, Adrien Weingärtner, Lucas Bethge, Judith Hauptmann, Marie Wikström Lindholm","doi":"10.1016/j.omtn.2024.102336","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102336","url":null,"abstract":"Phosphorothioates (PSs) can be essential in stabilizing therapeutic oligonucleotides against enzymatic degradation. However, unless synthesis is performed with stereodefined amidites, each PS introduces a chemically undefined stereocenter, resulting in 2<ce:sup loc=\"post\"><ce:italic>n</ce:italic></ce:sup> unique molecules in the final product and affecting downstream analytics and purification. Replacing the second non-bridging oxygen with sulfur results in phosphorodithioate (PS2) linkages, thereby removing the stereocenter. We describe synthesis and analytical data for <ce:italic>N</ce:italic>-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) with PS2 in the GalNAc cluster and at the siRNA termini. All siRNA conjugates with PS2 internucleotide linkages were produced with good yield and showed improved analytical properties. PS2 in the GalNAc cluster had no, or only minor, effect on <ce:italic>in vitro</ce:italic> and <ce:italic>in vivo</ce:italic> activity. Except for the 5′-antisense position, PS2 modifications were well tolerated at the siRNA termini, and a single PS2 internucleotide linkage gave similar or improved stabilization and <ce:italic>in vitro</ce:italic> activity as the two PSs typically used for end stabilization. Surprisingly, several of the PS2-containing siRNA conjugates resulted in increased <ce:italic>in vivo</ce:italic> activity and duration of action compared to the same siRNA sequence stabilized with PS linkages, suggesting PS2 linkages as interesting options for siRNA strand design with a reduced number of undefined stereocenters.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142247944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

RAB18 regulates extrahepatic SiRNA-mediated gene silencing efficacy RAB18 调节肝外 SiRNA 介导的基因沉默功效

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-10 DOI: 10.1016/j.omtn.2024.102335

Jiamiao Lu, Jasper Lee, Eric Yuan, Devin L. Wakefield, Matt Kanke, Danielle Pruitt, Jose Barreda, Ingrid C. Rulifson, Jiansong Xie, John Ferbas, Jason Long, Bryan Meade, Oliver Homann, Wei Guo, Tina Gomes, Hong Zhou, Bin Wu, Jixin Cui, Songli Wang

{"title":"RAB18 regulates extrahepatic SiRNA-mediated gene silencing efficacy","authors":"Jiamiao Lu, Jasper Lee, Eric Yuan, Devin L. Wakefield, Matt Kanke, Danielle Pruitt, Jose Barreda, Ingrid C. Rulifson, Jiansong Xie, John Ferbas, Jason Long, Bryan Meade, Oliver Homann, Wei Guo, Tina Gomes, Hong Zhou, Bin Wu, Jixin Cui, Songli Wang","doi":"10.1016/j.omtn.2024.102335","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102335","url":null,"abstract":"Small interfering RNAs (siRNAs) hold considerable therapeutic potential to selectively silence previously “undruggable” disease-associated targets, offering new opportunities to fight human diseases. This therapeutic strategy, however, is limited by the inability of naked siRNAs to passively diffuse across cellular membranes due to their large molecular size and negative charge. Delivery of siRNAs to liver through conjugation of siRNA to N-acetylgalactosamine (GalNAc) has been a success, providing robust and durable gene knockdown, specifically in hepatocytes. However, the poor delivery and silencing efficacy of siRNAs in other cell types has hindered their applications outside the liver. We previously reported that a genome-wide pooled knockout screen identified <ce:italic>RAB18</ce:italic> as a major modulator of GalNAc-siRNA conjugates. Herein, we demonstrate <ce:italic>RAB18</ce:italic> knockout/knockdown efficaciously enhances siRNA-mediated gene silencing in hepatic and extrahepatic cell lines and <ce:italic>in vivo</ce:italic>. Our results reveal a mechanism by which retrograde Golgi-endoplasmic reticulum (ER) transport and the intracellular lipid droplets (LDs) positively regulate siRNA-mediated gene silencing.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142247947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA 靶向病毒 RNA 的肽结合吗啉寡聚体抑制 SARS-CoV-2 在小鼠肺部的生长

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-10 DOI: 10.1016/j.omtn.2024.102331

Alexandra Sakai, Gagandeep Singh, Mahsa Khoshbakht, Scott Bittner, Christiane V. Löhr, Randy Diaz-Tapia, Prajakta Warang, Kris White, Luke Le Luo, Blanton Tolbert, Mario Blanco, Amy Chow, Mitchell Guttman, Cuiping Li, Yiming Bao, Joses Ho, Sebastian Maurer-Stroh, Arnab Chatterjee, Sumit Chanda, Adolfo García-Sastre, Michael Schotsaert, John R. Teijaro, Hong M. Moulton, David A. Stein

{"title":"Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA","authors":"Alexandra Sakai, Gagandeep Singh, Mahsa Khoshbakht, Scott Bittner, Christiane V. Löhr, Randy Diaz-Tapia, Prajakta Warang, Kris White, Luke Le Luo, Blanton Tolbert, Mario Blanco, Amy Chow, Mitchell Guttman, Cuiping Li, Yiming Bao, Joses Ho, Sebastian Maurer-Stroh, Arnab Chatterjee, Sumit Chanda, Adolfo García-Sastre, Michael Schotsaert, John R. Teijaro, Hong M. Moulton, David A. Stein","doi":"10.1016/j.omtn.2024.102331","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102331","url":null,"abstract":"Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO) called 5′END-2, targeting a highly conserved sequence in the 5′ UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth <ce:italic>in vitro</ce:italic> and <ce:italic>in vivo</ce:italic>. In HeLa-ACE 2 cells, 5′END-2 produced IC<ce:inf loc=\"post\">50</ce:inf> values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. <ce:italic>In vivo</ce:italic>, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5′END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0–2.0 mg/kg 5′END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5′END-2 to its complementary target RNA. Furthermore, using reporter constructs containing SARS-CoV-2 5′ UTR leader sequence, in an in-cell system, we observed that 5′END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5′END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142248300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Retraction Notice to: Circular RNA circFOXM1 Plays a Role in Papillary Thyroid Carcinoma by Sponging miR-1179 and Regulating HMGB1 Expression 撤稿通知环状 RNA circFOXM1 通过疏导 miR-1179 和调控 HMGB1 表达在甲状腺乳头状癌中发挥作用

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-06 DOI: 10.1016/j.omtn.2024.102216

Mao Ye, Haitao Hou, Minghai Shen, Shu Dong, Tao Zhang

引用次数: 0

Highly efficient nucleic acid encapsulation method for targeted gene therapy using antibody conjugation system 利用抗体共轭系统进行靶向基因治疗的高效核酸封装方法

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-05 DOI: 10.1016/j.omtn.2024.102322

Seokbong Hong, Seung-Hwan Jeong, Jang Hee Han, Hyung Dong Yuk, Chang Wook Jeong, Ja Hyeon Ku, Cheol Kwak

{"title":"Highly efficient nucleic acid encapsulation method for targeted gene therapy using antibody conjugation system","authors":"Seokbong Hong, Seung-Hwan Jeong, Jang Hee Han, Hyung Dong Yuk, Chang Wook Jeong, Ja Hyeon Ku, Cheol Kwak","doi":"10.1016/j.omtn.2024.102322","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102322","url":null,"abstract":"Gene therapy has surfaced as a promising avenue for treating cancers, offering the advantage of deliberate adjustment of targeted genes. Nonetheless, the swift degradation of nucleic acids in the bloodstream necessitates an effective and secure delivery system. The widespread utilization of poly(lactic-co-glycolic acid) (PLGA) nanoparticles as drug delivery systems has highlighted challenges in controlling particle size and release properties. Moreover, the encapsulation of nucleic acids exacerbates these difficulties due to the negatively charged surface of PLGA nanoparticles. In this study, we aimed to improve the encapsulation efficiency of nucleic acids by employing negatively charged microbeads and optimizing the timing of the specific formulation steps. Furthermore, by conjugating PSMA-617, a ligand for the prostate-specific membrane antigen (PSMA), with PLGA nanoparticles, we assessed the antitumor effects and the efficacy of a nucleic acid delivery system on a prostate cancer model. The employed technique within the nucleic acid encapsulation system represents a novel approach that could be adapted to encapsulate various kinds of nucleic acids. Moreover, it enables the attachment of targeting moieties to different cell membrane proteins, thereby unveiling new prospects for precise therapeutics in cancer therapy.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142247946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Identification of a histone deacetylase inhibitor as a therapeutic candidate for congenital central hypoventilation syndrome 鉴定组蛋白去乙酰化酶抑制剂作为先天性中枢通气不足综合征的候选疗法

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-03 DOI: 10.1016/j.omtn.2024.102319

Chiara Africano, Tiziana Bachetti, Paolo Uva, Gabriel Pitollat, Genny Del Zotto, Francesca Giacopelli, Giada Recchi, Nicolas Lenfant, Amélia Madani, Nathan Beckouche, Muriel Thoby-Brisson, Isabella Ceccherini

{"title":"Identification of a histone deacetylase inhibitor as a therapeutic candidate for congenital central hypoventilation syndrome","authors":"Chiara Africano, Tiziana Bachetti, Paolo Uva, Gabriel Pitollat, Genny Del Zotto, Francesca Giacopelli, Giada Recchi, Nicolas Lenfant, Amélia Madani, Nathan Beckouche, Muriel Thoby-Brisson, Isabella Ceccherini","doi":"10.1016/j.omtn.2024.102319","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102319","url":null,"abstract":"Congenital central hypoventilation syndrome (CCHS), a rare genetic disease caused by heterozygous mutations, is characterized by life-threatening breathing deficiencies. PHOX2B is a transcription factor required for the specification of the autonomic nervous system, which contains, in particular, brain stem respiratory centers. In CCHS, mutations lead to cytoplasmic PHOX2B protein aggregations, thus compromising its transcriptional capability. Currently, the only available treatment for CCHS is assisted mechanical ventilation. Therefore, identifying molecules with alleviating effects on CCHS-related breathing impairments is of primary importance. A transcriptomic analysis of cells transfected with different constructs was used to identify compounds of interest with the CMap tool. Using fluorescence microscopy and luciferase assay, the selected molecules were further tested for their ability to restore the nuclear location and function of PHOX2B. Finally, an electrophysiological approach was used to investigate the effects of the most promising molecule on respiratory activities of -mutant mouse isolated brain stem. The histone deacetylase inhibitor SAHA was found to have low toxicity , to restore the proper location and function of PHOX2B protein, and to improve respiratory rhythm-related parameters . Thus, our results identify SAHA as a promising agent to treat CCHS-associated breathing deficiencies.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci 高表达肌肉基因座中 RNA 引导 DNA 整合的精确性和有效性

IF 8.8 2区医学

Molecular Therapy. Nucleic Acids Pub Date : 2024-09-02 DOI: 10.1016/j.omtn.2024.102320

Made Harumi Padmaswari, Gabrielle Bulliard, Shilpi Agrawal, Mary S. Jia, Sabin Khadgi, Kevin A. Murach, Christopher E. Nelson

{"title":"Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci","authors":"Made Harumi Padmaswari, Gabrielle Bulliard, Shilpi Agrawal, Mary S. Jia, Sabin Khadgi, Kevin A. Murach, Christopher E. Nelson","doi":"10.1016/j.omtn.2024.102320","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102320","url":null,"abstract":"Gene replacement therapies primarily rely on adeno-associated virus (AAV) vectors for transgene expression. However, episomal expression can decline over time due to vector loss or epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to low invasiveness of intramuscular injections, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two highly expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts and skeletal muscle tissue. We hijacked the muscle creatine kinase () and myoglobin () promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":8.8,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0