Pub Date : 2024-09-10DOI: 10.1016/j.omtn.2024.102334
Brandon Levian, Yingping Hou, Xin Tang, Liat Bainvoll, Kate Zheng, Vasu Badarinarayana, Soheil Aghamohammadzadeh, Mei Chen
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 (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in LAMB3 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.
{"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":"16 1","pages":""},"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}
Pub Date : 2024-09-10DOI: 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
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 2n 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 N-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 in vitro and in vivo 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 in vitro activity as the two PSs typically used for end stabilization. Surprisingly, several of the PS2-containing siRNA conjugates resulted in increased in vivo 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.
{"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":"36 1","pages":""},"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}
Pub Date : 2024-09-10DOI: 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
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 RAB18 as a major modulator of GalNAc-siRNA conjugates. Herein, we demonstrate RAB18 knockout/knockdown efficaciously enhances siRNA-mediated gene silencing in hepatic and extrahepatic cell lines and in vivo. 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.
{"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":"14 1","pages":""},"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}
Pub Date : 2024-09-10DOI: 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
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 in vitro and in vivo. In HeLa-ACE 2 cells, 5′END-2 produced IC50 values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, 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.
{"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":"14 1","pages":""},"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}
Pub Date : 2024-09-06DOI: 10.1016/j.omtn.2024.102216
Mao Ye, Haitao Hou, Minghai Shen, Shu Dong, Tao Zhang
{"title":"Retraction Notice to: Circular RNA circFOXM1 Plays a Role in Papillary Thyroid Carcinoma by Sponging miR-1179 and Regulating HMGB1 Expression","authors":"Mao Ye, Haitao Hou, Minghai Shen, Shu Dong, Tao Zhang","doi":"10.1016/j.omtn.2024.102216","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102216","url":null,"abstract":"","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"114 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142227589","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 : 2024-09-05DOI: 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
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.
{"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":"11 1","pages":""},"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}
Pub Date : 2024-09-03DOI: 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
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.
{"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":"17 1","pages":""},"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}
Pub Date : 2024-09-02DOI: 10.1016/j.omtn.2024.102320
Made Harumi Padmaswari, Gabrielle Bulliard, Shilpi Agrawal, Mary S. Jia, Sabin Khadgi, Kevin A. Murach, Christopher E. Nelson
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.
{"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":"40 1","pages":""},"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}
Pub Date : 2024-08-31DOI: 10.1016/j.omtn.2024.102317
Kelly J. Fagan, Guillem Chillon, Ellie M. Carrell, Elisa A. Waxman, Beverly L. Davidson
Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by an expansion of the CAG repeat region of the gene. Currently there are no disease-modifying treatments; however, previous work has shown the potential of gene therapy, specifically RNAi, as a potential modality. Cas9 editing offers potential for these patients but has yet to be evaluated in SCA1 models. To test this, we first characterized the number of transgenes harbored in the common B05 mouse model of SCA1. Despite having five copies of the human mutant transgene, a 20% reduction of improved behavior deficits without increases in inflammatory markers. Importantly, the editing approach was confirmed in induced pluripotent stem cell (iPSC) neurons derived from patients with SCA1, promoting the translatability of the approach to patients.
{"title":"Cas9 editing of ATXN1 in a spinocerebellar ataxia type 1 mice and human iPSC-derived neurons","authors":"Kelly J. Fagan, Guillem Chillon, Ellie M. Carrell, Elisa A. Waxman, Beverly L. Davidson","doi":"10.1016/j.omtn.2024.102317","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102317","url":null,"abstract":"Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by an expansion of the CAG repeat region of the gene. Currently there are no disease-modifying treatments; however, previous work has shown the potential of gene therapy, specifically RNAi, as a potential modality. Cas9 editing offers potential for these patients but has yet to be evaluated in SCA1 models. To test this, we first characterized the number of transgenes harbored in the common B05 mouse model of SCA1. Despite having five copies of the human mutant transgene, a 20% reduction of improved behavior deficits without increases in inflammatory markers. Importantly, the editing approach was confirmed in induced pluripotent stem cell (iPSC) neurons derived from patients with SCA1, promoting the translatability of the approach to patients.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"17 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222588","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 : 2024-08-31DOI: 10.1016/j.omtn.2024.102318
Ian Helstrup Nielsen, Anne Bruun Rovsing, Jacob Hørlück Janns, Emil Aagaard Thomsen, Albert Ruzo, Andreas Bøggild, Frederikke Nedergaard, Charlotte Thornild Møller, Thomas Boesen, Søren Egedal Degn, Jagesh V. Shah, Jacob Giehm Mikkelsen
To fully utilize the potential of CRISPR-Cas9-mediated genome editing, time-restricted and targeted delivery is crucial. By modulating the pseudotype of engineered lentivirus-derived nanoparticles (LVNPs), we demonstrate efficient cell-targeted delivery of Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes, supporting gene modification in a defined subset of cells in mixed cell populations. LVNPs pseudotyped with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein resulted in angiotensin-converting enzyme 2 (ACE2)-dependent insertion or deletion (indel) formation in an ACE2/ACE2 population of cells, whereas Nipah virus glycoprotein pseudotyping resulted in Ephrin-B2/B3-specific gene knockout. Additionally, LVNPs pseudotyped with Edmonston strain measles virus glycoproteins (MV-H/F) delivered Cas9/sgRNA RNPs to CD46 cells with and without additional expression of SLAM (signaling lymphocytic activation molecule; CD150). However, an engineered SLAM-specific measles virus pseudotype (measles virus-hemagglutinin/fusion [MV-H/F]-SLAM) efficiently targeted LVNPs to SLAM cells. Lentiviral vectors (LVs) pseudotyped with MV-H/F-SLAM efficiently transduced >80% of interleukin (IL)-4/IL-21-stimulated primary B cells cultured on CD40 ligand (CD40L)-expressing feeder cells. Notably, LVNPs pseudotyped with MV-H/F and MV-H/F-SLAM reached indel rates of >80% and >60% in stimulated primary B cells, respectively. Collectively, our findings demonstrate the modularity of LVNP-directed delivery of ready-to-function Cas9/sgRNA complexes. Using a panel of different pseudotypes, we provide evidence that LVNPs can be engineered to induce effective indel formation in a subpopulation of cells defined by the expression of surface receptors.
{"title":"Cell-targeted gene modification by delivery of CRISPR-Cas9 ribonucleoprotein complexes in pseudotyped lentivirus-derived nanoparticles","authors":"Ian Helstrup Nielsen, Anne Bruun Rovsing, Jacob Hørlück Janns, Emil Aagaard Thomsen, Albert Ruzo, Andreas Bøggild, Frederikke Nedergaard, Charlotte Thornild Møller, Thomas Boesen, Søren Egedal Degn, Jagesh V. Shah, Jacob Giehm Mikkelsen","doi":"10.1016/j.omtn.2024.102318","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102318","url":null,"abstract":"To fully utilize the potential of CRISPR-Cas9-mediated genome editing, time-restricted and targeted delivery is crucial. By modulating the pseudotype of engineered lentivirus-derived nanoparticles (LVNPs), we demonstrate efficient cell-targeted delivery of Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes, supporting gene modification in a defined subset of cells in mixed cell populations. LVNPs pseudotyped with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein resulted in angiotensin-converting enzyme 2 (ACE2)-dependent insertion or deletion (indel) formation in an ACE2/ACE2 population of cells, whereas Nipah virus glycoprotein pseudotyping resulted in Ephrin-B2/B3-specific gene knockout. Additionally, LVNPs pseudotyped with Edmonston strain measles virus glycoproteins (MV-H/F) delivered Cas9/sgRNA RNPs to CD46 cells with and without additional expression of SLAM (signaling lymphocytic activation molecule; CD150). However, an engineered SLAM-specific measles virus pseudotype (measles virus-hemagglutinin/fusion [MV-H/F]-SLAM) efficiently targeted LVNPs to SLAM cells. Lentiviral vectors (LVs) pseudotyped with MV-H/F-SLAM efficiently transduced >80% of interleukin (IL)-4/IL-21-stimulated primary B cells cultured on CD40 ligand (CD40L)-expressing feeder cells. Notably, LVNPs pseudotyped with MV-H/F and MV-H/F-SLAM reached indel rates of >80% and >60% in stimulated primary B cells, respectively. Collectively, our findings demonstrate the modularity of LVNP-directed delivery of ready-to-function Cas9/sgRNA complexes. Using a panel of different pseudotypes, we provide evidence that LVNPs can be engineered to induce effective indel formation in a subpopulation of cells defined by the expression of surface receptors.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"29 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222584","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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