Pub Date : 2024-08-15DOI: 10.1016/j.omtn.2024.102308
Pedro Justicia-Lirio, María Tristán-Manzano, Noelia Maldonado-Pérez, Carmen Barbero-Jiménez, Marina Cortijo-Gutiérrez, Kristina Pavlovic, Francisco J. Molina-Estevez, Pilar Muñoz, Ana Hinckley-Boned, Juan R. Rodriguez-Madoz, Felipe Prosper, Carmen Griñán-Lison, Saúl A. Navarro-Marchal, Julia Muñoz-Ballester, Pedro A. González-Sierra, Concha Herrera, Juan A. Marchal, Francisco Martín
Although chimeric antigen receptor (CAR) T cell therapy has revolutionized type B cancer treatment, efficacy remains limited in various lymphomas and solid tumors. Reinforcing conventional CAR-T cells to release cytokines can improve their efficacy but also increase safety concerns. Several strategies have been developed to regulate their secretion using minimal promoters that are controlled by chimeric proteins harboring transactivators. However, these chimeric proteins can disrupt the normal physiology of T cells. Here, we present the first transactivator-free anti-CD19 CAR-T cells able to control IL-18 expression (iTRUCK19.18) under ultra-low doses of doxycycline and without altering cellular fitness. Interestingly, IL-18 secretion requires T cell activation in addition to doxycycline, allowing the external regulation of CAR-T cell potency. This effect was translated into an increased CAR-T cell antitumor activity against aggressive hematologic and solid tumor models. In a clinically relevant context, we generated patient-derived iTRUCK19.18 cells capable of eradicating primary B cells tumors in a doxycycline-dependent manner. Furthermore, IL-18-releasing CAR-T cells polarized pro-tumoral macrophages toward an antitumoral phenotype, suggesting potential for modulating the tumor microenvironment. In summary, we showed that our platform can generate exogenously controlled CAR-T cells with enhanced potency and in the absence of transactivators.
虽然嵌合抗原受体（CAR）T 细胞疗法彻底改变了 B 型癌症的治疗，但在各种淋巴瘤和实体瘤中的疗效仍然有限。加强传统 CAR-T 细胞释放细胞因子可以提高疗效，但也会增加安全性问题。目前已开发出几种策略，利用由携带转录因子的嵌合蛋白控制的最小启动子来调节细胞因子的分泌。然而，这些嵌合蛋白会破坏 T 细胞的正常生理机能。在这里，我们首次发现了不含转座因子的抗 CD19 CAR-T 细胞，它们能在超低剂量强力霉素作用下控制 IL-18 的表达（iTRUCK19.18），且不会改变细胞的适应性。有趣的是，IL-18 的分泌除了需要强力霉素外，还需要 T 细胞的活化，从而实现了对 CAR-T 细胞效力的外部调节。这种效应转化为 CAR-T 细胞对侵袭性血液肿瘤和实体肿瘤模型的抗肿瘤活性。在与临床相关的背景下，我们生成了源自患者的 iTRUCK19.18 细胞，它能够以多西环素依赖的方式根除原发性 B 细胞肿瘤。此外，释放IL-18的CAR-T细胞还能将亲肿瘤巨噬细胞极化为抗肿瘤表型，这表明CAR-T细胞具有调节肿瘤微环境的潜力。总之，我们的研究表明，我们的平台可以在没有反式激活剂的情况下生成外源控制的 CAR-T 细胞，并增强其效力。
{"title":"First-in-class transactivator-free, doxycycline-inducible IL-18-engineered CAR-T cells for relapsed/refractory B cell lymphomas","authors":"Pedro Justicia-Lirio, María Tristán-Manzano, Noelia Maldonado-Pérez, Carmen Barbero-Jiménez, Marina Cortijo-Gutiérrez, Kristina Pavlovic, Francisco J. Molina-Estevez, Pilar Muñoz, Ana Hinckley-Boned, Juan R. Rodriguez-Madoz, Felipe Prosper, Carmen Griñán-Lison, Saúl A. Navarro-Marchal, Julia Muñoz-Ballester, Pedro A. González-Sierra, Concha Herrera, Juan A. Marchal, Francisco Martín","doi":"10.1016/j.omtn.2024.102308","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102308","url":null,"abstract":"Although chimeric antigen receptor (CAR) T cell therapy has revolutionized type B cancer treatment, efficacy remains limited in various lymphomas and solid tumors. Reinforcing conventional CAR-T cells to release cytokines can improve their efficacy but also increase safety concerns. Several strategies have been developed to regulate their secretion using minimal promoters that are controlled by chimeric proteins harboring transactivators. However, these chimeric proteins can disrupt the normal physiology of T cells. Here, we present the first transactivator-free anti-CD19 CAR-T cells able to control IL-18 expression (iTRUCK19.18) under ultra-low doses of doxycycline and without altering cellular fitness. Interestingly, IL-18 secretion requires T cell activation in addition to doxycycline, allowing the external regulation of CAR-T cell potency. This effect was translated into an increased CAR-T cell antitumor activity against aggressive hematologic and solid tumor models. In a clinically relevant context, we generated patient-derived iTRUCK19.18 cells capable of eradicating primary B cells tumors in a doxycycline-dependent manner. Furthermore, IL-18-releasing CAR-T cells polarized pro-tumoral macrophages toward an antitumoral phenotype, suggesting potential for modulating the tumor microenvironment. In summary, we showed that our platform can generate exogenously controlled CAR-T cells with enhanced potency and in the absence of transactivators.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"40 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222598","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 : 2024-08-15DOI: 10.1016/j.omtn.2024.102305
Laura Teodori, Sarah K. Ochoa, Marjan Omer, Veronica L. Andersen, Pernille Bech, Junyi Su, Jessica Bridoux, Jesper S. Nielsen, Mathias B. Bertelsen, Sophie Hernot, Kurt V. Gothelf, Jørgen Kjems
In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both diagnostics and cancer molecular imaging with prospects for future theranostic applications.
{"title":"Plug-and-play nucleic acid-mediated multimerization of biparatopic nanobodies for molecular imaging","authors":"Laura Teodori, Sarah K. Ochoa, Marjan Omer, Veronica L. Andersen, Pernille Bech, Junyi Su, Jessica Bridoux, Jesper S. Nielsen, Mathias B. Bertelsen, Sophie Hernot, Kurt V. Gothelf, Jørgen Kjems","doi":"10.1016/j.omtn.2024.102305","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102305","url":null,"abstract":"In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both diagnostics and cancer molecular imaging with prospects for future theranostic applications.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"30 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222604","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 : 2024-08-15DOI: 10.1016/j.omtn.2024.102304
Tomas Venit, Jeremy Blavier, Sibusiso B. Maseko, Sam Shu, Lilia Espada, Christopher Breunig, Hans-Peter Holthoff, Sabrina C. Desbordes, Martin Lohse, Gennaro Esposito, Jean-Claude Twizere, Piergiorgio Percipalle
Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of severe acute respiratory syndrome coronavirus s (SARS-CoV-2) entry in the host cell by targeting surface-exposed spike protein. Here we have established a pipeline that instead targets highly conserved viral proteins made only after viral entry into the host cell when the SARS-CoV-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein (Nsp)9, which is required for viral genome replication. One of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and nuclear magnetic resonance spectroscopy for epitope mapping, was expressed and found to block SARS-CoV-2 replication specifically. We next encapsulated 2NSP23 nanobody into lipid nanoparticles (LNPs) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in cells and suppresses multiple SARS-CoV-2 variants, as seen by qPCR and RNA deep sequencing. These results are corroborated in three-dimensional reconstituted human epithelium kept at air-liquid interface to mimic the outer surface of lung tissue. These observations indicate that LNP-mRNA-2NSP23 is internalized and, after translation, it inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative strategy to generate novel antiviral drugs highly efficient across coronaviruses.
{"title":"Nanobody against SARS-CoV-2 non-structural protein Nsp9 inhibits viral replication in human airway epithelia","authors":"Tomas Venit, Jeremy Blavier, Sibusiso B. Maseko, Sam Shu, Lilia Espada, Christopher Breunig, Hans-Peter Holthoff, Sabrina C. Desbordes, Martin Lohse, Gennaro Esposito, Jean-Claude Twizere, Piergiorgio Percipalle","doi":"10.1016/j.omtn.2024.102304","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102304","url":null,"abstract":"Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of severe acute respiratory syndrome coronavirus s (SARS-CoV-2) entry in the host cell by targeting surface-exposed spike protein. Here we have established a pipeline that instead targets highly conserved viral proteins made only after viral entry into the host cell when the SARS-CoV-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein (Nsp)9, which is required for viral genome replication. One of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and nuclear magnetic resonance spectroscopy for epitope mapping, was expressed and found to block SARS-CoV-2 replication specifically. We next encapsulated 2NSP23 nanobody into lipid nanoparticles (LNPs) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in cells and suppresses multiple SARS-CoV-2 variants, as seen by qPCR and RNA deep sequencing. These results are corroborated in three-dimensional reconstituted human epithelium kept at air-liquid interface to mimic the outer surface of lung tissue. These observations indicate that LNP-mRNA-2NSP23 is internalized and, after translation, it inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative strategy to generate novel antiviral drugs highly efficient across coronaviruses.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"60 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222634","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 : 2024-08-08DOI: 10.1016/j.omtn.2024.102295
Chiranjib Chakraborty, Manojit Bhattacharya, Sang-Soo Lee, Zhi-Hong Wen, Yi-Hao Lo
Due to the transformation of artificial intelligence (AI) tools and technologies, AI-driven drug discovery has come to the forefront. It reduces the time and expenditure. Due to these advantages, pharmaceutical industries are concentrating on AI-driven drug discovery. Several drug molecules have been discovered using AI-based techniques and tools, and several newly AI-discovered drug molecules have already entered clinical trials. In this review, we first present the data and their resources in the pharmaceutical sector for AI-driven drug discovery and illustrated some significant algorithms or techniques used for AI and ML which are used in this field. We gave an overview of the deep neural network (NN) models and compared them with artificial NNs. Then, we illustrate the recent advancement of the landscape of drug discovery using AI to deep learning, such as the identification of drug targets, prediction of their structure, estimation of drug-target interaction, estimation of drug-target binding affinity, design of drug, prediction of drug toxicity, estimation of absorption, distribution, metabolism, excretion, toxicity; and estimation of drug-drug interaction. Moreover, we highlighted the success stories of AI-driven drug discovery and discussed several collaboration and the challenges in this area. The discussions in the article will enrich the pharmaceutical industry.
由于人工智能（AI）工具和技术的变革，AI 驱动的药物发现已走到前沿。它缩短了时间，减少了开支。由于这些优势，制药行业正专注于人工智能驱动的药物发现。一些药物分子已经利用基于人工智能的技术和工具被发现，一些新发现的人工智能药物分子已经进入临床试验阶段。在这篇综述中，我们首先介绍了制药领域用于人工智能驱动药物发现的数据及其资源，并说明了该领域使用的一些重要的人工智能和 ML 算法或技术。我们概述了深度神经网络（NN）模型，并将其与人工神经网络进行了比较。然后，我们阐述了利用人工智能和深度学习进行药物发现的最新进展，如药物靶点的识别、药物靶点结构的预测、药物与靶点相互作用的估计、药物与靶点结合亲和力的估计、药物设计、药物毒性的预测、吸收、分布、代谢、排泄、毒性的估计以及药物与药物相互作用的估计。此外，我们还重点介绍了人工智能驱动药物发现的成功案例，并讨论了这一领域的若干合作与挑战。文章中的讨论将丰富制药行业的内容。
{"title":"The changing scenario of drug discovery using AI to deep learning: Recent advancement, success stories, collaborations, and challenges","authors":"Chiranjib Chakraborty, Manojit Bhattacharya, Sang-Soo Lee, Zhi-Hong Wen, Yi-Hao Lo","doi":"10.1016/j.omtn.2024.102295","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102295","url":null,"abstract":"Due to the transformation of artificial intelligence (AI) tools and technologies, AI-driven drug discovery has come to the forefront. It reduces the time and expenditure. Due to these advantages, pharmaceutical industries are concentrating on AI-driven drug discovery. Several drug molecules have been discovered using AI-based techniques and tools, and several newly AI-discovered drug molecules have already entered clinical trials. In this review, we first present the data and their resources in the pharmaceutical sector for AI-driven drug discovery and illustrated some significant algorithms or techniques used for AI and ML which are used in this field. We gave an overview of the deep neural network (NN) models and compared them with artificial NNs. Then, we illustrate the recent advancement of the landscape of drug discovery using AI to deep learning, such as the identification of drug targets, prediction of their structure, estimation of drug-target interaction, estimation of drug-target binding affinity, design of drug, prediction of drug toxicity, estimation of absorption, distribution, metabolism, excretion, toxicity; and estimation of drug-drug interaction. Moreover, we highlighted the success stories of AI-driven drug discovery and discussed several collaboration and the challenges in this area. The discussions in the article will enrich the pharmaceutical industry.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"60 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142222616","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 : 2024-07-31DOI: 10.1016/j.omtn.2024.102290
Maud Auger, Luis Sorroza-Martinez, Nadine Brahiti, Carole-Ann Huppé, Laurence Faucher-Giguère, Imen Arbi, Maxime Hervault, Xue Cheng, Bruno Gaillet, Frédéric Couture, David Guay, Al-Halifa Soultan
Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs and the endosomal entrapment phenomenon, which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogeneous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a pegylated (PEG) linker was the most beneficial strategy compared with direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.
{"title":"Enhancing peptide and PMO delivery to mouse airway epithelia by chemical conjugation with the amphiphilic peptide S10","authors":"Maud Auger, Luis Sorroza-Martinez, Nadine Brahiti, Carole-Ann Huppé, Laurence Faucher-Giguère, Imen Arbi, Maxime Hervault, Xue Cheng, Bruno Gaillet, Frédéric Couture, David Guay, Al-Halifa Soultan","doi":"10.1016/j.omtn.2024.102290","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102290","url":null,"abstract":"Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs and the endosomal entrapment phenomenon, which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogeneous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a pegylated (PEG) linker was the most beneficial strategy compared with direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"46 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141932108","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 : 2024-07-31DOI: 10.1016/j.omtn.2024.102291
James W. Gilbert, Zachary Kennedy, Bruno Godinho, Ashley Summers, Alexandra Weiss, Dimas Echeverria, Brianna Bramato, Nicholas McHugh, David Cooper, Ken Yamada, Matthew Hassler, Hélène Tran, Fen Biao Gao, Robert H. Brown Jr., Anastasia Khvorova
A hexanucleotide (GC) repeat expansion (HRE) within intron one of is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing mRNA variants are found to preferentially localize to the nucleus, and thus mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting mRNA variants specifically or non-selectively reduce the expression of mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing mRNA transcripts. Combined, these data support RNAi-based degradation of as a potential therapeutic paradigm.
{"title":"Identification of selective and non-selective C9ORF72 targeting in vivo active siRNAs","authors":"James W. Gilbert, Zachary Kennedy, Bruno Godinho, Ashley Summers, Alexandra Weiss, Dimas Echeverria, Brianna Bramato, Nicholas McHugh, David Cooper, Ken Yamada, Matthew Hassler, Hélène Tran, Fen Biao Gao, Robert H. Brown Jr., Anastasia Khvorova","doi":"10.1016/j.omtn.2024.102291","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102291","url":null,"abstract":"A hexanucleotide (GC) repeat expansion (HRE) within intron one of is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing mRNA variants are found to preferentially localize to the nucleus, and thus mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting mRNA variants specifically or non-selectively reduce the expression of mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing mRNA transcripts. Combined, these data support RNAi-based degradation of as a potential therapeutic paradigm.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"2 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141932107","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 : 2024-07-31DOI: 10.1016/j.omtn.2024.102292
Aishwarya Saraswat, Hari Priya Vemana, Vikas Dukhande, Ketan Patel
Patients suffering from BRAF mutant melanoma have tumor recurrence within merely 7 months of treatment with a potent BRAF inhibitor (BRAFi) like vemurafenib. It has been proven that diverse molecular pathways driving BRAFi resistance converge to activation of c-Myc in melanoma. Therefore, we identified a novel combinatorial therapeutic strategy by targeting loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene and upregulated BRD4 oncoprotein as Myc-dependent vulnerabilities of drug-resistant melanoma. Being promising therapeutic targets, we decided to concomitantly deliver PTEN plasmid and BRD4 targeted PROteolysis-TArgeting Chimera (ARV) to drug the “undruggable” c-Myc in BRAFi-resistant melanoma. Since PTEN plasmid and ARV are distinct in their physicochemical properties, we fabricated PTEN-plasmid loaded lipid nanoparticles (PL-NANO) and ARV-825-loaded nanoliposomes (AL-NANO) to yield a mean particle size of less than 100 nm and greater than 99% encapsulation efficiency for each therapeutic payload. Combination of PL-NANO and AL-NANO displayed synergistic tumor growth inhibition and substantial apoptosis in two-dimensional and three-dimensional models. Importantly, simultaneous delivery of PL-NANO and AL-NANO achieved significant upregulation of PTEN expression levels and degradation of BRD4 protein to ultimately downregulate c-Myc levels in BRAFi-resistant melanoma cells. Altogether, lipid nanocarriers delivering this novel lethal cocktail stands as one-of-a-kind gene therapy to target undruggable c-Myc oncogene in BRAFi-resistant melanoma.
{"title":"Novel gene therapy for drug-resistant melanoma: Synergistic combination of PTEN plasmid and BRD4 PROTAC-loaded lipid nanocarriers","authors":"Aishwarya Saraswat, Hari Priya Vemana, Vikas Dukhande, Ketan Patel","doi":"10.1016/j.omtn.2024.102292","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102292","url":null,"abstract":"Patients suffering from BRAF mutant melanoma have tumor recurrence within merely 7 months of treatment with a potent BRAF inhibitor (BRAFi) like vemurafenib. It has been proven that diverse molecular pathways driving BRAFi resistance converge to activation of c-Myc in melanoma. Therefore, we identified a novel combinatorial therapeutic strategy by targeting loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene and upregulated BRD4 oncoprotein as Myc-dependent vulnerabilities of drug-resistant melanoma. Being promising therapeutic targets, we decided to concomitantly deliver PTEN plasmid and BRD4 targeted PROteolysis-TArgeting Chimera (ARV) to drug the “undruggable” c-Myc in BRAFi-resistant melanoma. Since PTEN plasmid and ARV are distinct in their physicochemical properties, we fabricated PTEN-plasmid loaded lipid nanoparticles (PL-NANO) and ARV-825-loaded nanoliposomes (AL-NANO) to yield a mean particle size of less than 100 nm and greater than 99% encapsulation efficiency for each therapeutic payload. Combination of PL-NANO and AL-NANO displayed synergistic tumor growth inhibition and substantial apoptosis in two-dimensional and three-dimensional models. Importantly, simultaneous delivery of PL-NANO and AL-NANO achieved significant upregulation of PTEN expression levels and degradation of BRD4 protein to ultimately downregulate c-Myc levels in BRAFi-resistant melanoma cells. Altogether, lipid nanocarriers delivering this novel lethal cocktail stands as one-of-a-kind gene therapy to target undruggable c-Myc oncogene in BRAFi-resistant melanoma.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"39 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141932071","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}
Antisense oligonucleotides (ASOs) are a therapeutic modality for incurable diseases. However, systemic injection of gapmer-type ASOs causes class-related toxicities, including prolongation of activated partial thromboplastin time (aPTT) and thrombocytopenia. We previously reported that cholesterol-conjugated DNA/RNA heteroduplex oligonucleotides (Chol-HDOs) exhibit significantly enhanced gene-silencing effects compared to ASOs, even in the central nervous system, by crossing the blood-brain barrier. In the present study, we initially evaluated the effect of the HDO structure on class-related toxicities. The HDO structure ameliorated the class-related toxicities associated with ASOs, but they remained to some extent. As a further antidote, we have developed artificial cationic oligopeptides, L-2,4-diaminobutanoic acid oligomers (DabOs), which bind to the phosphates in the major groove of the A-type double-helical structure of HDOs. The DabO/Chol-HDO complex showed significantly improved aPTT prolongation and thrombocytopenia in mice while maintaining gene-silencing efficacy. Moreover, the conjugation with DabOs effectively prevented cerebral infarction, a condition frequently observed in mice intravenously injected with high-dose Chol-HDO. These approaches, combining HDO technology with DabOs, offer distinct advantages over conventional strategies in reducing toxicities. Consequently, the DabO/HDO complex represents a promising platform for overcoming the class-related toxicities associated with therapeutic ASOs.
反义寡核苷酸（ASO）是一种治疗不治之症的方法。然而，全身注射间隙型反义寡核苷酸会引起与类相关的毒性，包括活化部分凝血活酶时间（aPTT）延长和血小板减少。我们以前曾报道过，胆固醇共轭 DNA/RNA 异质双链寡核苷酸（Chol-HDOs）通过穿越血脑屏障，即使在中枢神经系统中也能表现出比 ASOs 明显更强的基因沉默效应。在本研究中，我们初步评估了 HDO 结构对同类相关毒性的影响。HDO 结构改善了与 ASOs 相关的类相关毒性，但在一定程度上仍然存在。作为进一步的解毒剂，我们开发了人工阳离子寡肽--L-2,4-二氨基丁酸寡聚体（DabOs），它能与 HDOs 的 A 型双螺旋结构主要沟槽中的磷酸盐结合。DabO/Chol-HDO 复合物能显著改善小鼠的 aPTT 延长和血小板减少，同时保持基因沉默的功效。此外，与 DabOs 的共轭还能有效防止脑梗塞，而脑梗塞是静脉注射大剂量 Chol-HDO 的小鼠经常出现的症状。与传统策略相比，这些将 HDO 技术与 DabOs 相结合的方法在降低毒性方面具有明显优势。因此，DabO/HDO 复合物是克服与治疗性 ASO 有关的类相关毒性的一个前景广阔的平台。
{"title":"DNA/RNA heteroduplex technology with cationic oligopeptide reduces class-related adverse effects of nucleic acid drugs","authors":"Masahiro Ohara, Tetsuya Nagata, Rintaro Iwata Hara, Kie Yoshida-Tanaka, Nozomi Toide, Kazunori Takagi, Kazuki Sato, Tomoya Takenaka, Masanori Nakakariya, Kenichi Miyata, Maeda Yusuke, Kazuko Toh, Takeshi Wada, Takanori Yokota","doi":"10.1016/j.omtn.2024.102289","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102289","url":null,"abstract":"Antisense oligonucleotides (ASOs) are a therapeutic modality for incurable diseases. However, systemic injection of gapmer-type ASOs causes class-related toxicities, including prolongation of activated partial thromboplastin time (aPTT) and thrombocytopenia. We previously reported that cholesterol-conjugated DNA/RNA heteroduplex oligonucleotides (Chol-HDOs) exhibit significantly enhanced gene-silencing effects compared to ASOs, even in the central nervous system, by crossing the blood-brain barrier. In the present study, we initially evaluated the effect of the HDO structure on class-related toxicities. The HDO structure ameliorated the class-related toxicities associated with ASOs, but they remained to some extent. As a further antidote, we have developed artificial cationic oligopeptides, L-2,4-diaminobutanoic acid oligomers (DabOs), which bind to the phosphates in the major groove of the A-type double-helical structure of HDOs. The DabO/Chol-HDO complex showed significantly improved aPTT prolongation and thrombocytopenia in mice while maintaining gene-silencing efficacy. Moreover, the conjugation with DabOs effectively prevented cerebral infarction, a condition frequently observed in mice intravenously injected with high-dose Chol-HDO. These approaches, combining HDO technology with DabOs, offer distinct advantages over conventional strategies in reducing toxicities. Consequently, the DabO/HDO complex represents a promising platform for overcoming the class-related toxicities associated with therapeutic ASOs.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"98 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141932111","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 : 2024-07-19DOI: 10.1016/j.omtn.2024.102282
Anna Bellizzi, Senem Çakır, Martina Donadoni, Rahsan Sariyer, Shuren Liao, Hong Liu, Guo-Xiang Ruan, Jennifer Gordon, Kamel Khalili, Ilker K. Sariyer
Although our understanding of herpes simplex virus type 1 (HSV-1) biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR-Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D human induced pluripotent stem cell-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 genes. We found that targeting the ICP0 or ICP27 genes with AAV2-CRISPR-Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR-Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR-Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1-associated complications.
{"title":"Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models","authors":"Anna Bellizzi, Senem Çakır, Martina Donadoni, Rahsan Sariyer, Shuren Liao, Hong Liu, Guo-Xiang Ruan, Jennifer Gordon, Kamel Khalili, Ilker K. Sariyer","doi":"10.1016/j.omtn.2024.102282","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102282","url":null,"abstract":"Although our understanding of herpes simplex virus type 1 (HSV-1) biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR-Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D human induced pluripotent stem cell-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 genes. We found that targeting the ICP0 or ICP27 genes with AAV2-CRISPR-Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR-Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR-Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1-associated complications.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"69 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782960","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 : 2024-07-19DOI: 10.1016/j.omtn.2024.102285
Megumi Shigematsu, Takuya Kawamura, Deepak A. Deshpande, Yohei Kirino
Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA sequencing (RNA-seq) method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5′-phosphate and 3′-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5′-tRNA halves containing a 2′,3′-cyclic phosphate. We discovered a remarkable accumulation of the 5′-half derived from tRNA in plasma from COPD patients, whereas the 5′-tRNA half is predominant in healthy donors. Further, the 5′-tRNA half activates human macrophages via Toll-like receptor 7 and induces cytokine production. Additionally, we identified circulating rRNA-derived fragments that were upregulated in COPD patients and demonstrated their ability to induce cytokine production in macrophages. Our findings provide evidence of circulating, immune-active sncRNAs in patients with COPD, suggesting that they serve as inflammatory mediators in the pathogenesis of COPD.
{"title":"Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease","authors":"Megumi Shigematsu, Takuya Kawamura, Deepak A. Deshpande, Yohei Kirino","doi":"10.1016/j.omtn.2024.102285","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102285","url":null,"abstract":"Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA sequencing (RNA-seq) method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5′-phosphate and 3′-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5′-tRNA halves containing a 2′,3′-cyclic phosphate. We discovered a remarkable accumulation of the 5′-half derived from tRNA in plasma from COPD patients, whereas the 5′-tRNA half is predominant in healthy donors. Further, the 5′-tRNA half activates human macrophages via Toll-like receptor 7 and induces cytokine production. Additionally, we identified circulating rRNA-derived fragments that were upregulated in COPD patients and demonstrated their ability to induce cytokine production in macrophages. Our findings provide evidence of circulating, immune-active sncRNAs in patients with COPD, suggesting that they serve as inflammatory mediators in the pathogenesis of COPD.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"20 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141872466","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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