mRNA is a promising modality for expressing a protein . Drug delivery systems are required for the efficient transfection of mRNA into cells. In this study, we evaluated several drug delivery systems for transfecting mRNA into tumors. A lipid nanoparticle delivered mRNA to the draining lymph nodes and liver, even by intratumoral injection. A liposome-based system did not consistently provide mRNA for different types of tumor cells. We found that PBS introduced mRNA into several tumors, and calcium ions enhanced the efficiency, particularly in male mice. The circular dichroism spectrometer suggested a structural change in mRNA in PBS. Transmission electron microscopy revealed that calcium ions promoted the formation of mRNA nanoparticles in PBS. Transfection of mRNAs coding OX40-ligand, interleukin (IL)-36γ, and IL-23 by PBS + calcium ions attenuated tumor growth. Our results indicate that combining PBS with calcium ions promotes the transfection of mRNA into tumors. These data provide information for the development of methods for transfection of mRNA for cancer therapy.
{"title":"Combining mRNA with PBS and calcium ions improves the efficiency of the transfection of mRNA into tumors","authors":"Noriko Ohta, Takashi Matsuzaki, Masayoshi Nakai, Yasuhiko Tabata, Keisuke Nimura","doi":"10.1016/j.omtn.2024.102273","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102273","url":null,"abstract":"mRNA is a promising modality for expressing a protein . Drug delivery systems are required for the efficient transfection of mRNA into cells. In this study, we evaluated several drug delivery systems for transfecting mRNA into tumors. A lipid nanoparticle delivered mRNA to the draining lymph nodes and liver, even by intratumoral injection. A liposome-based system did not consistently provide mRNA for different types of tumor cells. We found that PBS introduced mRNA into several tumors, and calcium ions enhanced the efficiency, particularly in male mice. The circular dichroism spectrometer suggested a structural change in mRNA in PBS. Transmission electron microscopy revealed that calcium ions promoted the formation of mRNA nanoparticles in PBS. Transfection of mRNAs coding OX40-ligand, interleukin (IL)-36γ, and IL-23 by PBS + calcium ions attenuated tumor growth. Our results indicate that combining PBS with calcium ions promotes the transfection of mRNA into tumors. These data provide information for the development of methods for transfection of mRNA for cancer therapy.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"329 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782962","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 : 2024-07-17DOI: 10.1016/j.omtn.2024.102271
Hussain Al Dera, Bdour Al Qahtani
Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, presents considerable challenges in both diagnosis and treatment. It is categorized into sporadic and familial amyotrophic lateral sclerosis (fALS); the latter accounts for approximately 10% of cases and is primarily inherited in an autosomal dominant manner. This review summarizes the molecular genetics of fALS, highlighting key mutations that contribute to its pathogenesis, such as mutations in , , and . Central to this discourse is exploring antisense oligonucleotides (ASOs) that target these genetic aberrations, providing a promising therapeutic strategy. This review provides a detailed overview of the molecular mechanisms underlying fALS and the potential therapeutic value of ASOs, offering new insights into treating neurodegenerative diseases.
{"title":"Molecular mechanisms and antisense oligonucleotide therapies of familial amyotrophic lateral sclerosis","authors":"Hussain Al Dera, Bdour Al Qahtani","doi":"10.1016/j.omtn.2024.102271","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102271","url":null,"abstract":"Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, presents considerable challenges in both diagnosis and treatment. It is categorized into sporadic and familial amyotrophic lateral sclerosis (fALS); the latter accounts for approximately 10% of cases and is primarily inherited in an autosomal dominant manner. This review summarizes the molecular genetics of fALS, highlighting key mutations that contribute to its pathogenesis, such as mutations in , , and . Central to this discourse is exploring antisense oligonucleotides (ASOs) that target these genetic aberrations, providing a promising therapeutic strategy. This review provides a detailed overview of the molecular mechanisms underlying fALS and the potential therapeutic value of ASOs, offering new insights into treating neurodegenerative diseases.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"40 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782964","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 : 2024-07-17DOI: 10.1016/j.omtn.2024.102274
Sun-Ji Park, Seo Jung Park, Yang Woo Kwon, Eui-Hwan Choi
CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.
CRISPR-Cas9 已成为基因组编辑的强大工具。然而，Cas9 基因组编辑面临着低效率和脱靶效应等挑战。在这里，我们报告了在人类胚胎肾脏 293T 和人类诱导多能干细胞中，RAD51（同源重组的关键因子）和 SCR7（一种 DNA 连接酶 IV 小分子抑制剂）的联合处理提高了 CRISPR-Cas9 介导的基因组编辑效率，细胞透射电子显微镜和功能分析证实了这一点。首先，我们的研究结果揭示了 RAD51 在同源重组（HR）介导的 DNA 修复过程中的关键作用。外源 RAD51 水平的升高促进了单链 DNA 间隙修复过程的复制后步骤，确保了 DNA 复制的完成。其次，利用一体化的CRISPR-Cas9-RAD51系统，高表达的RAD51通过与SCR7协同激活基于HR的修复途径，提高了多个内源基因的敲除/剔除效率和插入/缺失（InDel）突变。桑格测序显示，RAD51-SCR7 在多个基因组位点的 InDel 突变比例方面有不同的结果。第三，RAD51-SCR7的组合可以通过增强HR过程诱导高效的R环解析和DNA修复，从而导致DNA复制停滞，这对基于CRISPR-Cas9的稳定基因组编辑是有利的。我们的研究表明，通过RAD51和SCR7提高CRISPR-Cas9的效率，有望应用于基因组编辑，为生物技术和治疗提供潜在的进步。
{"title":"Synergistic combination of RAD51-SCR7 enhances HR repair system and improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation","authors":"Sun-Ji Park, Seo Jung Park, Yang Woo Kwon, Eui-Hwan Choi","doi":"10.1016/j.omtn.2024.102274","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102274","url":null,"abstract":"CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"180 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782961","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}
RNase H-dependent antisense oligonucleotides (gapmer ASOs) represent a class of nucleic acid therapeutics that bind to target RNA to facilitate RNase H-mediated RNA cleavage, thereby regulating the expression of disease-associated proteins. Integrating artificial nucleic acids into gapmer ASOs enhances their therapeutic efficacy. Among these, amido-bridged nucleic acid (AmNA) stands out for its potential to confer high affinity and stability to ASOs. However, a significant challenge in the design of gapmer ASOs incorporating artificial nucleic acids, such as AmNA, is the accurate prediction of their melting temperature (). The is a critical parameter for designing effective gapmer ASOs to ensure proper functioning. However, predicting accurate values for oligonucleotides containing artificial nucleic acids remains problematic. We developed a prediction model using a library of AmNA-containing ASOs to address this issue. We measured the values of 157 oligonucleotides through differential scanning calorimetry, enabling the construction of an accurate prediction model. Additionally, molecular dynamics simulations were used to elucidate the molecular mechanisms by which AmNA modifications elevate , thereby informing the design strategies of gapmer ASOs.
{"title":"Construction of a Tm-value prediction model and molecular dynamics study of AmNA-containing gapmer antisense oligonucleotide","authors":"Masataka Kuroda, Yuuya Kasahara, Masako Hirose, Harumi Yamaguma, Masayuki Oda, Chioko Nagao, Kenji Mizuguchi","doi":"10.1016/j.omtn.2024.102272","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102272","url":null,"abstract":"RNase H-dependent antisense oligonucleotides (gapmer ASOs) represent a class of nucleic acid therapeutics that bind to target RNA to facilitate RNase H-mediated RNA cleavage, thereby regulating the expression of disease-associated proteins. Integrating artificial nucleic acids into gapmer ASOs enhances their therapeutic efficacy. Among these, amido-bridged nucleic acid (AmNA) stands out for its potential to confer high affinity and stability to ASOs. However, a significant challenge in the design of gapmer ASOs incorporating artificial nucleic acids, such as AmNA, is the accurate prediction of their melting temperature (). The is a critical parameter for designing effective gapmer ASOs to ensure proper functioning. However, predicting accurate values for oligonucleotides containing artificial nucleic acids remains problematic. We developed a prediction model using a library of AmNA-containing ASOs to address this issue. We measured the values of 157 oligonucleotides through differential scanning calorimetry, enabling the construction of an accurate prediction model. Additionally, molecular dynamics simulations were used to elucidate the molecular mechanisms by which AmNA modifications elevate , thereby informing the design strategies of gapmer ASOs.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"3 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782963","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 : 2024-07-16DOI: 10.1016/j.omtn.2024.102269
Véronique Bolduc, Katherine Sizov, Astrid Brull, Eric Esposito, Grace S. Chen, Prech Uapinyoying, Apurva Sarathy, Kory Johnson, Carsten G. Bönnemann
The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR-Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at an SNV in the gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed SpCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep sequencing for two gRNAs tested showed that single-nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.
{"title":"Allele-specific CRISPR-Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy","authors":"Véronique Bolduc, Katherine Sizov, Astrid Brull, Eric Esposito, Grace S. Chen, Prech Uapinyoying, Apurva Sarathy, Kory Johnson, Carsten G. Bönnemann","doi":"10.1016/j.omtn.2024.102269","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102269","url":null,"abstract":"The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR-Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at an SNV in the gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed SpCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep sequencing for two gRNAs tested showed that single-nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"41 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782966","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 : 2024-07-16DOI: 10.1016/j.omtn.2024.102267
Loree C. Heller, Guilan Shi, Amanda Sales Conniff, Julie Singh, Samantha Mannarino, Jody Synowiec, Richard Heller
Interleukin-12 (IL-12) gene electrotransfer (GET) delivery is highly effective in inducing long-term, complete regression in mouse and human melanoma and other solid tumors. Therapeutic efficacy is enhanced by immune checkpoint inhibitors, and the combination of IL-12 plasmid GET (pIL-12 GET) and anti-programmed cell death protein 1 (PD-1) monoclonal antibodies has reached clinical trials. In this study, we designed peptides and plasmids encoding the mouse homologs of the pembrolizumab and nivolumab programmed cell death 1 ligand 1 (PD-L1) binding regions. We hypothesized that intratumor autocrine/paracrine peptide expression would block PD-1/PD-L1 binding and provide cancer patients with an effective and cost-efficient treatment alternative. We demonstrated that the mouse homolog to pembrolizumab was effective at blocking PD-1/PD-L1 . After intratumor plasmid delivery, both peptides bound PD-L1 on tumor cells. We established that plasmid DNA delivery to tumors or to tumor cells upregulated several immune modulators and PD-L1 mRNA and protein, potentiating this therapy. Finally, we tested the combination of pIL-12 GET therapy and peptide plasmids. We determined that pIL-12 GET therapeutic efficacy could be enhanced by combination with the plasmid encoding the pembrolizumab mouse homolog.
{"title":"IL-12 and PD-1 peptide combination gene therapy for the treatment of melanoma","authors":"Loree C. Heller, Guilan Shi, Amanda Sales Conniff, Julie Singh, Samantha Mannarino, Jody Synowiec, Richard Heller","doi":"10.1016/j.omtn.2024.102267","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102267","url":null,"abstract":"Interleukin-12 (IL-12) gene electrotransfer (GET) delivery is highly effective in inducing long-term, complete regression in mouse and human melanoma and other solid tumors. Therapeutic efficacy is enhanced by immune checkpoint inhibitors, and the combination of IL-12 plasmid GET (pIL-12 GET) and anti-programmed cell death protein 1 (PD-1) monoclonal antibodies has reached clinical trials. In this study, we designed peptides and plasmids encoding the mouse homologs of the pembrolizumab and nivolumab programmed cell death 1 ligand 1 (PD-L1) binding regions. We hypothesized that intratumor autocrine/paracrine peptide expression would block PD-1/PD-L1 binding and provide cancer patients with an effective and cost-efficient treatment alternative. We demonstrated that the mouse homolog to pembrolizumab was effective at blocking PD-1/PD-L1 . After intratumor plasmid delivery, both peptides bound PD-L1 on tumor cells. We established that plasmid DNA delivery to tumors or to tumor cells upregulated several immune modulators and PD-L1 mRNA and protein, potentiating this therapy. Finally, we tested the combination of pIL-12 GET therapy and peptide plasmids. We determined that pIL-12 GET therapeutic efficacy could be enhanced by combination with the plasmid encoding the pembrolizumab mouse homolog.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"52 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782967","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 : 2024-07-16DOI: 10.1016/j.omtn.2024.102268
Dongfang Wang, Damian Kaniowski, Karol Jacek, Yu-Lin Su, Chunsong Yu, Jeremy Hall, Haiqing Li, Mingye Feng, Susanta Hui, Bożena Kaminska, Vittorio DeFranciscis, Carla Lucia Esposito, Annalisa DiRuscio, Bin Zhang, Guido Marcucci, Ya-Huei Kuo, Marcin Kortylewski
Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation . At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation () and antigen-presentation ()-related genes with concomitant reduction of leukemia-promoting . Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible silencing , IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.
尽管先天性免疫受体（如 Toll 样受体 9 (TLR9)）高表达，急性髓性白血病（AML）细胞仍会抵抗分化刺激。我们以前曾证实，使用 TLR9 靶向诱饵寡核苷酸（CpG-STAT3d）靶向信号转导子和转录激活子 3（STAT3）可增加人和小鼠 AML 细胞的免疫原性。在这里，我们阐明了 STAT3 抑制/TLR9 激活驱动 inv(16) AML 重编程的分子机制。在转录水平上，静脉注射 CpG-STAT3d 或白血病靶向沉默和 TLR9 共同刺激后，从小鼠体内分离出的 AML 细胞显示出类似的髓细胞分化（）和抗原递呈（）相关基因的上调，同时白血病促进基因的减少。单细胞转录组学显示，CpG-STAT3d 能诱导 AML 细胞多线分化为单核细胞/巨噬细胞、红细胞和 B 细胞亚群。诱导性沉默显示，IRF8 的上调对白血病细胞的单核-巨噬细胞分化至关重要。TLR9驱动的AML细胞重编程可能是通过下调STAT3控制的甲基化调节因子（如DNMT1和DNMT3）实现的。事实上，使用阿扎胞苷抑制DNA甲基转移酶（DNMT）和单独使用CpG寡核苷酸可模拟CpG-STAT3d效应，从而导致AML细胞分化、T细胞活化和全身性白血病消退。这些发现凸显了双功能寡核苷酸的免疫治疗潜力，通过同时抑制 STAT3 和/或 DNMT，释放 TLR9 驱动的白血病细胞分化。
{"title":"Bi-functional CpG-STAT3 decoy oligonucleotide triggers multilineage differentiation of acute myeloid leukemia in mice","authors":"Dongfang Wang, Damian Kaniowski, Karol Jacek, Yu-Lin Su, Chunsong Yu, Jeremy Hall, Haiqing Li, Mingye Feng, Susanta Hui, Bożena Kaminska, Vittorio DeFranciscis, Carla Lucia Esposito, Annalisa DiRuscio, Bin Zhang, Guido Marcucci, Ya-Huei Kuo, Marcin Kortylewski","doi":"10.1016/j.omtn.2024.102268","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102268","url":null,"abstract":"Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation . At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation () and antigen-presentation ()-related genes with concomitant reduction of leukemia-promoting . Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible silencing , IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"354 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782969","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 : 2024-07-16DOI: 10.1016/j.omtn.2024.102270
Enkhjin Saruuldalai, Hwi-Ho Lee, Yeon-Su Lee, Eun Kyung Hong, Soyoun Ro, Yeochan Kim, TaeJin Ahn, Jong-Lyul Park, Seon-Young Kim, Seung-Phil Shin, Wonkyun Ronny Im, Eunjung Cho, Beom K. Choi, Jiyoung Joan Jang, Byung-Han Choi, Yuh-Seog Jung, In-Hoo Kim, Sang-Jin Lee, Yong Sun Lee
Recombinant adenovirus (rAdV) vector is the most promising vehicle to deliver an exogenous gene into target cells and is preferred for gene therapy. Exogenous gene expression from rAdV is often too inefficient to induce phenotypic changes and the amount of administered rAdV must be very high to achieve a therapeutic dose. However, it is often hampered because a high dose of rAdV is likely to induce cytotoxicity by activating immune responses. nc886, a 102-nucleotide non-coding RNA that is transcribed by RNA polymerase III, acts as an immune suppressor and a facilitator of AdV entry into the nucleus. Therefore, in this study, we have constructed an rAdV expressing nc886 (AdV:nc886) to explore whether AdV:nc886 overcomes the aforementioned drawbacks of conventional rAdV vectors. When infected into mouse cell lines and mice, AdV:nc886 expresses a sufficient amount of nc886, which suppresses the induction of interferon-stimulated genes and apoptotic pathways triggered by AdV infection. As a result, AdV:nc886 is less cytotoxic and produces more rAdV-delivered gene products, compared with the parental rAdV vector lacking nc886. In conclusion, this study demonstrates that the nc886-expressing rAdV could become a superior gene delivery vehicle with greater safety and higher efficiency for gene therapy.
{"title":"Adenovirus expressing nc886, an anti-interferon and anti-apoptotic non-coding RNA, is an improved gene delivery vector","authors":"Enkhjin Saruuldalai, Hwi-Ho Lee, Yeon-Su Lee, Eun Kyung Hong, Soyoun Ro, Yeochan Kim, TaeJin Ahn, Jong-Lyul Park, Seon-Young Kim, Seung-Phil Shin, Wonkyun Ronny Im, Eunjung Cho, Beom K. Choi, Jiyoung Joan Jang, Byung-Han Choi, Yuh-Seog Jung, In-Hoo Kim, Sang-Jin Lee, Yong Sun Lee","doi":"10.1016/j.omtn.2024.102270","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102270","url":null,"abstract":"Recombinant adenovirus (rAdV) vector is the most promising vehicle to deliver an exogenous gene into target cells and is preferred for gene therapy. Exogenous gene expression from rAdV is often too inefficient to induce phenotypic changes and the amount of administered rAdV must be very high to achieve a therapeutic dose. However, it is often hampered because a high dose of rAdV is likely to induce cytotoxicity by activating immune responses. nc886, a 102-nucleotide non-coding RNA that is transcribed by RNA polymerase III, acts as an immune suppressor and a facilitator of AdV entry into the nucleus. Therefore, in this study, we have constructed an rAdV expressing nc886 (AdV:nc886) to explore whether AdV:nc886 overcomes the aforementioned drawbacks of conventional rAdV vectors. When infected into mouse cell lines and mice, AdV:nc886 expresses a sufficient amount of nc886, which suppresses the induction of interferon-stimulated genes and apoptotic pathways triggered by AdV infection. As a result, AdV:nc886 is less cytotoxic and produces more rAdV-delivered gene products, compared with the parental rAdV vector lacking nc886. In conclusion, this study demonstrates that the nc886-expressing rAdV could become a superior gene delivery vehicle with greater safety and higher efficiency for gene therapy.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"5 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782965","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 : 2024-06-28DOI: 10.1016/j.omtn.2024.102264
Matthieu Drouyer, Jessica Merjane, Teodora Nedelkoska, Adrian Westhaus, Suzanne Scott, Scott Lee, Peter G.R. Burke, Simon McMullan, Jose L. Lanciego, Ana F. Vicente, Ricardo Bugallo, Carmen Unzu, Gloria González-Aseguinolaza, Anai Gonzalez-Cordero, Leszek Lisowski
Viral vectors based on recombinant adeno-associated virus (rAAV) have become the most widely used system for therapeutic gene delivery in the CNS. Despite clinical safety and efficacy in neurological applications, a barrier to adoption of the current generation of vectors lies in their limited efficiency, resulting in limited transduction of CNS target cells. To address this limitation, researchers have bioengineered fit-for-purpose AAVs with improved CNS tropism and tissue penetration. While the preclinical assessment of these novel AAVs is primarily conducted in animal models, human induced pluripotent stem cell (hiPSC)-derived organoids offer a unique opportunity to functionally evaluate novel AAV variants in a human context. In this study, we performed a comprehensive and unbiased evaluation of a large number of wild-type and bioengineered AAV capsids for their transduction efficiency in hiPSC-derived brain organoids. We demonstrate that efficient AAV transduction observed in organoids was recapitulated in both mouse and non-human primate models after cerebrospinal fluid (CSF) delivery. In summary, our study showcases the use of brain organoid systems for the pre-screening of novel AAV vectors. Additionally, we report data for novel AAV variants that exhibit improved CNS transduction efficiency when delivered via the CSF in preclinical models.
{"title":"Enhanced AAV-mediated transduction across preclinical CNS models: A comparative study in human brain organoids with cross-species evaluations","authors":"Matthieu Drouyer, Jessica Merjane, Teodora Nedelkoska, Adrian Westhaus, Suzanne Scott, Scott Lee, Peter G.R. Burke, Simon McMullan, Jose L. Lanciego, Ana F. Vicente, Ricardo Bugallo, Carmen Unzu, Gloria González-Aseguinolaza, Anai Gonzalez-Cordero, Leszek Lisowski","doi":"10.1016/j.omtn.2024.102264","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102264","url":null,"abstract":"Viral vectors based on recombinant adeno-associated virus (rAAV) have become the most widely used system for therapeutic gene delivery in the CNS. Despite clinical safety and efficacy in neurological applications, a barrier to adoption of the current generation of vectors lies in their limited efficiency, resulting in limited transduction of CNS target cells. To address this limitation, researchers have bioengineered fit-for-purpose AAVs with improved CNS tropism and tissue penetration. While the preclinical assessment of these novel AAVs is primarily conducted in animal models, human induced pluripotent stem cell (hiPSC)-derived organoids offer a unique opportunity to functionally evaluate novel AAV variants in a human context. In this study, we performed a comprehensive and unbiased evaluation of a large number of wild-type and bioengineered AAV capsids for their transduction efficiency in hiPSC-derived brain organoids. We demonstrate that efficient AAV transduction observed in organoids was recapitulated in both mouse and non-human primate models after cerebrospinal fluid (CSF) delivery. In summary, our study showcases the use of brain organoid systems for the pre-screening of novel AAV vectors. Additionally, we report data for novel AAV variants that exhibit improved CNS transduction efficiency when delivered via the CSF in preclinical models.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"51 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141587665","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 : 2024-06-26DOI: 10.1016/j.omtn.2024.102262
Amela Jusic, Zoi Erpapazoglou, Louise Torp Dalgaard, Païvi Lakkisto, David de Gonzalo Calvo, Bettina Benczik, Bence Ágg, Péter Ferdinandy, Katarzyna Fiedorowicz, Blanche Schroen, Antigone Lazou, Yvan Devaux, on behalf of EU-CardioRNA COST Action CA17129, AtheroNET COST Action CA21153
Mitochondria are the energy-producing organelles of mammalian cells with critical involvement in metabolism and signaling. Studying their regulation in pathological conditions may lead to the discovery of novel drugs to treat, for instance, cardiovascular or neurological diseases, which affect high-energy-consuming cells such as cardiomyocytes, hepatocytes, or neurons. Mitochondria possess both protein-coding and noncoding RNAs, such as microRNAs, long noncoding RNAs, circular RNAs, and piwi-interacting RNAs, encoded by the mitochondria or the nuclear genome. Mitochondrial RNAs are involved in anterograde-retrograde communication between the nucleus and mitochondria and play an important role in physiological and pathological conditions. Despite accumulating evidence on the presence and biogenesis of mitochondrial RNAs, their study continues to pose significant challenges. Currently, there are no standardized protocols and guidelines to conduct deep functional characterization and expression profiling of mitochondrial RNAs. To overcome major obstacles in this emerging field, the EU-CardioRNA and AtheroNET COST Action networks summarize currently available techniques and emphasize critical points that may constitute sources of variability and explain discrepancies between published results. Standardized methods and adherence to guidelines to quantify and study mitochondrial RNAs in normal and disease states will improve research outputs, their reproducibility, and translation potential to clinical application.
{"title":"Guidelines for mitochondrial RNA analysis","authors":"Amela Jusic, Zoi Erpapazoglou, Louise Torp Dalgaard, Païvi Lakkisto, David de Gonzalo Calvo, Bettina Benczik, Bence Ágg, Péter Ferdinandy, Katarzyna Fiedorowicz, Blanche Schroen, Antigone Lazou, Yvan Devaux, on behalf of EU-CardioRNA COST Action CA17129, AtheroNET COST Action CA21153","doi":"10.1016/j.omtn.2024.102262","DOIUrl":"https://doi.org/10.1016/j.omtn.2024.102262","url":null,"abstract":"Mitochondria are the energy-producing organelles of mammalian cells with critical involvement in metabolism and signaling. Studying their regulation in pathological conditions may lead to the discovery of novel drugs to treat, for instance, cardiovascular or neurological diseases, which affect high-energy-consuming cells such as cardiomyocytes, hepatocytes, or neurons. Mitochondria possess both protein-coding and noncoding RNAs, such as microRNAs, long noncoding RNAs, circular RNAs, and piwi-interacting RNAs, encoded by the mitochondria or the nuclear genome. Mitochondrial RNAs are involved in anterograde-retrograde communication between the nucleus and mitochondria and play an important role in physiological and pathological conditions. Despite accumulating evidence on the presence and biogenesis of mitochondrial RNAs, their study continues to pose significant challenges. Currently, there are no standardized protocols and guidelines to conduct deep functional characterization and expression profiling of mitochondrial RNAs. To overcome major obstacles in this emerging field, the EU-CardioRNA and AtheroNET COST Action networks summarize currently available techniques and emphasize critical points that may constitute sources of variability and explain discrepancies between published results. Standardized methods and adherence to guidelines to quantify and study mitochondrial RNAs in normal and disease states will improve research outputs, their reproducibility, and translation potential to clinical application.","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. Nucleic Acids","volume":"487 1","pages":""},"PeriodicalIF":8.8,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141551494","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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