Molecular Therapy-Methods & Clinical Development最新文献_第5页

Toward a large-batch manufacturing process for silicon-stabilized lipid nanoparticles: A highly customizable RNA delivery platform 硅稳定脂质纳米颗粒的大批量制造工艺：高度可定制的 RNA 运送平台

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-17 DOI: 10.1016/j.omtm.2024.101299

Suzanne Saffie-Siebert, Nissim Torabi-Pour, Andrew Gibson, Flavia Maria Sutera, Ashkan Dehsorkhi, Paulina Baran-Rachwalska, Skye Quinn

{"title":"Toward a large-batch manufacturing process for silicon-stabilized lipid nanoparticles: A highly customizable RNA delivery platform","authors":"Suzanne Saffie-Siebert, Nissim Torabi-Pour, Andrew Gibson, Flavia Maria Sutera, Ashkan Dehsorkhi, Paulina Baran-Rachwalska, Skye Quinn","doi":"10.1016/j.omtm.2024.101299","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101299","url":null,"abstract":"While lipid nanoparticles (LNPs) are a key enabling technology for RNA-based therapeutics, some outstanding challenges hinder their wider clinical translation and use, particularly in terms of RNA stability and limited shelf life. In response to these limitations, we developed silicon-stabilized hybrid lipid nanoparticles (sshLNPs) as a next-generation nanocarrier with improved physical and temperature stability, as well as the highly advantageous capacity for “post-hoc loading” of RNA. Nevertheless, previously reported sshLNP formulations were produced using lipid thin film hydration, making scale-up impractical. To realize the potential of this emerging delivery platform, a manufacturing process enabling multikilogram batch sizes was required for successful clinical translation and deployment at scale. This was achieved by developing a revised protocol based on solvent injection mixing and incorporating other process adjustments to enable in-flow extrusion of multiliter volumes, while ensuring sshLNPs with the desired characteristics. Optimized procedures for nanoparticle formation, extrusion, and tangential flow filtration (to remove residual organic solvent) currently enable production of 2 kg finished batches. Importantly, sshLNPs produced via the modified large-scale workflow show equivalent physical and functional properties to those derived from the earlier small-scale methods, paving the way for GMP manufacturing protocols to enable vital translational clinical studies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141782920","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Subacute liver injury in two young infants following gene replacement therapy for spinal muscular atrophy 两名婴儿在接受脊髓性肌萎缩症基因替代疗法后出现亚急性肝损伤

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-02 DOI: 10.1016/j.omtm.2024.101296

Cassie Ables, Catalina Jaramillo, E. Lynne Wood, Sara Stern, Mouied Alashari, Linda Book, Russell J. Butterfield

{"title":"Subacute liver injury in two young infants following gene replacement therapy for spinal muscular atrophy","authors":"Cassie Ables, Catalina Jaramillo, E. Lynne Wood, Sara Stern, Mouied Alashari, Linda Book, Russell J. Butterfield","doi":"10.1016/j.omtm.2024.101296","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101296","url":null,"abstract":"Spinal muscular atrophy is a neurodegenerative disorder resulting from the irreversible loss of anterior horn cells secondary to homozygous mutations in the survival motor neuron gene . Gene replacement therapy using a recombinant adeno-associated virus 9 vector containing an gene construct, onasemnogene abeparvovec-xioi, was approved by the US Food and Drug Administration in May 2019. Subacute mild elevation of liver function tests following infusion has since been shown to be a common adverse event. Additionally, there have been case reports of liver failure following administration of this therapy and two reported patient deaths. While these adverse events are relatively common, they have not been reported in the youngest treated patients. We present two cases of subacute severe elevation of liver function tests >10–20 times the upper limit of normal, without progression to liver failure, following onasemnogene abeparvovec administration in young infants less than 4 weeks old. Potential mechanisms of injury, management, and implications for future treatment with onasemnogene abeparvovec and other adeno-associated virus vector gene therapies are discussed.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141586163","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A self-complementary AAV proviral plasmid system to reduce aberrant cross-packaging and ITR promoter activity in AAV vector preparations 减少 AAV 载体制备中异常交叉包装和 ITR 启动子活性的自补体 AAV 后裔质粒系统

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-26 DOI: 10.1016/j.omtm.2024.101295

Noah K. Taylor, Matthew J. Guggenbiller, Pranali P. Mistry, Oliver D. King, Scott Q. Harper

{"title":"A self-complementary AAV proviral plasmid system to reduce aberrant cross-packaging and ITR promoter activity in AAV vector preparations","authors":"Noah K. Taylor, Matthew J. Guggenbiller, Pranali P. Mistry, Oliver D. King, Scott Q. Harper","doi":"10.1016/j.omtm.2024.101295","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101295","url":null,"abstract":"Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced , thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552022","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice 小鼠 AAV5 向量整合的纵向动力学特征和克隆扩增评估

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-26 DOI: 10.1016/j.omtm.2024.101294

Ashrafali Mohamed Ismail, Evan Witt, Taren Bouwman, Wyatt Clark, Bridget Yates, Matteo Franco, Sylvia Fong

{"title":"The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice","authors":"Ashrafali Mohamed Ismail, Evan Witt, Taren Bouwman, Wyatt Clark, Bridget Yates, Matteo Franco, Sylvia Fong","doi":"10.1016/j.omtm.2024.101294","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101294","url":null,"abstract":"Adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes integrate into the host genome, but the theoretical risk of tumorigenesis depends on vector regulatory features. A mouse model was used to investigate integration profiles of an AAV serotype 5 (AAV5) vector produced using and HEK293 cells that mimic key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads were derived from episomes, and mean (± standard deviation) integration frequency was 2.70 ± 1.26 and 1.79 ± 0.86 integrations per 1,000 cells for - and HEK293-produced vector. Longitudinal integration analysis suggested integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal and randomly distributed. No major differences in integration profiles were observed for either vector production platform, and no integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver ( = 1 × 10) and less enriched for genes of lower expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry 用质量光度法检测重组 AAV 包囊的完整性和热应力后的基因组释放情况

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101293

Eduard H.T.M. Ebberink, Alisa Ruisinger, Markus Nuebel, Helena Meyer-Berg, Irene R.S. Ferreira, Marco Thomann, Albert J.R. Heck

{"title":"Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry","authors":"Eduard H.T.M. Ebberink, Alisa Ruisinger, Markus Nuebel, Helena Meyer-Berg, Irene R.S. Ferreira, Marco Thomann, Albert J.R. Heck","doi":"10.1016/j.omtm.2024.101293","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101293","url":null,"abstract":"Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated () through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quantification of full and empty particles of adeno-associated virus vectors via a novel dual fluorescence-linked immunosorbent assay 通过新型双荧光联免疫吸附测定法量化腺相关病毒载体的全颗粒和空颗粒

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101291

Sereirath Soth, Mikako Takakura, Masahiro Suekawa, Takayuki Onishi, Kiichi Hirohata, Tamami Hashimoto, Takahiro Maruno, Mitsuko Fukuhara, Yasuo Tsunaka, Tetsuo Torisu, Susumu Uchiyama

{"title":"Quantification of full and empty particles of adeno-associated virus vectors via a novel dual fluorescence-linked immunosorbent assay","authors":"Sereirath Soth, Mikako Takakura, Masahiro Suekawa, Takayuki Onishi, Kiichi Hirohata, Tamami Hashimoto, Takahiro Maruno, Mitsuko Fukuhara, Yasuo Tsunaka, Tetsuo Torisu, Susumu Uchiyama","doi":"10.1016/j.omtm.2024.101291","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101291","url":null,"abstract":"The adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established. In dFLISA, capture of AAV particles, detection of capsid proteins, and release and detection of the viral genome are performed in the same well. We demonstrated that the capsid and genomic titers determined by dFLISA were comparable with those of analytical ultracentrifugation. The FP ratios determined by dFLISA were in good agreement with the expected values. In addition, we showed that dFLISA can quantify the genomic and capsid titers of crude samples. dFLISA can be easily modified for measuring other AAV vector serotypes and AAV vectors with different genome lengths. These features make dFLISA a valuable tool for the future development of AAV-based gene therapies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141551961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering single-cycle MeV vector for CRISPR-Cas9 gene editing 为 CRISPR-Cas9 基因编辑设计单循环 MeV 载体

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101290

Ramya Rallabandi, Brenna Sharp, Spencer Majerus, Austin Royster, Sarrianna Hoffer, Mia Ikeda, Patricia Devaux

引用次数: 0

Highly branched poly β-amino ester/CpG-depleted CFTR plasmid nanoparticles for non-viral gene therapy in lung cystic fibrosis disease 用于肺囊性纤维化疾病非病毒基因疗法的高支化聚 β-氨基酯/CpG-depleted CFTR 质粒纳米颗粒

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101292

Bei Qiu, Darío Manzanares, Yinghao Li, Xianqing Wang, Zishan Li, Sébastien Terreau, Zhonglei He, Jing Lyu, Wenxin Wang, Irene Lara-Sáez

{"title":"Highly branched poly β-amino ester/CpG-depleted CFTR plasmid nanoparticles for non-viral gene therapy in lung cystic fibrosis disease","authors":"Bei Qiu, Darío Manzanares, Yinghao Li, Xianqing Wang, Zishan Li, Sébastien Terreau, Zhonglei He, Jing Lyu, Wenxin Wang, Irene Lara-Sáez","doi":"10.1016/j.omtm.2024.101292","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101292","url":null,"abstract":"Lung cystic fibrosis (CF) is a lethal inherited disease caused by mutations in the CF transmembrane conductance regulator () gene, leading to a dysfunctional CFTR protein. Gene therapy offers promise for the treatment of lung CF. However, the development and clinical application of CF gene therapy have long been hampered by the absence of safe and highly efficient delivery vectors. In this work, a novel polymer-based gene replacement treatment approach was developed. A series of poly (β-amino esters) (PAEs) with various topological structures and chemical compositions were screened to create non-viral therapeutic systems for CFTR restoration in lung CF disease. A nanoparticle, formed by the selected highly branched PAE (HPAE) with a CpG-depleted CFTR plasmid, demonstrated gene expression and biocompatibility in lung epithelial cells, outperforming leading commercial gene transfection reagents such as Lipofectamine 3000 and Xfect. The newly developed gene therapy system successfully restored functional CFTR protein production in lung CF epithelial monolayers. This therapeutic approach holds great potential for use as an efficient and safe non-viral treatment for CF patients.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141552024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Lipid nanoparticle-encapsulated DOCK11-siRNA efficiently reduces hepatitis B virus cccDNA level in infected mice 脂质纳米粒包裹的 DOCK11-siRNA 可有效降低感染小鼠体内的乙型肝炎病毒 cccDNA 水平

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101289

Hikari Okada, Takeharu Sakamoto, Kouki Nio, Yingyi Li, Kazuyuki Kuroki, Saiho Sugimoto, Tetsuro Shimakami, Nobuhide Doi, Masao Honda, Motoharu Seiki, Shuichi Kaneko, Taro Yamashita

{"title":"Lipid nanoparticle-encapsulated DOCK11-siRNA efficiently reduces hepatitis B virus cccDNA level in infected mice","authors":"Hikari Okada, Takeharu Sakamoto, Kouki Nio, Yingyi Li, Kazuyuki Kuroki, Saiho Sugimoto, Tetsuro Shimakami, Nobuhide Doi, Masao Honda, Motoharu Seiki, Shuichi Kaneko, Taro Yamashita","doi":"10.1016/j.omtm.2024.101289","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101289","url":null,"abstract":"The hepatitis B virus (HBV) infects many people worldwide. As HBV infection frequently leads to liver fibrosis and carcinogenesis, developing anti-HBV therapeutic drugs is urgent. Therapeutic drugs for preventing covalently closed circular DNA (cccDNA) production, which can eliminate HBV infection, are unavailable. The host factor dedicator of cytokinesis 11 (DOCK11) is involved in the synthesis and maintenance of HBV cccDNA . However, the effectiveness of DOCK11 as a target for the elimination of HBV cccDNA remains unclear. In this study, we assess whether DOCK11 inhibitors suppress HBV cccDNA production in mouse models of HBV infection. The tocopherol-conjugate hetero gapmer, a DNA/RNA duplex of gapmer/complementary RNA targeting the DOCK11 sequence, partially reduces the expression of , but not that of HBV cccDNA, in the livers of HBV-infected human hepatocyte chimeric mice, along with weight loss and decreased serum human albumin levels. Lipid nanoparticle-encapsulated chemically modified siRNAs specific for suppress expression and decrease HBV cccDNA levels without adverse effects in the mice. Therefore, nucleic acid-based drugs targeting DOCK11 in hepatocytes are potentially effective anti-HBV therapeutics that can reduce HBV cccDNA levels .","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141551959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

5′ Transgenes drive leaky expression of 3′ transgenes in Cre-inducible bi-cistronic vectors 5′ 转基因驱动 Cre 诱导的双组分载体中 3′ 转基因的泄漏表达

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI: 10.1016/j.omtm.2024.101288

Yasuyuki Osanai, Yao Lulu Xing, Shinya Mochizuki, Kenta Kobayashi, Jihane Homman-Ludiye, Amali Cooray, Jasmine Poh, Ayumu Inutsuka, Nobuhiko Ohno, Tobias D. Merson

{"title":"5′ Transgenes drive leaky expression of 3′ transgenes in Cre-inducible bi-cistronic vectors","authors":"Yasuyuki Osanai, Yao Lulu Xing, Shinya Mochizuki, Kenta Kobayashi, Jihane Homman-Ludiye, Amali Cooray, Jasmine Poh, Ayumu Inutsuka, Nobuhiko Ohno, Tobias D. Merson","doi":"10.1016/j.omtm.2024.101288","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101288","url":null,"abstract":"Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences provide an invaluable tool for understanding the molecular regulation of cellular functions. The Cre-lox system is used for inducing the expression of recombinant proteins encoded within a bi-/poly-cistronic cassette. However, leak expression of transgenes is often observed in the absence of Cre recombinase activity, compromising the utility of this approach. To investigate the mechanism of leak expression, we generated Cre-inducible bi-cistronic vectors to monitor the expression of transgenes positioned either 5′ or 3′ of a 2A peptide or internal ribosomal entry site (IRES) sequence. Cells transfected with these bi-cistronic vectors exhibited Cre-independent leak expression specifically of transgenes positioned 3′ of the 2A peptide or IRES sequence. Similarly, AAV- vectors encoding bi-cistronic cassettes or fusion proteins revealed the selective Cre-independent leak expression of transgenes positioned at the 3′ end of the open reading frame. Our data demonstrate that 5′ transgenes confer promoter-like activity that drives the expression of 3′ transgenes. An additional STOP cassette between the 2A sequence and 3′ transgene dramatically decreased Cre-independent transgene expression. Our findings highlight the need for appropriate experimental controls when using Cre-inducible bi-/poly-cistronic constructs and inform improved design of vectors for more tightly regulated inducible transgene expression.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141553040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0