Molecular Therapy-Methods & Clinical Development最新文献_第2页

Magnetic bead-sensitized optoporation coupled with antibodies-based activation for mRNA CAR-T cell manufacturing.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-02-04 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101428

Noelia Maldonado-Pérez, Marie-Agnès Doucey, Dzhangar Dzhumashev, Darel Martínez Bedoya, Luis Castillo Cantero, Caroline Boudousquie, Yann Pierson, Luc Henry, Valérie Dutoit, Denis Migliorini

{"title":"Magnetic bead-sensitized optoporation coupled with antibodies-based activation for mRNA CAR-T cell manufacturing.","authors":"Noelia Maldonado-Pérez, Marie-Agnès Doucey, Dzhangar Dzhumashev, Darel Martínez Bedoya, Luis Castillo Cantero, Caroline Boudousquie, Yann Pierson, Luc Henry, Valérie Dutoit, Denis Migliorini","doi":"10.1016/j.omtm.2025.101428","DOIUrl":"https://doi.org/10.1016/j.omtm.2025.101428","url":null,"abstract":"Immunotherapy is facing a revolution with the advent of immune cell engineering. Chimeric antigen receptor (CAR)-T cell therapy has shown unprecedented efficacy in B cell malignancies and is now being evaluated in other disease areas. Viral transduction is the most common method for immune cell genetic engineering, but presents important limitations, such as high reagent costs and regulatory concerns due to mutagenesis risk. One prevailing non-viral gene delivery strategy relies on the electroporation of non-integrating RNA. However, most modern electroporation technologies also require high reagent costs and rely on the use of proprietary software and transfection buffers. Nanoparticle-sensitized optoporation represents an alternative method for transient permeabilization of cells. Here, we introduce magnetic bead-sensitized optoporation, in which commercially available superparamagnetic beads coupled with anti-human CD3 and CD28 antibodies are used as photosensitizers for efficient genetic cargo delivery into human primary T cells and other immune cells. We show that magnetic bead-sensitized optoporation of human T cells generates functional mRNA-based CAR-T cells without affecting T cell product memory phenotype or activation potential. Importantly, optoporated T cells exhibited a greater proliferation capacity relative to electroporated T cells. In conclusion, our findings suggest that magnetic bead-sensitized optoporation holds promise as mRNA delivery strategy for immune cell therapy.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101428"},"PeriodicalIF":4.6,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11910140/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143651894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Preclinical development of TAK-754, a high-performance AAV8-based vector expressing coagulation factor VIII.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-01-28 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101424

Johannes Lengler, Markus Weiller, Franziska Horling, Josef Mayrhofer, Maria Schuster, Falko G Falkner, Irene Gil-Farina, Matthias Klugmann, Friedrich Scheiflinger, Werner Hoellriegl, Hanspeter Rottensteiner

{"title":"Preclinical development of TAK-754, a high-performance AAV8-based vector expressing coagulation factor VIII.","authors":"Johannes Lengler, Markus Weiller, Franziska Horling, Josef Mayrhofer, Maria Schuster, Falko G Falkner, Irene Gil-Farina, Matthias Klugmann, Friedrich Scheiflinger, Werner Hoellriegl, Hanspeter Rottensteiner","doi":"10.1016/j.omtm.2025.101424","DOIUrl":"10.1016/j.omtm.2025.101424","url":null,"abstract":"This report concerns the preclinical development of TAK-754, an AAV8-based human factor VIII (FVIII) vector designed to deliver a codon-optimized and CpG-depleted B domain-deleted F8 transgene under the control of a liver-specific promoter for gene therapy in patients with hemophilia A. A dose-dependent increase in plasma FVIII activity was detected in FVIII knockout mice at a dose of 1.0 × 1012 TAK-754 capsid particles (CP)/kg or higher. This increase was shown to be in accordance with a dose-dependent decrease in blood loss in a hemostatic efficacy assay. TAK-754 (3.1 × 1012 CP/kg) mediated long-term and stable FVIII expression in immunologically tolerant transgenic human FVIII mice. Toxicology and biodistribution assessments with a single administration of TAK-754 ranging between 1.9 × 1012 and 5.0 × 1013 CP/kg were conducted in male C57BL/6J mice. The highest TAK-754 dose occurred without TAK-754-related adverse clinical signs. Biodistribution profiling showed predominant detection in the liver with a low occurrence of vector DNA in other tissues. Integration site analysis revealed minimal vector integration, with no observations of clonal outgrowth or preferred integrations in genes previously implicated in hepatocellular carcinoma formation within the observation period. These preclinical studies demonstrate a good safety and efficacy profile for TAK-754.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101424"},"PeriodicalIF":4.6,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11929063/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143694398","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Validation of a quantitative cell-based relative potency assay for LUXTURNA.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-01-25 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101423

Katherine A High, Dave Le Blond, Karen Doucette, Dezhong Liu, Rafal Farjo, Irena Ignatova, George Buchlis, Daniel Chung, Linda B Couto

{"title":"Validation of a quantitative cell-based relative potency assay for LUXTURNA.","authors":"Katherine A High, Dave Le Blond, Karen Doucette, Dezhong Liu, Rafal Farjo, Irena Ignatova, George Buchlis, Daniel Chung, Linda B Couto","doi":"10.1016/j.omtm.2025.101423","DOIUrl":"10.1016/j.omtm.2025.101423","url":null,"abstract":"Voretigene neparvovec-rzyl (Luxturna) is an AAV2 vector (AAV2-hRPE65v2) that expresses a cDNA encoding the human retinal pigment epithelium-specific 65 kDa protein (RPE65). It has been approved for the treatment of visual deficits associated with biallelic mutations in human RPE65 in the US, European Union (EU), and multiple other countries. To achieve regulatory approval, it was necessary to validate an assay demonstrating its biological activity or potency. The assay measures AAV2-hRPE65v2 transduction in HEK293 cells and the subsequent biological activity of the vector-encoded RPE65 protein in cell lysates. RPE65 converts all-trans-retinol to 11-cis-retinol, which is quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The assay was validated for seven characteristics, namely system and sample suitability, specificity, linearity, precision, relative accuracy, range, and robustness. The validated assay can be used to confirm the relative potency levels of different lots of Luxturna in the range of 50%-150% of a reference standard (defined as 100% potent). This represents the first report of validation studies supporting an in vitro cell-based relative potency assay for an AAV vector, which was used to evaluate lot-to-lot consistency, stability, and comparability following manufacturing changes and to successfully launch Luxturna, the first gene therapy approved in the US for a genetic disease.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101423"},"PeriodicalIF":4.6,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11914788/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143659686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Immune-driven gene expression loss following intramuscular AAV delivery to non-human primates is only transient.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-01-19 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2025.101409

Malo Journou, Marie Devaux, Nicolas Jaulin, Virginie Pichard, Mercedes Segovia, Aurélie Moreau, Johanne Le Duff, Maria Cristina Cuturi, Mickaël Guilbaud, Oumeya Adjali

{"title":"Immune-driven gene expression loss following intramuscular AAV delivery to non-human primates is only transient.","authors":"Malo Journou, Marie Devaux, Nicolas Jaulin, Virginie Pichard, Mercedes Segovia, Aurélie Moreau, Johanne Le Duff, Maria Cristina Cuturi, Mickaël Guilbaud, Oumeya Adjali","doi":"10.1016/j.omtm.2025.101409","DOIUrl":"10.1016/j.omtm.2025.101409","url":null,"abstract":"Recombinant adeno-associated virus (rAAV) vectors stand out as highly promising for in vivo gene transfer, particularly in targeting the skeletal muscle for treating muscular genetic diseases or secreting therapeutic factors. Despite the simplicity and efficacy of the established intramuscular (IM) route, it has been often associated with an immune-induced rapid loss of transgene expression, in particular in large animal models, and generally considered irreversible as a consequence of a cytotoxic elimination of transduced cells. Here, we report in a non-human primate model that transgene expression loss after IM delivery of an rAAV1 expressing an immunogenic protein is only transient, with the re-expression of the transgene lasting up to 5 years post-injection. We show that the recovery of transgene expression is due to persisting viral genomes in the injected muscles despite the detection of peripheral anti-transgene cellular immunity. Persisting genomes were observed in the presence of infiltrated mononuclear CD8 and CD4 T lymphocytes, among which we were able to detect FoxP3+ regulatory cells. This is to our knowledge the first report of a transient immune-mediated loss of gene expression in a large animal model after rAAV delivery that should shed new light on the issue of rAAV vector immunogenicity.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101409"},"PeriodicalIF":4.6,"publicationDate":"2025-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11914520/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143659681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Antibodies to recombinant human alpha-L-iduronidase prevent disease correction in cortical bone in MPS I mice.

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2025-01-02 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2024.101405

Sarah C Hurt, Steven Q Le, Shih-Hsin Kan, Quang D Bui, Michael D Brodt, Patricia I Dickson

{"title":"Antibodies to recombinant human alpha-L-iduronidase prevent disease correction in cortical bone in MPS I mice.","authors":"Sarah C Hurt, Steven Q Le, Shih-Hsin Kan, Quang D Bui, Michael D Brodt, Patricia I Dickson","doi":"10.1016/j.omtm.2024.101405","DOIUrl":"10.1016/j.omtm.2024.101405","url":null,"abstract":"Mucopolysaccharidosis I (MPS I) is a lysosomal storage disorder caused by deficiency of the enzyme α-l-iduronidase (IDUA). Failure of enzyme replacement therapy (ERT) to treat skeletal disease may be due to development of anti-IDUA antibodies, found previously to alter tissue distribution of ERT in animal models. To test this hypothesis, immunocompromised (non-obese diabetic [NOD]-severe combined immunodeficiency [SCID]) MPS I mice were treated with weekly ERT from birth (ERT alone). Some mice also received weekly injections of rabbit immunoglobulin G (IgG) against IDUA (immunized rabbit immune globulin [IRIG]) concomitant with ERT, imitating antibodies developed in patients (ERT+IRIG). Mice treated with ERT+IRIG showed lower IDUA activity and higher disease burden than mice treated with ERT alone in most tissues. Femora were harvested at 20 weeks for ex vivo microcomputed tomography (μCT). Femoral cortical bone thickness and cortical bone area in MPS I mice were greater than in unaffected mice. Mice treated with ERT alone had values that were statistically indistinguishable from carrier mice, while mice that received ERT+IRIG had no significant differences compared to vehicle-treated MPS I mice. The data suggests that immune-modulatory or immune-suppressive therapy to prevent or reduce the humoral immune response against ERT may improve treatment of skeletal disease due to MPS I.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101405"},"PeriodicalIF":4.6,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11928967/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143694360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Reducing off-target expression of mRNA therapeutics and vaccines in the liver with microRNA binding sites. 利用 microRNA 结合位点减少 mRNA 疗法和疫苗在肝脏中的脱靶表达。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-12-19 eCollection Date: 2025-03-13 DOI: 10.1016/j.omtm.2024.101402

Brian J Parrett, Satoko Yamaoka, Michael A Barry

{"title":"Reducing off-target expression of mRNA therapeutics and vaccines in the liver with microRNA binding sites.","authors":"Brian J Parrett, Satoko Yamaoka, Michael A Barry","doi":"10.1016/j.omtm.2024.101402","DOIUrl":"10.1016/j.omtm.2024.101402","url":null,"abstract":"Lipid nanoparticles (LNPs) are often liver tropic, presenting challenges for LNP-delivered mRNA therapeutics intended for other tissues, as off-target expression in the liver may increase side effects and modulate immune responses. To avoid off-target expression in the liver, miR-122 binding sites have been used by others in viral and non-viral therapeutics. Here, we use a luciferase reporter system to compare different copy numbers and insertion locations of miR-122 binding sequences to restrict liver expression. We inserted one to five miR-122 binding sites into the 5' or 3' untranslated regions (UTRs) of luciferase mRNAs and tested them in LNPs in vitro and in vivo via systemic intravenous and local intramuscular injections in mice. Our results showed no significant differences in de-targeting efficacy between mRNAs harboring one or multiple miR-122 binding sites or between those with 5' or 3' UTR placements. To test the impact of miR-122 binding sites on antibody response to a mRNA vaccine, Ebola virus matrix protein VP40 mRNAs were modified with or without miR-122 binding sites and injected in mice intramuscularly. This work reinforces the utility of miR-122 binding sites while providing a comparison of these sites to aid the future development of LNP-mRNA therapies for non-hepatic tissues.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"33 1","pages":"101402"},"PeriodicalIF":4.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758401/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143048574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimizing regulatory frameworks for gene therapies in rare diseases: Challenges and solutions. 优化罕见病基因治疗的监管框架：挑战和解决方案。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-12-05 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101386

Diane Berry, Kate Donigan, Lisa Kahlman, James Long, Christina Markus, Caitlin K McCombs

{"title":"Optimizing regulatory frameworks for gene therapies in rare diseases: Challenges and solutions.","authors":"Diane Berry, Kate Donigan, Lisa Kahlman, James Long, Christina Markus, Caitlin K McCombs","doi":"10.1016/j.omtm.2024.101386","DOIUrl":"10.1016/j.omtm.2024.101386","url":null,"abstract":"The advent of genetic medicines and advanced diagnostics has revolutionized the treatment landscape for rare diseases and, with over 10,000 identified conditions affecting millions globally, has the potential to improve many lives. Despite this progress, only 5% of rare diseases have FDA-approved therapies, highlighting a significant unmet need. This article examines the critical need for optimizing the regulatory environment to support the development and approval of gene therapies for rare and ultrarare diseases, which often face unique challenges due to their complexity in the midst of a rapidly evolving field. Key issues discussed include the mismatch between traditional regulatory paradigms and the nature of gene therapies, the need for innovative clinical trial designs, and the importance of flexible manufacturing processes. The article proposes targeted reforms to align regulatory frameworks with the needs of patients with rare diseases and the pace of science, emphasizing the value of a holistic evidence approach, platform technologies, and iterative manufacturing evaluations. By addressing these challenges, we can accelerate the development of life-changing therapies in order to realize the opportunity to provide treatments to patients with rare genetic disorders in their lifetime.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101386"},"PeriodicalIF":4.6,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11666948/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886332","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Thank you to our 2024 reviewers. 感谢我们的 2024 评论员。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-12-02 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101387

引用次数: 0

Determining recombinant AAV capsid extracellular and intracellular biodistribution by dual radioisotope labeling. 双放射性同位素标记法测定重组AAV衣壳细胞内外生物分布。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-21 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101373

Randy J Chandler

引用次数: 0

Size-exclusion chromatography as a multi-attribute method for process and product characterization of adeno-associated virus. 尺寸排阻色谱法作为一种多属性方法，用于表征腺相关病毒的过程和产品特征。

IF 4.6 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-11-19 eCollection Date: 2024-12-12 DOI: 10.1016/j.omtm.2024.101382

Sri Hari Raju Mulagapati, Arun Parupudi, Tomasz Witkos, Nick Bond, Xiaoyu Chen, Thomas Linke, Guoling Xi, Albert Ethan Schmelzer, Wei Xu

{"title":"Size-exclusion chromatography as a multi-attribute method for process and product characterization of adeno-associated virus.","authors":"Sri Hari Raju Mulagapati, Arun Parupudi, Tomasz Witkos, Nick Bond, Xiaoyu Chen, Thomas Linke, Guoling Xi, Albert Ethan Schmelzer, Wei Xu","doi":"10.1016/j.omtm.2024.101382","DOIUrl":"10.1016/j.omtm.2024.101382","url":null,"abstract":"Adeno-associated viruses (AAVs) have recently emerged as a leading platform for gene therapy. Due to the complex manufacturing process and structural features of AAVs, extensive process and product characterization studies are required to better understand product quality and batch-to-batch variability. It is, therefore, critical to develop a fast and reliable analytical method to monitor different product quality attributes (PQAs) of AAVs. In this study, we developed a multiple-attribute monitoring (MAM) method for the characterization of AAV PQAs. The MAM method was developed using the separation capability of size-exclusion chromatography (SEC) in connection with multiple in-line detectors: ultraviolet (UV), fluorescence (FLD), multi-angle light scattering (MALS), and refractive index (RI). We demonstrate that our SEC-based MAM method can be used to measure different PQAs, including genome and capsid titer, purity, aggregation, and full/empty capsid ratios in a single assay. Our SEC-based MAM method achieves similar results when compared side by side with orthogonal, individual assays such as quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and anion-exchange chromatography (AEX). Moreover, here we demonstrate that a simple, label-free, cost-effective, minimum sample requirement, and a high-throughput method can be applied to support process development, product characterization, release, and stability testing.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101382"},"PeriodicalIF":4.6,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11647602/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142839257","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0