Molecular Therapy-Methods & Clinical Development最新文献_第3页

Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2 通过组成型表达 tHMGA2 使体内 HDAd 转导的造血干细胞自动扩增

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-13 DOI: 10.1016/j.omtm.2024.101319

Hongjie Wang, Aphrodite Georgakopoulou, Evangelos Nizamis, Ka Wai Mok, Raïssa Eluère, Robert A. Policastro, Paul N. Valdmanis, André Lieber

{"title":"Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2","authors":"Hongjie Wang, Aphrodite Georgakopoulou, Evangelos Nizamis, Ka Wai Mok, Raïssa Eluère, Robert A. Policastro, Paul N. Valdmanis, André Lieber","doi":"10.1016/j.omtm.2024.101319","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101319","url":null,"abstract":"We developed an hematopoietic stem cell (HSC) gene therapy approach that does not require cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we constructed HSC-tropic HDAd5/35++ vectors expressing a 3′ UTR truncated HMGA2 gene and a GFP reporter gene. A SB100x transposase vector mediated random integration of the tHMGA2/GFP transgene cassette. HSCs in mice were mobilized by subcutaneous injections of G-CSF and AMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resulted in a slow but progressive, competitive expansion of GFP PBMCs, reaching about 50% by week 44 with further expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at the levels of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly, based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonal dominance. Whole-exome sequencing did not show significant differences in the genomic instability indices between tHGMGA2/GFP mice and untreated control mice. Auto-expansion by tHMGA2 was validated in humanized mice. This is the first demonstration that simple injections of mobilization drugs and HDAd vectors can trigger auto-expansion of transduced HSCs resulting in transgene-marking rates that, theoretically, are curative for hemoglobinopathies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197305","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

An autonucleolytic suspension HEK293F host cell line for high-titre serum-free AAV5 and AAV9 production with reduced levels of DNA impurity 用于生产高活性无血清 AAV5 和 AAV9 的自溶悬浮 HEK293F 宿主细胞系，可降低 DNA 杂质含量

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-12 DOI: 10.1016/j.omtm.2024.101317

{"title":"An autonucleolytic suspension HEK293F host cell line for high-titre serum-free AAV5 and AAV9 production with reduced levels of DNA impurity","authors":"","doi":"10.1016/j.omtm.2024.101317","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101317","url":null,"abstract":"We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293-F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, 'NuPro-1S'. When cultivated in serum-free media, NuPro-1S cells yielded 3.06x1010 AAV5 viral genomes (vg) / mL via transient transfection, compared to 3.85x109 vg /mL from the parental HEK293-F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8x1013 vg /mL from NuPro-1S cells compared to 7.35x1012 vg /mL from HEK293-F cells. AAV9 from both HEK293-F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue in-vivo. Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared to HEK293-F cells. A similar reduction in HEK293-F cells was only achievable with a 50 U / mL Benzonase® treatment.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944522","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

Validation of high-sensitivity fluorometric assays to quantitate cerebrospinal fluid and serum β-galactosidase activity in patients with GM1-gangliosidosis 验证高灵敏度荧光测定法，以定量检测 GM1-神经节苷脂病患者脑脊液和血清中的β-半乳糖苷酶活性

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-10 DOI: 10.1016/j.omtm.2024.101318

{"title":"Validation of high-sensitivity fluorometric assays to quantitate cerebrospinal fluid and serum β-galactosidase activity in patients with GM1-gangliosidosis","authors":"","doi":"10.1016/j.omtm.2024.101318","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101318","url":null,"abstract":"GM1-gangliosidosis (GM1) is a lysosomal storage disorder caused by mutations in the galactosidase beta 1 gene (GLB1) that leads to reduced β-galactosidase (β-gal) activity. This enzyme deficiency results in neuronal degeneration, developmental delay, and early death. A sensitive assay for the measurement of β-gal enzyme activity is required for the development of disease-modifying therapies. We have optimized fluorometric assays for quantitative analysis of β-gal activity in human cerebrospinal fluid (CSF) and serum for the development of a GLB1 gene replacement therapy. Assay analytical performance was characterized by assessing sensitivity, precision, accuracy, parallelism, specificity, and sample stability. Sensitivity of the CSF and serum β-gal activity assays were 0.05 nmol/mL/3hr and 0.20 nmol/mL/3hr, respectively. Assay precision represented by inter-assay percent coefficient of variation of the human CSF and serum was <15% and <20%, respectively. The effect of pre-analytical factors on β-gal activity was examined, and rapid processing and freezing of samples post-collection was critical to preserve enzyme activity. These assays enabled measurement of CSF and serum β-gal activities in both healthy individuals and patients with GM1-gangliosidosis. This CSF β-gal activity assay is the first of its kind with sufficient sensitivity to quantitatively measure β-gal enzyme activity in CSF samples from GM1 patients.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944507","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

Lentiviral vector packaging and producer cell lines yield titres equivalent to the industry-standard four-plasmid process Stable cells for lentiviral vector manufacturing 慢病毒载体包装和生产细胞系的滴度相当于行业标准的四质粒工艺用于慢病毒载体生产的稳定细胞

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-08 DOI: 10.1016/j.omtm.2024.101315

{"title":"Lentiviral vector packaging and producer cell lines yield titres equivalent to the industry-standard four-plasmid process Stable cells for lentiviral vector manufacturing","authors":"","doi":"10.1016/j.omtm.2024.101315","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101315","url":null,"abstract":"Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing and transfection all contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearised plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVV that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944523","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

Persistent tailoring of MSC activation through genetic priming 通过基因引物持续定制间充质干细胞活化

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-08 DOI: 10.1016/j.omtm.2024.101316

Michael A. Beauregard, Guy C. Bedford, Daniel A. Brenner, Leonardo D. Sanchez Solis, Tomoki Nishiguchi, Abhimanyu, Santiago Carrero Longlax, Barun Mahata, Omid Veiseh, Pamela L. Wenzel, Andrew R. DiNardo, Isaac B. Hilton, Michael R. Diehl

{"title":"Persistent tailoring of MSC activation through genetic priming","authors":"Michael A. Beauregard, Guy C. Bedford, Daniel A. Brenner, Leonardo D. Sanchez Solis, Tomoki Nishiguchi, Abhimanyu, Santiago Carrero Longlax, Barun Mahata, Omid Veiseh, Pamela L. Wenzel, Andrew R. DiNardo, Isaac B. Hilton, Michael R. Diehl","doi":"10.1016/j.omtm.2024.101316","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101316","url":null,"abstract":"Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce the expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a genetic priming method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor interferon response factor 1 (IRF1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFN-γ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFN-γ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197298","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

Combination AAV Gene Therapy with Galectin-1 and SOD1 Downregulation Demonstrates Superior Therapeutic Effect in a severe mouse model of ALS AAV 基因疗法与 Galectin-1 和 SOD1 下调相结合，在严重 ALS 小鼠模型中显示出卓越疗效

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-06 DOI: 10.1016/j.omtm.2024.101312

{"title":"Combination AAV Gene Therapy with Galectin-1 and SOD1 Downregulation Demonstrates Superior Therapeutic Effect in a severe mouse model of ALS","authors":"","doi":"10.1016/j.omtm.2024.101312","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101312","url":null,"abstract":"Neuroinflammation is a miscreant in accelerating progression of many neurodegenerative diseases, including ALS. However, treatments targeting neuroinflammation alone have led to disappointing results in clinical trials. Both neuronal and non-neuronal cell types have been implicated in pathogenesis of ALS, and multiple studies have shown correction of each cell type has beneficial effect on disease outcome. Previously, we showed that AAV9-mediated SOD1 suppression in motor neurons and astrocytes significantly improves motor function and extends survival in ALS mouse models. Despite neuron and astrocyte correction, ALS mice still succumb to death with microgliosis observed in endpoint tissue. Therefore, we hypothesized that the optimal therapeutic approach will target and simultaneously correct motor neurons, astrocytes, and microglia. Here, we developed a novel approach to indirectly target microglia with Galectin-1 and combined this with our previously established AAV9.SOD1.shRNA treatment. We show Galectin-1 conditioning of SOD1G93A microglia reduces inflammatory markers and rescues motor neuron death in vitro. When paired with SOD1 downregulation, we found a synergistic effect of combination treatment in vivo and show a significant extension of survival of SOD1G93A mice over SOD1 suppression alone. These results highlight the importance of targeting inflammatory microglia as a critical component in future therapeutic development.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944524","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

Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine 猪静脉注射 mRNA 脂质纳米颗粒后，蛋白质在所有主要器官中均有表达

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-06 DOI: 10.1016/j.omtm.2024.101314

{"title":"Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine","authors":"","doi":"10.1016/j.omtm.2024.101314","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101314","url":null,"abstract":"In vivo delivery of mRNA is promising for the study of gene expression and the treatment of diseases. Lipid nanoparticles (LNP) enable efficient delivery of mRNA constructs, but protein expression has been assumed to be limited to the liver. With specialized LNP, delivery to extrahepatic tissue occurs in small animal models, however it is unclear if global delivery of mRNA to all major organs is possible in humans, because delivery may be affected by differences in innate immune response and relative organ size. Furthermore, limited studies with LNP have been performed in large animal models, such as swine, due to their sensitivity to complement activation-related pseudoallergy (CARPA). In this study, we found that exogenous protein expression occurred in all major organs when swine were injected intravenously with a relatively low dose of mRNA encapsulated in a clinically relevant LNP formulation. Exogenous protein was detected in the liver, spleen, lung, heart, uterus, colon, stomach, kidney, small intestine, and brain of the swine without inducing CARPA. Furthermore, protein expression was detected in the bone marrow, including megakaryocytes, hematopoietic stem cells, granulocytes, and in circulating white blood cells and platelets. These results show that nearly all major organs contain exogenous protein expression and are viable targets for mRNA therapies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944506","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

Preclinical development of hematopoietic stem cell-gene therapy for Mucopolysaccharidosis type IVB using a GLB1 transgene with enhanced therapeutic potential. 利用具有更大治疗潜力的 GLB1 转基因对 IVB 型粘多糖病进行造血干细胞基因疗法的临床前开发。

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-06 DOI: 10.1016/j.omtm.2024.101313

{"title":"Preclinical development of hematopoietic stem cell-gene therapy for Mucopolysaccharidosis type IVB using a GLB1 transgene with enhanced therapeutic potential.","authors":"","doi":"10.1016/j.omtm.2024.101313","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101313","url":null,"abstract":"Mucopolysaccharidosis type IVB (MPSIVB) is a lysosomal storage disorder caused by β-galactosidase (β-GAL) deficiency characterized by severe skeletal and neurological alterations without approved treatments. To develop hematopoietic stem progenitor cell-gene therapy (HSPC-GT) for MPSIVB, we designed lentiviral vectors (LVs) encoding human β-GAL to achieve supraphysiological release of the therapeutic enzyme in human HSPCs and metabolic correction of diseased cells. Transduced HSPCs displayed proper colony formation, proliferation, and differentiation capacity, but their progeny failed to release the enzyme at supraphysiological levels. Therefore, we tested alternative LVs to overexpress an enhanced β-GAL deriving from murine (LV-enhGLB1) and human selectively mutated GLB1 sequences (LV-mutGLB1). Only human HSPCs transduced with LV-enhGLB1 overexpressed β-GAL in vitro and in vivo without evidence of overexpression-related toxicity. Their hematopoietic progeny efficiently released β-GAL, allowing the cross-correction of defective cells, including skeletal cells. We found that the low levels of human GLB1 mRNA in human hematopoietic cells and the improved stability of the enhanced β-GAL contribute to the increased efficacy of LV-enhGLB1. Importantly, the enhanced β-GAL enzyme showed physiological lysosomal trafficking in human cells and was not associated with increased immunogenicity in vitro. These results support the use of LV-enhGLB1 for further HSPC-GT development and future clinical translation to treat MPSIVB multisystem disease.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944508","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

Macrophage manufacturing and engineering with 5’-Cap1 and N1-methylpseudouridine-modified mRNA 利用 5'-Cap1 和 N1-甲基假尿苷修饰的 mRNA 制造和改造巨噬细胞

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-31 DOI: 10.1016/j.omtm.2024.101307

{"title":"Macrophage manufacturing and engineering with 5’-Cap1 and N1-methylpseudouridine-modified mRNA","authors":"","doi":"10.1016/j.omtm.2024.101307","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101307","url":null,"abstract":"Macrophage-based cell therapeutics are an emerging modality to treat cancer and repair tissue damage. A reproducible manufacturing and engineering process is central to fulfill their therapeutical potential. Here, we establish a robust macrophage manufacturing platform (Mo-Mac), and demonstrate that macrophage functionality can be enhanced by N1-methylpseudouridine (m1Ψ)-modified mRNA. Using single-cell transcriptomic analysis as an unbiased approach, we found that >90% cells in the final product were macrophages, and the rest primarily comprised T cells, B cells, natural killer cells, promyelocytes, promonocytes and hematopoietic stem cells. This analysis also guided the development of flow cytometry strategies to assess cell compositions in the manufactured product to meet requirements by the National Medical Products Administration. To modulate macrophage functionality, as an illustrative example, we examined whether the engulfment capability of macrophages could be enhanced by mRNA technology. We found that efferocytosis was increased in vitro when macrophages were electroporated with m1Ψ-modified mRNA encoding CD300LF (CD300LF-mRNA-macrophage). Consistently, in a mouse model of acute liver failure, CD300LF-mRNA-macrophage facilitated organ recovery from acetaminophen-induced hepatotoxicity. These results demonstrate a GMP-compliant macrophage manufacturing process, and indicate that macrophage can be engineered by versatile mRNA technology to achieve therapeutic goals.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141864125","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 cellular disease model towards gene therapy of TGM1-dependent Lamellar Ichthyosis 基因治疗 TGM1 依赖性鳞状鱼鳞病的细胞疾病模型

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-07-31 DOI: 10.1016/j.omtm.2024.101311

{"title":"A cellular disease model towards gene therapy of TGM1-dependent Lamellar Ichthyosis","authors":"","doi":"10.1016/j.omtm.2024.101311","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101311","url":null,"abstract":"Lamellar Ichthyosis (LI) is a chronic disease, mostly caused by mutations in TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene edited cells showed complete absence of Transglutaminase-1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a SINγ-RV vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity and cornified envelopes formation, hence restored proper epidermal architecture. Single cell multiomic analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial to epidermal regeneration. This study serves as a proof-of-concept for assessing the potential of this therapeutic approach in treating TGM1 dependent LI.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141864080","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