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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL 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
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.
我们开发了一种无需细胞移植的造血干细胞(HSC)基因治疗方法。为了获得治疗相关数量的校正细胞,我们构建了表达3′UTR截短HMGA2基因和GFP报告基因的造血干细胞转HDAd5/35++载体。SB100x转座酶载体介导了tHMGA2/GFP转基因盒的随机整合。通过皮下注射 G-CSF 和 AMD3100/Plerixafor 动员小鼠的造血干细胞,然后静脉注射整合的 tHMGA2/GFP 载体。这导致了 GFP PBMC 缓慢但渐进的竞争性扩增,在第 44 周达到约 50%,并在二次受体中进一步扩增。扩增发生在造血干细胞水平以及骨髓和脾脏中的髓系、淋巴系和红系祖细胞水平。重要的是,根据全基因组整合位点分析,扩增是多克隆性的,没有任何克隆优势的迹象。全外显子组测序结果显示,tHGMGA2/GFP小鼠与未经处理的对照组小鼠之间的基因组不稳定性指数没有明显差异。tHMGA2的自动扩增功能在人源化小鼠中得到了验证。这是首次证明,只需注射动员药物和 HDAd 载体,就能引发转导的造血干细胞自动扩增,从而产生转基因标记率,理论上可治疗血红蛋白病。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-08-12 DOI: 10.1016/j.omtm.2024.101317

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.

我们试图改造哺乳动物细胞,使其分泌核酸酶活性,从而在 AAV5 和 AAV9 作为基因治疗载体进行生物处理时,不再需要购买商业核酸酶作为加工添加剂。用血清型核酸酶转基因改造 HeLa 细胞不会在周围培养基中产生核酸酶活性,而用金黄色葡萄球菌核酸酶转基因改造无血清、悬浮适配的 HEK293-F 细胞则会在由此产生的稳定转染细胞系 "NuPro-1S "的周围培养基中产生可检测到的核酸酶活性。在无血清培养基中培养时,NuPro-1S 细胞通过瞬时转染产生了 3.06x1010 AAV5 病毒基因组(vg)/毫升,而亲代 HEK293-F 细胞系产生的病毒基因组为 3.85x109 vg/毫升。通过超速离心法纯化 AAV9 后,NuPro-1S 细胞的产量为 1.8x1013 vg /毫升,而 HEK293-F 细胞的产量为 7.35x1012 vg /毫升。HEK293-F 和 NuPro-1S 的 AAV9 在体内转导嵌入模拟支架组织的细胞或小鼠新生脑组织细胞的能力几乎相同。琼脂糖凝胶数据比较表明,与 HEK293-F 细胞相比,来自 NuPro-1S 细胞的 AAV5 和 AAV9 过程流的 DNA 含量减少了约 60%。HEK293-F 细胞中的 DNA 含量只有在 50 U / mL Benzonase® 处理后才能达到类似的降低。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-08-10 DOI: 10.1016/j.omtm.2024.101318

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.

GM1-神经节苷脂病(GM1)是一种溶酶体贮积症,由β-半乳糖苷酶1基因(GLB1)突变引起,导致β-半乳糖苷酶(β-gal)活性降低。这种酶的缺乏会导致神经元变性、发育迟缓和早期死亡。开发疾病调节疗法需要一种灵敏的β-gal酶活性测定方法。我们优化了荧光测定法,用于定量分析人脑脊液(CSF)和血清中的β-gal活性,以开发GLB1基因替代疗法。通过评估灵敏度、精确度、准确性、平行性、特异性和样品稳定性,对测定分析性能进行了鉴定。CSF和血清β-gal活性测定的灵敏度分别为0.05 nmol/mL/3hr和0.20 nmol/mL/3hr。人CSF和血清的测定精度(以测定间变异系数百分比表示)分别为15%和20%。研究还考察了分析前因素对β-gal活性的影响,样品采集后的快速处理和冷冻对保持酶的活性至关重要。通过这些检测方法,可以测量健康人和 GM1-神经节苷脂病患者的脑脊液和血清中的β-gal 活性。这种脑脊液β-gal活性测定法是同类测定中首个灵敏度足以定量测定GM1患者脑脊液样本中β-gal酶活性的方法。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-08-08 DOI: 10.1016/j.omtm.2024.101315

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.

慢病毒载体(LVV)介导的细胞和基因疗法有可能治愈目前需要终身干预的疾病。然而,质粒转染的要求阻碍了慢病毒载体的大规模生产。此外,大规模的质粒生产、测试和转染都会增加操作风险,并导致这种治疗方法的成本居高不下。因此,我们开发了 LVV 包装细胞系和生产细胞系,减少或消除了 LVV 生产过程中质粒转染的需要。为了开发包装细胞系,我们通过随机整合线性化质粒 DNA 来稳定整合慢病毒包装基因。然后,为了培育 EGFP 和抗 CD19 嵌合抗原受体编码生产细胞系,通过转座酶介导的整合来整合转运质粒。通过单细胞分离和测试,分离出性能最好的克隆包装细胞系和生产细胞系。与行业标准的四质粒转染方法相比,生产编码各种货物基因的 LVV 发现包装细胞系和生产细胞系的生产性能具有一致性。通过减少或消除质粒转染的要求,同时实现与当前行业标准一致的生产性能,本文开发的包装和生产细胞系可以降低LVV生产的成本和操作风险,从而增加患者获得LVV介导的细胞和基因疗法的机会。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL 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
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.
间充质干/基质细胞(间充质干细胞)因其安全性和分泌多种免疫调节和再生分子的独特能力,成为一种极具吸引力的细胞治疗平台。然而,众所周知,间充质干细胞需要预处理或诱导才能提高疗效。目前的诱导方法对间叶干细胞活化的控制有限,只能产生短暂的效果,而且往往会诱导促炎效应因子的表达,从而增强免疫原性。在这里,我们介绍了一种基因诱导方法,它能通过控制炎症刺激反应转录因子干扰素反应因子1(IRF1)的表达,选择性地、持续地提高间充质干细胞的效力。超表达 IRF1 的间充质干细胞能重现许多核心反应,这些反应可通过使用促炎细胞因子干扰素-γ(IFN-γ)进行生化诱导来获得。这包括抗炎效应分子的上调和间充质干细胞抑制 T 细胞活化能力的增强。然而,我们的研究表明,IRF1 介导的遗传启动比生化启动更持久,并能规避 IFN-γ 依赖性的免疫原性 MHC II 类分子的表达。可持续激活和选择性定制间充质干细胞引物反应的能力为更全面地激活间充质干细胞用于治疗创造了可能性。
{"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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-08-06 DOI: 10.1016/j.omtm.2024.101312

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.

神经炎症是加速包括渐冻人症在内的多种神经退行性疾病进展的罪魁祸首。然而,仅针对神经炎症的治疗方法在临床试验中的结果却令人失望。神经元和非神经元细胞类型都与 ALS 的发病机制有关,多项研究表明,纠正每种细胞类型都会对疾病预后产生有益影响。此前,我们研究发现,AAV9 介导的运动神经元和星形胶质细胞 SOD1 抑制能显著改善 ALS 小鼠模型的运动功能并延长存活时间。尽管对神经元和星形胶质细胞进行了矫正,但 ALS 小鼠仍会死亡,并在终点组织中观察到小胶质细胞增生。因此,我们假设最佳的治疗方法是同时针对并纠正运动神经元、星形胶质细胞和小胶质细胞。在这里,我们开发了一种用 Galectin-1 间接靶向小胶质细胞的新方法,并将其与之前建立的 AAV9.SOD1.shRNA 治疗相结合。我们的研究表明,Galectin-1 调节 SOD1G93A 小胶质细胞可减少炎症标志物,并挽救体外运动神经元的死亡。当与 SOD1 下调配对时,我们发现联合治疗在体内具有协同作用,并显示 SOD1G93A 小鼠的存活期比单独抑制 SOD1 有显著延长。这些结果凸显了靶向炎性小胶质细胞作为未来治疗开发中关键组成部分的重要性。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-08-06 DOI: 10.1016/j.omtm.2024.101314

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.

在体内输送 mRNA 有助于研究基因表达和治疗疾病。脂质纳米颗粒(LNP)能有效地传递 mRNA 构建体,但蛋白质的表达一直被认为仅限于肝脏。在小型动物模型中,使用专门的 LNP 可以将 mRNA 运送到肝外组织,但目前还不清楚人类是否可以将 mRNA 全面运送到所有主要器官,因为运送可能会受到先天性免疫反应差异和相对器官大小的影响。此外,由于猪对补体活化相关假过敏(CARPA)的敏感性,在猪等大型动物模型中进行的 LNP 研究十分有限。在这项研究中,我们发现给猪静脉注射相对低剂量的封装在临床相关 LNP 配方中的 mRNA 时,所有主要器官都会出现外源蛋白表达。在猪的肝、脾、肺、心脏、子宫、结肠、胃、肾、小肠和大脑中都检测到了外源性蛋白,而不会诱发 CARPA。此外,在骨髓(包括巨核细胞、造血干细胞、粒细胞)以及循环白细胞和血小板中也检测到了蛋白表达。这些结果表明,几乎所有主要器官都含有外源蛋白表达,是 mRNA 疗法的可行靶点。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-08-06 DOI: 10.1016/j.omtm.2024.101313

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.

ⅣB型粘多糖病(MPSIVB)是一种溶酶体贮积症,由β-半乳糖苷酶(β-GAL)缺乏症引起,以严重的骨骼和神经系统改变为特征,目前尚无有效的治疗方法。为了开发针对 MPSIVB 的造血干祖细胞基因疗法(HSPC-GT),我们设计了编码人 β-GAL 的慢病毒载体(LVs),以实现人 HSPCs 中治疗酶的超生理释放和病变细胞的代谢矫正。转导的 HSPCs 显示出适当的集落形成、增殖和分化能力,但其后代却不能以超生理水平释放酶。因此,我们测试了替代性 LV,以过表达来自鼠类(LV-enhGLB1)和人类选择性突变 GLB1 序列(LV-mutGLB1)的增强型 β-GAL。只有用 LV-enhGLB1 转导的人类 HSPCs 在体外和体内过表达 β-GAL,且没有证据表明存在与过表达相关的毒性。它们的造血后代能有效释放β-GAL,从而交叉矫正包括骨骼细胞在内的缺陷细胞。我们发现,人造血细胞中的人 GLB1 mRNA 水平较低,而增强型 β-GAL 的稳定性更强,这有助于提高 LV-enhGLB1 的功效。重要的是,增强型β-GAL酶在人体细胞中表现出生理性溶酶体转运,而且在体外与免疫原性增加无关。这些结果支持将 LV-enhGLB1 用于 HSPC-GT 的进一步开发和未来治疗 MPSIVB 多系统疾病的临床转化。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-31 DOI: 10.1016/j.omtm.2024.101307

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.

基于巨噬细胞的细胞疗法是一种治疗癌症和修复组织损伤的新兴模式。可重复的制造和工程过程是发挥其治疗潜力的关键。在这里,我们建立了一个强大的巨噬细胞制造平台(Mo-Mac),并证明巨噬细胞的功能可以通过N1-甲基假尿苷(m1Ψ)修饰的mRNA来增强。利用单细胞转录组分析这一无偏见的方法,我们发现最终产品中 90% 的细胞是巨噬细胞,其余主要包括 T 细胞、B 细胞、自然杀伤细胞、原核细胞、原核细胞和造血干细胞。这项分析还指导了流式细胞术策略的开发,以评估制成品中的细胞组成,从而满足国家医药产品管理局的要求。为了调节巨噬细胞的功能,我们研究了是否可以通过 mRNA 技术增强巨噬细胞的吞噬能力。我们发现,在体外用 m1Ψ 修饰的编码 CD300LF 的 mRNA(CD300LF-mRNA-巨噬细胞)电穿孔巨噬细胞时,其吞噬能力会增强。同样,在急性肝功能衰竭的小鼠模型中,CD300LF-mRNA-巨噬细胞有助于器官从对乙酰氨基酚诱导的肝毒性中恢复过来。这些结果证明了符合 GMP 标准的巨噬细胞制造工艺,并表明巨噬细胞可以通过多功能 mRNA 技术来实现治疗目标。
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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 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-07-31 DOI: 10.1016/j.omtm.2024.101311

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.

鳞状鱼鳞病(LI)是一种慢性疾病,主要由 TGM1 基因突变引起,其特征是皮肤屏障形成受损。目前尚无确切的治疗方法,目前的治疗主要是缓解症状。LI小鼠模型往往不能忠实再现人类皮肤病的临床和组织病理学特征。为了开发先进的治疗方法,如体内外细胞和基因联合疗法,我们在人类原代克隆性角质形成细胞中通过 CRISPR-Cas9 介导的 TGM1 基因高效消融建立了 LI 的人体细胞模型。基因编辑后的细胞完全没有转谷氨酰胺酶-1(TG1)的表达,并在体外重现了具有大多数 LI 分子特征的过度角化表型。我们使用一种在自身启动子控制下表达转基因 TGM1 的 SINγ-RV 载体,测试了一种体内外基因治疗方法,并验证了这种 LI 模型可作为临床前评估研究的平台。经基因校正的TGM1无效角质细胞显示出正常的TG1表达、酶活性和粟粒状包膜形成,从而恢复了正常的表皮结构。单细胞多组学分析表明,病毒整合存在于整体形成的表皮干细胞中,这对表皮再生至关重要。这项研究为评估这种治疗方法在治疗TGM1依赖性LI方面的潜力提供了概念验证。
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Molecular Therapy-Methods & Clinical Development
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