Molecular Therapy-Methods & Clinical Development最新文献_第8页

Lentiviral vectors for precise expression to treat X-linked lymphoproliferative disease 用于精确表达的慢病毒载体治疗 X 连锁淋巴细胞增生症

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-20 DOI: 10.1016/j.omtm.2024.101323

Paul G. Ayoub, Julia Gensheimer, Lindsay Lathrop, Colin Juett, Jason Quintos, Kevin Tam, Jack Reid, Feiyang Ma, Curtis Tam, Grace E. McAuley, Devin Brown, Xiaomeng Wu, Ruixue Zhang, Kathryn Bradford, Roger P. Hollis, Gay M. Crooks, Donald B. Kohn

{"title":"Lentiviral vectors for precise expression to treat X-linked lymphoproliferative disease","authors":"Paul G. Ayoub, Julia Gensheimer, Lindsay Lathrop, Colin Juett, Jason Quintos, Kevin Tam, Jack Reid, Feiyang Ma, Curtis Tam, Grace E. McAuley, Devin Brown, Xiaomeng Wu, Ruixue Zhang, Kathryn Bradford, Roger P. Hollis, Gay M. Crooks, Donald B. Kohn","doi":"10.1016/j.omtm.2024.101323","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101323","url":null,"abstract":"X-linked lymphoproliferative disease (XLP1) results from gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations . NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ T cells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"2 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197292","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

Predictive power of deleterious single amino acid changes to infer on AAV2 and AAV2-13 capsids fitness 单个氨基酸的有害变化对推断 AAV2 和 AAV2-13 外壳适应性的预测能力

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-20 DOI: 10.1016/j.omtm.2024.101327

Tiziana La Bella, Bérangère Bertin, Ante Mihaljevic, Justine Nozi, Patrice Vidal, Sandrine Imbeaud, Jean-Charles Nault, Jessica Zucman-Rossi, Giuseppe Ronzitti

{"title":"Predictive power of deleterious single amino acid changes to infer on AAV2 and AAV2-13 capsids fitness","authors":"Tiziana La Bella, Bérangère Bertin, Ante Mihaljevic, Justine Nozi, Patrice Vidal, Sandrine Imbeaud, Jean-Charles Nault, Jessica Zucman-Rossi, Giuseppe Ronzitti","doi":"10.1016/j.omtm.2024.101327","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101327","url":null,"abstract":"Adeno-associated virus (AAV) is the most widely used vector for gene transfer. A major limitation of capsid engineering is the incomplete understanding of the consequences of multiple amino acid variations on AAV capsid stability resulting in high frequency of non-viable capsids. In this context, the study of natural AAV variants can provide valuable insights into capsid regions that exhibit greater tolerance to mutations. Here, the characterization of AAV2 variants and the analysis of two public capsid libraries highlighted common features associated with deleterious mutations, suggesting that the impact of mutations on capsid viability is strictly dependent on their 3D location within the capsid structure. We developed a novel prediction method to infer the fitness of AAV2 variants containing multiple amino acid variations with 98% sensitivity, 98% accuracy, and 95% specificity. This novel approach might streamline the development of AAV vector libraries enriched in viable capsids, thus accelerating the identification of therapeutic candidates among engineered capsids.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"28 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197293","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 novel approach to quantitate biodistribution and transduction of adeno-associated virus gene therapy using radiolabeled AAV vectors in mice 使用放射性标记的 AAV 载体量化小鼠腺相关病毒基因疗法的生物分布和转导的新方法

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-19 DOI: 10.1016/j.omtm.2024.101326

Hongzhi Wang, Ran Li, Shraddha Sadekar, Amrita V. Kamath, Ben-Quan Shen

{"title":"A novel approach to quantitate biodistribution and transduction of adeno-associated virus gene therapy using radiolabeled AAV vectors in mice","authors":"Hongzhi Wang, Ran Li, Shraddha Sadekar, Amrita V. Kamath, Ben-Quan Shen","doi":"10.1016/j.omtm.2024.101326","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101326","url":null,"abstract":"An understanding of recombinant adeno-associated virus (AAV) biodistribution profiles is an important element of a preclinical development program. Here, we have developed a radiolabeling strategy utilizing the co-delivery of I (non-residualizing) and In (residualizing) radionuclide-conjugated AAVs to provide a detailed distribution quantification at tissue level delineating between the cellular internalized AAV (degraded, In-I) and AAV remaining in the extracellular matrix (intact, I). This labeling method has been successfully applied to AAV9 and AAV-PHP.eB as tool molecules without altering the physical properties and biological activities of the AAVs. Upon labeling with either of the radioactive probes, these molecules were systemically injected into C57BL/6 mice. The biodistribution results indicate that AAVs, with a fast distribution profile, were mainly located in the extracellular matrix of highly perfused organs such as liver and spleen at early time points, leading to a difference between capsid quantification and vector genome quantification. The results suggest that the I-AAV/In-AAV co-delivery approach offers a robust and efficient analytical strategy to investigate the detailed tissue distribution of AAV vectors, including both vector genome and protein capsids. This novel method has the potential to be applied to capsid optimization, selection, and lead candidate development.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"202 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197294","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 encapsulation of a Delta spike-CD40L DNA vaccine improves effectiveness against Omicron challenge in Syrian hamsters 用脂质纳米颗粒封装 Delta spike-CD40L DNA 疫苗可提高叙利亚仓鼠抵抗 Omicron 挑战的效力

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-19 DOI: 10.1016/j.omtm.2024.101325

Levi Tamming, Diana Duque, Anh Tran, Casey Lansdell, Grant Frahm, Jianguo Wu, Emily E.F. Fekete, Marybeth Creskey, Sathya N. Thulasi Raman, Emmanuel Laryea, Wanyue Zhang, Annabelle Pfeifle, Caroline Gravel, Andrew Stalker, Anwar M. Hashem, Wangxue Chen, Matthew Stuible, Yves Durocher, David Safronetz, Jingxin Cao, Lisheng Wang, Simon Sauve, Michael Rosu-Myles, Xu Zhang, Michael J.W. Johnston, Xuguang Li

{"title":"Lipid nanoparticle encapsulation of a Delta spike-CD40L DNA vaccine improves effectiveness against Omicron challenge in Syrian hamsters","authors":"Levi Tamming, Diana Duque, Anh Tran, Casey Lansdell, Grant Frahm, Jianguo Wu, Emily E.F. Fekete, Marybeth Creskey, Sathya N. Thulasi Raman, Emmanuel Laryea, Wanyue Zhang, Annabelle Pfeifle, Caroline Gravel, Andrew Stalker, Anwar M. Hashem, Wangxue Chen, Matthew Stuible, Yves Durocher, David Safronetz, Jingxin Cao, Lisheng Wang, Simon Sauve, Michael Rosu-Myles, Xu Zhang, Michael J.W. Johnston, Xuguang Li","doi":"10.1016/j.omtm.2024.101325","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101325","url":null,"abstract":"The effectiveness of mRNA vaccines largely depends on their lipid nanoparticle (LNP) component. Herein, we investigate the effectiveness of DLin-KC2-DMA (KC2) and SM-102-based LNPs for the intramuscular delivery of a plasmid encoding B.1.617.2 (Delta) spike fused with CD40 ligand. LNP encapsulation of this CD40L-adjuvanted DNA vaccine with either LNP formulation drastically enhanced antibody responses, enabling neutralization of heterologous Omicron variants. The DNA-LNP formulations provided excellent protection from homologous challenge, reducing viral replication, and preventing histopathological changes in the pulmonary tissues. Moreover, the DNA-LNP vaccines maintained a high level of protection against heterologous Omicron BA.5 challenge despite a reduced neutralizing response. In addition, we observed that DNA-LNP vaccination led to the pulmonary downregulation of interferon signaling, interleukin-12 signaling, and macrophage response pathways following SARS-CoV-2 challenge, shedding some light on the mechanisms underlying the prevention of pulmonary injury. These results highlight the potential combination of molecular adjuvants with LNP-based vaccine delivery to induce greater and broader immune responses capable of preventing inflammatory damage and protecting against emerging variants. These findings could be informative for the future design of both DNA and mRNA vaccines.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"27 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197295","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

Production of recombinant adeno-associated virus 5 using a novel self-attenuating adenovirus production platform 利用新型自增强腺病毒生产平台生产重组腺相关病毒 5

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-14 DOI: 10.1016/j.omtm.2024.101320

Matthew K. Roach, Phillip Wirz, Jeremy Rouse, Allison Schorzman, Clayton W. Beard, David Scott

{"title":"Production of recombinant adeno-associated virus 5 using a novel self-attenuating adenovirus production platform","authors":"Matthew K. Roach, Phillip Wirz, Jeremy Rouse, Allison Schorzman, Clayton W. Beard, David Scott","doi":"10.1016/j.omtm.2024.101320","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101320","url":null,"abstract":"Recombinant adeno-associated virus (rAAV) has become a prominent vector for clinical use. Despite an increase in successful clinical outcomes, the amount of high-quality rAAVs required for clinical trials and eventual commercial demand is difficult to produce, especially for genetic diseases that are prevalent or require high doses. Many groups are focused on establishing production processes that can produce sufficient rAAV while maintaining potency and quality. Our group used a novel production platform to increase our yield of rAAV5. This production platform uses tetracycline-enabled self-silencing adenovirus (TESSA) to deliver the wild-type AAV replication and capsid genes alongside the adenovirus helper genes necessary for production. Here, we describe our efforts to evaluate the TESSA platform in house. We conducted numerous experiments to determine the optimal conditions for producing rAAV5 from the TESSA production system. We then produced rAAV5 from the TESSA system to compare against rAAV5 produced from triple transfection. Ultimately, we generated data that showed that the vector genome yield of rAAV5 produced with TESSA was >20-fold higher than rAAV5 produced with triple transfection. Additionally, our data show that quality as well as potency in mice of rAAV5 produced with the TESSA system and by triple transfection are equivalent.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"46 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197296","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

CRISPR-Cas9-mediated genome editing delivered by a single AAV9 vector inhibits HSV-1 reactivation in a latent rabbit keratitis model 由单一 AAV9 载体传递的 CRISPR-Cas9 介导的基因组编辑可抑制潜伏兔角膜炎模型中的 HSV-1 再激活

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-14 DOI: 10.1016/j.omtm.2024.101303

Nadia Amrani, Kevin Luk, Pankaj Singh, Mason Shipley, Meltem Isik, Martina Donadoni, Anna Bellizzi, Kamel Khalili, Ilker K. Sariyer, Donna Neumann, Jennifer Gordon, Guo-Xiang Ruan

{"title":"CRISPR-Cas9-mediated genome editing delivered by a single AAV9 vector inhibits HSV-1 reactivation in a latent rabbit keratitis model","authors":"Nadia Amrani, Kevin Luk, Pankaj Singh, Mason Shipley, Meltem Isik, Martina Donadoni, Anna Bellizzi, Kamel Khalili, Ilker K. Sariyer, Donna Neumann, Jennifer Gordon, Guo-Xiang Ruan","doi":"10.1016/j.omtm.2024.101303","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101303","url":null,"abstract":"Herpes simples virus 1 (HSV-1) keratitis is a major cause of blindness globally. During primary infection, HSV-1 travels to the trigeminal ganglia and establishes lifelong latency. Although some treatments can reduce symptom severity and recurrence, there is no cure for HSV-1 keratitis. We used CRISPR-Cas9 to co-target gene sequences encoding two essential HSV-1 proteins, ICP0 and ICP27, as a potential therapy for HSV-1 keratitis. In HSV-1-infected Vero cells, the HSV-1 viral load and titer were significantly reduced by plasmid transfection or AAV2 vector transduction expressing Cas9 nuclease from (SaCas9) and paired guide RNAs (gRNAs). Off-target assessment showed minimal off-target editing activity from the selected gRNAs. We then tested our CRISPR-Cas9 gene editing approach in a latent rabbit model of HSV-1 keratitis. Corneal scarification with all-in-one AAV8(Y733F)-SaCas9 or AAV9-SaCas9 vector reduced viral shedding by over 50%. Interestingly, intravenous administration of the same AAV9-SaCas9 vector eliminated viral shedding in 92% of treated eyes. In addition, treated trigeminal ganglia showed a reduction in HSV-1 DNA and RNA expression. Our results support the utility of single-dose AAV9 all-in-one CRISPR-Cas9 gene editing as a safe and effective strategy for treating HSV-1 keratitis.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"62 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142225203","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

Assessment of adeno-associated virus purity by capillary electrophoresis-based western 用毛细管电泳法评估腺相关病毒的纯度

IF 4.7 2区医学

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-08-14 DOI: 10.1016/j.omtm.2024.101321

Julyana Acevedo, Yiling Bi, Jessica Gee, Santoshkumar L. Khatwani

{"title":"Assessment of adeno-associated virus purity by capillary electrophoresis-based western","authors":"Julyana Acevedo, Yiling Bi, Jessica Gee, Santoshkumar L. Khatwani","doi":"10.1016/j.omtm.2024.101321","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101321","url":null,"abstract":"A rigorous analytical assessment of recombinant adeno-associated virus (rAAV)-based drug products is critical for their successful development as clinical candidates. It is especially important to ascertain high purity while simultaneously ensuring low levels of impurities in the final drug product. One approach to evaluate the purity of rAAV drug products is to determine the relative stoichiometry of the three viral proteins (VPs) that comprise an rAAV capsid, and the levels of impurities in the final drug product. Here we present two capillary electrophoresis-western (CE-western) assays for quantifying (1) the relative stoichiometry of VP using the anti-AAV B1 antibody, and (2) residual levels of a baculovirus protein impurity, GP64, using the anti-GP64 antibody. In each assay, various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results obtained from both assays were further supported by liquid chromatography-mass spectrometry analyses. Overall, we report that CE-western is a high-throughput platform that utilizes low sample volumes for a rapid, sensitive, and robust assessment of the identity, composition, and purity of rAAV drug products.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"103 4 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197297","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

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":"202 1","pages":""},"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":"81 1","pages":""},"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":"9 1","pages":""},"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