Pub Date : 2024-08-26DOI: 10.1016/j.omtm.2024.101329
Konstantina Tzimou, David Catalán-Tatjer, Lars K. Nielsen, Jesús Lavado-García
Producing recombinant adeno-associated virus (rAAV) for gene therapy via triple transfection is an intricate process involving many cellular interactions. Each of the different elements encoded in the three required plasmids—pHelper, pRepCap, and pGOI—plays a distinct role, affecting different cellular pathways when producing rAAVs. The required expression balance emphasizes the critical need to fine-tune the concentration of all these different elements. The use of design of experiments (DOE) to find optimal ratios is a powerful method to streamline the process. However, the choice of the DOE method and design construction is crucial to avoid misleading results. In this work, we examined and compared four distinct DOE approaches: rotatable central composite design (RCCD), Box-Behnken design (BBD), face-centered central composite design (FCCD), and mixture design (MD). We compared the abilities of the different models to predict optimal ratios and interactions among the plasmids and the transfection reagent. Our findings revealed that blocking is essential to reduce the variability caused by uncontrolled random effects and that MD coupled with FCCD outperformed all other approaches, improving volumetric productivity 109-fold. These outcomes underscore the importance of selecting a model that can effectively account for the biological context, ultimately yielding superior results in optimizing rAAV production.
{"title":"Unlocking DOE potential by selecting the most appropriate design for rAAV optimization","authors":"Konstantina Tzimou, David Catalán-Tatjer, Lars K. Nielsen, Jesús Lavado-García","doi":"10.1016/j.omtm.2024.101329","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101329","url":null,"abstract":"Producing recombinant adeno-associated virus (rAAV) for gene therapy via triple transfection is an intricate process involving many cellular interactions. Each of the different elements encoded in the three required plasmids—pHelper, pRepCap, and pGOI—plays a distinct role, affecting different cellular pathways when producing rAAVs. The required expression balance emphasizes the critical need to fine-tune the concentration of all these different elements. The use of design of experiments (DOE) to find optimal ratios is a powerful method to streamline the process. However, the choice of the DOE method and design construction is crucial to avoid misleading results. In this work, we examined and compared four distinct DOE approaches: rotatable central composite design (RCCD), Box-Behnken design (BBD), face-centered central composite design (FCCD), and mixture design (MD). We compared the abilities of the different models to predict optimal ratios and interactions among the plasmids and the transfection reagent. Our findings revealed that blocking is essential to reduce the variability caused by uncontrolled random effects and that MD coupled with FCCD outperformed all other approaches, improving volumetric productivity 109-fold. These outcomes underscore the importance of selecting a model that can effectively account for the biological context, ultimately yielding superior results in optimizing rAAV production.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197291","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}
Pub Date : 2024-08-22DOI: 10.1016/j.omtm.2024.101328
Katharina Schindler, Katharina Eva Ruppel, Claudia Müller, Ulrike Koehl, Stephan Fricke, Dominik Schmiedel
Chimeric antigen receptor (CAR) T cell therapies have demonstrated significant successes in treating cancer. Currently, there are six approved CAR T cell products available on the market that target different malignancies of the B cell lineage. However, to overcome the limitations of CAR T cell therapies, other immune cells are being investigated for CAR-based cell therapies. CAR natural killer (NK) cells can be applied as allogeneic cell therapy, providing an economical, safe, and efficient alternative to autologous CAR T cells. To improve CAR research and future in-patient monitoring of cell therapeutics, a simple, reliable, and versatile CAR detection reagent is crucial. As most existing CARs contain a single-chain variable fragment (scFv) with either a Whitlow or a G4S linker site, linker-specific monoclonal antibodies (mAbs) can detect a broad range of CARs. This study demonstrates that these linker-specific mAbs can detect different CAR NK cells , spiked in whole blood, and within patient-derived tumor spheroids with high specificity and sensitivity, providing an effective and almost universal alternative for scFv-based CAR detection. Additionally, we confirm that linker-specific antibodies can be used for functional testing and enrichment of CAR NK cells, thereby providing a useful research tool to fast-track the development of novel CAR-based therapies.
嵌合抗原受体（CAR）T 细胞疗法在治疗癌症方面取得了巨大成功。目前，市场上有六种获批的 CAR T 细胞产品，针对 B 细胞系的不同恶性肿瘤。然而，为了克服 CAR T 细胞疗法的局限性，目前正在研究其他免疫细胞的 CAR 细胞疗法。CAR 自然杀伤（NK）细胞可用作异体细胞疗法，为自体 CAR T 细胞提供了一种经济、安全、高效的替代选择。为了改进 CAR 研究和未来对细胞疗法的住院监测，一种简单、可靠、多功能的 CAR 检测试剂至关重要。由于现有的大多数 CAR 都含有一个带有 Whitlow 或 G4S 连接位点的单链可变片段 (scFv)，因此连接位点特异性单克隆抗体 (mAbs) 可以检测多种 CAR。本研究证明，这些连接子特异性 mAbs 能以高特异性和高灵敏度检测全血中的不同 CAR NK 细胞和患者来源的肿瘤球体内的 CAR NK 细胞，为基于 scFv 的 CAR 检测提供了一种有效且几乎通用的替代方法。此外，我们还证实了连接子特异性抗体可用于 CAR NK 细胞的功能测试和富集，从而为快速开发基于 CAR 的新型疗法提供了有用的研究工具。
{"title":"Linker-specific monoclonal antibodies present a simple and reliable detection method for scFv-based CARNK cells","authors":"Katharina Schindler, Katharina Eva Ruppel, Claudia Müller, Ulrike Koehl, Stephan Fricke, Dominik Schmiedel","doi":"10.1016/j.omtm.2024.101328","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101328","url":null,"abstract":"Chimeric antigen receptor (CAR) T cell therapies have demonstrated significant successes in treating cancer. Currently, there are six approved CAR T cell products available on the market that target different malignancies of the B cell lineage. However, to overcome the limitations of CAR T cell therapies, other immune cells are being investigated for CAR-based cell therapies. CAR natural killer (NK) cells can be applied as allogeneic cell therapy, providing an economical, safe, and efficient alternative to autologous CAR T cells. To improve CAR research and future in-patient monitoring of cell therapeutics, a simple, reliable, and versatile CAR detection reagent is crucial. As most existing CARs contain a single-chain variable fragment (scFv) with either a Whitlow or a G4S linker site, linker-specific monoclonal antibodies (mAbs) can detect a broad range of CARs. This study demonstrates that these linker-specific mAbs can detect different CAR NK cells , spiked in whole blood, and within patient-derived tumor spheroids with high specificity and sensitivity, providing an effective and almost universal alternative for scFv-based CAR detection. Additionally, we confirm that linker-specific antibodies can be used for functional testing and enrichment of CAR NK cells, thereby providing a useful research tool to fast-track the development of novel CAR-based therapies.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197290","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}
Pub Date : 2024-08-20DOI: 10.1016/j.omtm.2024.101324
Stefan Barisic, Elena Cherkasova, Rosa Nadal, Xin Tian, Long Chen, Angelina Parrizzi, Robert N. Reger, Gina M. Scurti, Michael I. Nishimura, Richard W. Childs
expansion of genetically modified T cells in cancer patients following adoptive transfer has been linked to both anti-tumor activity and T cell-mediated toxicities. The development of digital PCR has improved the accuracy in quantifying the status of adoptively infused T cells compared to qPCR or flow cytometry. Here, we developed and evaluated the feasibility and performance of nanoplate-based digital PCR (ndPCR) to quantify adoptively infused T cells engineered with a T cell receptor (TCR) that recognizes a human endogenous retrovirus type E (HERV-E) antigen. Analysis of blood samples collected from patients with metastatic kidney cancer following the infusion of HERV-E TCR-transduced T cells established the limit of detection of ndPCR to be 0.3 transgene copies/μL of reaction. The lower limit of quantification for ndPCR was one engineered T cell per 10,000 PBMCs, which outperformed both qPCR and flow cytometry by 1 log. High inter-test and test-retest reliability was confirmed by analyzing blood samples collected from multiple patients. In conclusion, we demonstrated the feasibility of ndPCR for detecting and monitoring the fate of TCR-engineered T cells in adoptive cell therapy.
在癌症患者体内进行基因修饰 T 细胞的采用性转移扩增与抗肿瘤活性和 T 细胞介导的毒性有关。与 qPCR 或流式细胞术相比，数字 PCR 的发展提高了量化被收养输注 T 细胞状态的准确性。在这里，我们开发并评估了基于纳米板的数字 PCR（ndPCR）的可行性和性能，以量化使用识别人类内源性逆转录病毒 E 型（HERV-E）抗原的 T 细胞受体（TCR）设计的被收养输注 T 细胞。对转移性肾癌患者输注 HERV-E TCR 转导 T 细胞后采集的血液样本进行分析，确定 ndPCR 的检测限为 0.3 个转基因拷贝/μL 反应液。ndPCR的定量下限是每10,000个PBMC中一个工程T细胞，比qPCR和流式细胞术都高出1个对数。通过分析从多名患者采集的血液样本，证实了测试间和测试后的高度可靠性。总之，我们证明了 ndPCR 在采用细胞疗法中检测和监测 TCR 工程 T 细胞命运的可行性。
{"title":"Quantification of circulating TCR-engineered T cells targeting a human endogenous retrovirus post-adoptive transfer using nanoplate digital PCR","authors":"Stefan Barisic, Elena Cherkasova, Rosa Nadal, Xin Tian, Long Chen, Angelina Parrizzi, Robert N. Reger, Gina M. Scurti, Michael I. Nishimura, Richard W. Childs","doi":"10.1016/j.omtm.2024.101324","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101324","url":null,"abstract":"expansion of genetically modified T cells in cancer patients following adoptive transfer has been linked to both anti-tumor activity and T cell-mediated toxicities. The development of digital PCR has improved the accuracy in quantifying the status of adoptively infused T cells compared to qPCR or flow cytometry. Here, we developed and evaluated the feasibility and performance of nanoplate-based digital PCR (ndPCR) to quantify adoptively infused T cells engineered with a T cell receptor (TCR) that recognizes a human endogenous retrovirus type E (HERV-E) antigen. Analysis of blood samples collected from patients with metastatic kidney cancer following the infusion of HERV-E TCR-transduced T cells established the limit of detection of ndPCR to be 0.3 transgene copies/μL of reaction. The lower limit of quantification for ndPCR was one engineered T cell per 10,000 PBMCs, which outperformed both qPCR and flow cytometry by 1 log. High inter-test and test-retest reliability was confirmed by analyzing blood samples collected from multiple patients. In conclusion, we demonstrated the feasibility of ndPCR for detecting and monitoring the fate of TCR-engineered T cells in adoptive cell therapy.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142225186","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}
Pub Date : 2024-08-20DOI: 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
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.
X连锁淋巴细胞增生症（XLP1）是由影响SLAM相关蛋白（SAP）的基因突变引起的。设计用于在 T、NK 和 NKT 细胞中以治疗水平表达 SAP 的调控慢病毒载体（LV）XLP-SMART LV 对于有效的基因治疗至关重要。我们通过实验确定了该基因的 34 个基因组调控元件，并设计了 XLP-SMART LV 来模拟 SAP 的系谱和阶段特异性调控。我们筛选了它们在 T 细胞、NK 细胞和 NKT 细胞中的靶上增强子活性，以及在 B 细胞和骨髓细胞群中的脱靶增强子活性。三个增强子元件组合在一起可使SAP启动子在靶上群体中的表达量增加4倍。使用XLP-SMART LV进行的NSG-Tg(Hu-IL15)异种移植研究表明，与对照组EFS-LV相比，SAP在靶细胞中的表达量增加了7倍，而且没有脱靶表达。XLP-SMART LV 在外周血、骨髓、脾脏和胸腺组织中表现出阶段特异性的 T 细胞和 NK 细胞表达（模拟 SAP 的表达模式）。用XLP-SMART LVs转导XLP1患者的CD8+ T细胞或BM CD34+细胞，可分别将刺激诱导的细胞死亡和NK细胞毒性恢复到野生型水平。这些数据表明，通过基因疗法创建一种系和阶段特异性 LV 来恢复 XLP1 表型是可行的。
{"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":null,"pages":null},"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}
Pub Date : 2024-08-20DOI: 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
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-08-19DOI: 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
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-08-19DOI: 10.1016/j.omtm.2024.101326
Hongzhi Wang, Ran Li, Shraddha Sadekar, Amrita V. Kamath, Ben-Quan Shen
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-08-14DOI: 10.1016/j.omtm.2024.101320
Matthew K. Roach, Phillip Wirz, Jeremy Rouse, Allison Schorzman, Clayton W. Beard, David Scott
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-08-14DOI: 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
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-08-14DOI: 10.1016/j.omtm.2024.101321
Julyana Acevedo, Yiling Bi, Jessica Gee, Santoshkumar L. Khatwani
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.
{"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":null,"pages":null},"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}
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