Pub Date : 2024-09-06DOI: 10.1016/j.omtm.2024.101333
Nadia Tavakolidakhrabadi, Wen Y. Ding, Moin A. Saleem, Gavin I. Welsh, Carl May
Chronic kidney disease (CKD) poses a significant global health challenge, projected to become one of the leading causes of death by 2040. Current treatments primarily manage complications and slow progression, highlighting the urgent need for personalized therapies targeting the disease-causing genes. Our increased understanding on the underlying genomic changes that leads to kidney diseases coupled with recent successful gene therapies targeting specific kidney cells have turned gene therapy and genome editing into a promising therapeutic approach for treating kidney disease. This review paper will reflect on different delivery routes and system that can be exploited to target specific kidney cells, and the ways that gene therapy can be used to improve kidney health.
{"title":"Gene Therapy and kidney diseases","authors":"Nadia Tavakolidakhrabadi, Wen Y. Ding, Moin A. Saleem, Gavin I. Welsh, Carl May","doi":"10.1016/j.omtm.2024.101333","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101333","url":null,"abstract":"<p>Chronic kidney disease (CKD) poses a significant global health challenge, projected to become one of the leading causes of death by 2040. Current treatments primarily manage complications and slow progression, highlighting the urgent need for personalized therapies targeting the disease-causing genes. Our increased understanding on the underlying genomic changes that leads to kidney diseases coupled with recent successful gene therapies targeting specific kidney cells have turned gene therapy and genome editing into a promising therapeutic approach for treating kidney disease. This review paper will reflect on different delivery routes and system that can be exploited to target specific kidney cells, and the ways that gene therapy can be used to improve kidney health.</p>","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"17 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197242","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 : 2024-08-31DOI: 10.1016/j.omtm.2024.101331
Erika M. Shaw, Alexander J. Tate, Ramesh Periasamy, Daniel M. Lipinski
Age-related macular degeneration (AMD) affects millions of individuals worldwide and is a leading cause of blindness in the elderly. In dry AMD, lipoproteinaceous deposits called drusen accumulate between the retinal pigment epithelium (RPE) and Bruch’s membrane, leading to impairment of oxygen and nutrient trafficking to the neural retina, and degeneration of the overlying photoreceptor cells. Owing to key differences in human and animal ocular anatomy and the slowly progressing nature of the disease, AMD is not easily modeled In this study, we further characterize a “drusen-in-a-dish” primary porcine RPE model system by employing vital lipid staining to monitor sub-RPE deposition over time in monolayers of cells cultured on porous transwell membranes. We demonstrate for the first time using a semi-automated image analysis pipeline that the number and size of sub-RPE deposits increases gradually but significantly over time and confirm that sub-RPE deposits grown in culture immunostain positive for multiple known components found in human drusen. As a result, we propose that drusen-in-a-dish cell culture models represent a high-throughput and cost-scalable alternative to animal models in which to study the pathobiology of drusen accumulation and may serve as useful tools for screening novel therapeutics aimed at treating dry AMD.
{"title":"Longitudinal characterization of sub-retinal pigment epithelium deposit formation in a primary porcine tissue culture model of dry age-related macular degeneration","authors":"Erika M. Shaw, Alexander J. Tate, Ramesh Periasamy, Daniel M. Lipinski","doi":"10.1016/j.omtm.2024.101331","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101331","url":null,"abstract":"Age-related macular degeneration (AMD) affects millions of individuals worldwide and is a leading cause of blindness in the elderly. In dry AMD, lipoproteinaceous deposits called drusen accumulate between the retinal pigment epithelium (RPE) and Bruch’s membrane, leading to impairment of oxygen and nutrient trafficking to the neural retina, and degeneration of the overlying photoreceptor cells. Owing to key differences in human and animal ocular anatomy and the slowly progressing nature of the disease, AMD is not easily modeled In this study, we further characterize a “drusen-in-a-dish” primary porcine RPE model system by employing vital lipid staining to monitor sub-RPE deposition over time in monolayers of cells cultured on porous transwell membranes. We demonstrate for the first time using a semi-automated image analysis pipeline that the number and size of sub-RPE deposits increases gradually but significantly over time and confirm that sub-RPE deposits grown in culture immunostain positive for multiple known components found in human drusen. As a result, we propose that drusen-in-a-dish cell culture models represent a high-throughput and cost-scalable alternative to animal models in which to study the pathobiology of drusen accumulation and may serve as useful tools for screening novel therapeutics aimed at treating dry AMD.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"19 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197243","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}
Therapeutic antibodies (Ab) have revolutionized the management of multiple illnesses including respiratory tract infections (RTIs). However, anti-infectious Ab displayed several limitations including antigen restrictiveness, narrowed therapeutic windows, and limited dose in the vicinity of the target when delivered by parenteral routes. Strategies enhancing further Ab-dependent containment of infection are currently needed. Here we showed that a combination of inhaled anti-infectious Ab and probiotics is an efficient formulation to protect against lung infection. Using a mouse model of Pseudomonas aeruginosa-induced pneumonia, we demonstrated a synergistic effect reducing both bacterial burden and pro-inflammatory response affording protection against primary and secondary infections. This is the first study showing that the local combination in the airways of anti-infective Ab and probiotics subverts suboptimal potency of Ab monotherapy and provides protection against respiratory pathogen.
{"title":"Synergy between Lactobacillus murinus and anti-PcrV antibody delivered in the airways to boost protection against Pseudomonas aeruginosa","authors":"Thomas Sécher, Mélanie Cortes, Chloé Boisseau, Marie-Thérèse Barba Goudiaby, Aubin Pitiot, Christelle Parent, Muriel Thomas, Nathalie Heuzé-Vourc’h","doi":"10.1016/j.omtm.2024.101330","DOIUrl":"https://doi.org/10.1016/j.omtm.2024.101330","url":null,"abstract":"<p>Therapeutic antibodies (Ab) have revolutionized the management of multiple illnesses including respiratory tract infections (RTIs). However, anti-infectious Ab displayed several limitations including antigen restrictiveness, narrowed therapeutic windows, and limited dose in the vicinity of the target when delivered by parenteral routes. Strategies enhancing further Ab-dependent containment of infection are currently needed. Here we showed that a combination of inhaled anti-infectious Ab and probiotics is an efficient formulation to protect against lung infection. Using a mouse model of <em>Pseudomonas aeruginosa</em>-induced pneumonia, we demonstrated a synergistic effect reducing both bacterial burden and pro-inflammatory response affording protection against primary and secondary infections. This is the first study showing that the local combination in the airways of anti-infective Ab and probiotics subverts suboptimal potency of Ab monotherapy and provides protection against respiratory pathogen.</p>","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"879 1","pages":""},"PeriodicalIF":4.7,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142197288","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 : 2024-08-26DOI: 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":"27 1","pages":""},"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":"11 1","pages":""},"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":"182 1","pages":""},"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":"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}
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":"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}
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