Pub Date : 2023-12-01Epub Date: 2023-10-18DOI: 10.1089/hum.2023.041
Yanan Zhang, Dingyue Tang, Li Wang, Jing Yang, Xia Wu, Xiao Xiao, Jian-She Wang
Zinc finger FYVE-type containing 19 (ZFYVE19) deficiency, caused by biallelic ZFYVE19 complete loss-of-function variants, is a recently identified chronic hepatobiliary disorder characterized by obvious portal-tract fibrosis, increased numbers of bile ducts with malformations, and abnormal levels of serum markers of hepatobiliary injury. As liver-targeted adeno-associated virus (AAV) gene therapy has been used successfully in hepatobiliary diseases, liver-targeted gene therapy has been explored in a mouse model of this disorder. Three ZFYVE19 AAV vectors (AAV-hZFYVE19, AAV-hZFYVE19-m, and AAV-hZFYVE19-co) were constructed and injected into Zfyve19-/- mice, which were treated with alpha-naphthyl isothiocyanate, a hepatobiliary toxin. Hematoxylin/eosin, immunohistochemical staining, immunofluorescence staining, Sirius Red staining, real-time quantitative PCR, and Western blotting of liver tissue, along with serum hepatobiliary injury marker analyses, were performed to evaluate the effects of gene therapy. AAV-hZFYVE19 decreased serum hepatobiliary injury markers, portal-tract inflammation, ductal hyperplasia, and portal-tract fibrosis in the Zfyve19-/- model mice most substantially at a relatively low dose (1 × 1011 vg/kg), whereas AAV-hZFYVE19 at a higher dose gradually lost the abovementioned benefits and even caused deterioration at the highest dose of 5 × 1012 vg/kg. These observations verified the pathogenicity of ZFYVE19 deficiency and suggested that the ZFYVE19 gene needs to function well at an optimal level of expression; both too low and too high a ZFYVE19 expression may be harmful.
{"title":"Prevention of Portal-Tract Fibrosis in <i>Zfyve19<sup>-/-</sup></i> Mouse Model with Adeno-Associated Virus Vector Delivering <i>ZFYVE19</i>.","authors":"Yanan Zhang, Dingyue Tang, Li Wang, Jing Yang, Xia Wu, Xiao Xiao, Jian-She Wang","doi":"10.1089/hum.2023.041","DOIUrl":"10.1089/hum.2023.041","url":null,"abstract":"<p><p>Zinc finger FYVE-type containing 19 (ZFYVE19) deficiency, caused by biallelic <i>ZFYVE19</i> complete loss-of-function variants, is a recently identified chronic hepatobiliary disorder characterized by obvious portal-tract fibrosis, increased numbers of bile ducts with malformations, and abnormal levels of serum markers of hepatobiliary injury. As liver-targeted adeno-associated virus (AAV) gene therapy has been used successfully in hepatobiliary diseases, liver-targeted gene therapy has been explored in a mouse model of this disorder. Three <i>ZFYVE19</i> AAV vectors (AAV-<i>hZFYVE19</i>, AAV-<i>hZFYVE19-m</i>, and AAV-<i>hZFYVE19-co</i>) were constructed and injected into <i>Zfyve19<sup>-/-</sup></i> mice, which were treated with alpha-naphthyl isothiocyanate, a hepatobiliary toxin. Hematoxylin/eosin, immunohistochemical staining, immunofluorescence staining, Sirius Red staining, real-time quantitative PCR, and Western blotting of liver tissue, along with serum hepatobiliary injury marker analyses, were performed to evaluate the effects of gene therapy. AAV-<i>hZFYVE19</i> decreased serum hepatobiliary injury markers, portal-tract inflammation, ductal hyperplasia, and portal-tract fibrosis in the <i>Zfyve19<sup>-/-</sup></i> model mice most substantially at a relatively low dose (1 × 10<sup>11</sup> vg/kg), whereas AAV-<i>hZFYVE19</i> at a higher dose gradually lost the abovementioned benefits and even caused deterioration at the highest dose of 5 × 10<sup>12</sup> vg/kg. These observations verified the pathogenicity of ZFYVE19 deficiency and suggested that the <i>ZFYVE19</i> gene needs to function well at an optimal level of expression; both too low and too high a <i>ZFYVE19</i> expression may be harmful.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10170223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chimeric antigen receptor T (CAR-T) cell therapy initiates new methods and turns the scale of clinical treatment on relapsed/refractory acute T lymphoblastic leukemia (T-ALL). In this study, we generated the second-generation CD7-targeting CAR-T cells with a new antigen-binding single-chain variable fragment sequence and made it universal via CRISPR-based knockout of TRAC and CD7 genes (termed UCAR-T). The CD7 UCAR-T cells can efficiently proliferate and lyse T-ALL tumor cell in vitro, along with prominent proinflammatory cytokines secretion. A Jurkat-based xenograft mouse model further verified the superior cytotoxicity of the UCAR-T cells in vivo. During the UCAR-T construction, we observed a CD4/CD8 ratio shift among CD7-/- T/CAR-T cells, which motivated us to further analyze the effects of CD7 antigen on T/CAR-T cells. We sorted out CD7+/- T or anti-CD19 CAR-T cells after partially CD7 knockout and performed functional, phenotypic detection, as well as translational analysis. CD7-/- CAR-T cells tended to be CD8 negative and showed slightly better cytotoxicity at long-term assay. RNA-seq further confirmed an elevation of activated CD4 memory cell subpopulation. However, limited distinction on crucial regulatory genes and pathways was revealed, suggesting the safety and feasibility of UCAR-T application as well as the potential translational rather than transcriptional regulation of CD7 antigen.
{"title":"Universal Anti-CD7 CAR-T Cells Targeting T-ALL and Functional Analysis of CD7 Antigen on T/CAR-T Cells.","authors":"Leling Xie, Runxia Gu, Xue Yang, Shaowei Qiu, Yingxi Xu, Junli Mou, Ying Wang, Haiyan Xing, Kejing Tang, Zheng Tian, Qing Rao, Min Wang, Jianxiang Wang","doi":"10.1089/hum.2023.029","DOIUrl":"10.1089/hum.2023.029","url":null,"abstract":"<p><p>Chimeric antigen receptor T (CAR-T) cell therapy initiates new methods and turns the scale of clinical treatment on relapsed/refractory acute T lymphoblastic leukemia (T-ALL). In this study, we generated the second-generation CD7-targeting CAR-T cells with a new antigen-binding single-chain variable fragment sequence and made it universal via CRISPR-based knockout of <i>TRAC</i> and <i>CD7</i> genes (termed UCAR-T). The CD7 UCAR-T cells can efficiently proliferate and lyse T-ALL tumor cell <i>in vitro</i>, along with prominent proinflammatory cytokines secretion. A Jurkat-based xenograft mouse model further verified the superior cytotoxicity of the UCAR-T cells <i>in vivo</i>. During the UCAR-T construction, we observed a CD4/CD8 ratio shift among CD7<sup>-/-</sup> T/CAR-T cells, which motivated us to further analyze the effects of CD7 antigen on T/CAR-T cells. We sorted out CD7<sup>+/-</sup> T or anti-CD19 CAR-T cells after partially CD7 knockout and performed functional, phenotypic detection, as well as translational analysis. CD7<sup>-/-</sup> CAR-T cells tended to be CD8 negative and showed slightly better cytotoxicity at long-term assay. RNA-seq further confirmed an elevation of activated CD4 memory cell subpopulation. However, limited distinction on crucial regulatory genes and pathways was revealed, suggesting the safety and feasibility of UCAR-T application as well as the potential translational rather than transcriptional regulation of CD7 antigen.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49676980","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Laura Kimberly, Cara Hunt, Katherine Beaverson, Emma James, Alison Bateman-House, Richard McGowan, Jennifer DeSante-Bertkau
Little is known about patients' and families' lived experiences of participating in pediatric gene therapy (GT) clinical trials. Currently, pediatric GT research targets a broad range of indications--including rare and ultra-rare diseases--which vary in severity and in the availability of alternative therapies. Pediatric GT differs meaningfully from adult GT because the decision to participate involves a dyad of both the child and parent or caregiver/s. It is critical to understand patients' and caregivers' perceptions and experiences of social, emotional, physical, and logistical burdens or benefits of participating in such trials, and how they weigh and prioritize these factors when deciding whether to participate. We conducted a scoping review of the current literature in this subject area with objectives to (1) provide an overview of existing literature, (2) identify gaps and areas for further research, and (3) better understand the lived impact of pediatric GT research on patients and their parents/caregivers. Four themes emerged, including (1) weighing risks and benefits (2) timing of GT trial participation, (3) value of clear communication, and (4) potential impact on quality of life. Notably, our sample surfaced articles about how patients/parents/caregivers were thinking about GT-their understanding of its safety, efficacy, and risks-rather than accounts of their experiences, which was our initial intention. Nevertheless, our findings offer useful insights to improve the informed consent process and promote a more patient- and family-centered approach. Moreover, our findings can contribute to patient advocacy organizations' efforts to develop educational materials tailored to patients' and families' expressed informational needs and perspectives, and can inform more patient- and family-centered policies from GT clinical trial sponsors.
{"title":"The Lived Experience of Pediatric Gene Therapy: A Scoping Review.","authors":"Laura Kimberly, Cara Hunt, Katherine Beaverson, Emma James, Alison Bateman-House, Richard McGowan, Jennifer DeSante-Bertkau","doi":"10.1089/hum.2023.157","DOIUrl":"10.1089/hum.2023.157","url":null,"abstract":"<p><p>Little is known about patients' and families' lived experiences of participating in pediatric gene therapy (GT) clinical trials. Currently, pediatric GT research targets a broad range of indications--including rare and ultra-rare diseases--which vary in severity and in the availability of alternative therapies. Pediatric GT differs meaningfully from adult GT because the decision to participate involves a dyad of both the child and parent or caregiver/s. It is critical to understand patients' and caregivers' perceptions and experiences of social, emotional, physical, and logistical burdens or benefits of participating in such trials, and how they weigh and prioritize these factors when deciding whether to participate. We conducted a scoping review of the current literature in this subject area with objectives to (1) provide an overview of existing literature, (2) identify gaps and areas for further research, and (3) better understand the lived impact of pediatric GT research on patients and their parents/caregivers. Four themes emerged, including (1) weighing risks and benefits (2) timing of GT trial participation, (3) value of clear communication, and (4) potential impact on quality of life. Notably, our sample surfaced articles about how patients/parents/caregivers <i>were thinking</i> about GT-their understanding of its safety, efficacy, and risks-rather than accounts of their <i>experiences</i>, which was our initial intention. Nevertheless, our findings offer useful insights to improve the informed consent process and promote a more patient- and family-centered approach. Moreover, our findings can contribute to patient advocacy organizations' efforts to develop educational materials tailored to patients' and families' expressed informational needs and perspectives, and can inform more patient- and family-centered policies from GT clinical trial sponsors.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"107591144","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dan Li, Lin Jiang, Weilin Zhou, Yong Huang, Yuening Yang, Jing Li, Jinrong Yang, Fengling Wang, Jiaqian Li, Yalan Zhang, Feiyang Yan, Haozhan Gao, Xianling Guo, Qing Xu, Shisheng Tan, Yu-Quan Wei, Wei Wang
Distant metastasis and primary tumor relapse are the two main hurdles to the success of surgical treatment for cancer patients. Circulating tumor cells (CTCs) and incomplete surgical resection are the primary cause of distant metastasis and local recurrence of tumors, respectively. Chimeric antigen receptor (CAR)-modified T cells target residual carcinomas and CTCs hold the potential to inhibit primary recurrence and reduce tumor metastasis, but the experimental evidence is lacking. Here, we developed a surgery-induced tumor metastasis model in immunocompetent mice to investigate the efficacy of CAR-T cells therapy in preventing metastasis and local recurrence. We observed that subcutaneous tumor resection has induced a large number of CTCs intravasated into circulation. EpCAM-specific CAR-T was effective in clearing CTCs following surgical removal of the tumor. This resulted in less pulmonary metastasis and longer survival in mice when compared to mice treated with surgery followed by Mock-T cells infusion. In addition, the local relapse was obviously inhibited at the surgical site followed by EpCAM-CAR-T cell treatment. This study demonstrated that CAR-T cell therapy can be an adjuvant treatment following surgery to prevent tumor metastasis and inhibit primary tumor relapse for cancer patients.
{"title":"Chimeric Antigen Receptor-T Cell Therapy Decreases Distant Metastasis and Inhibits Local Recurrence Post-surgery in Mice.","authors":"Dan Li, Lin Jiang, Weilin Zhou, Yong Huang, Yuening Yang, Jing Li, Jinrong Yang, Fengling Wang, Jiaqian Li, Yalan Zhang, Feiyang Yan, Haozhan Gao, Xianling Guo, Qing Xu, Shisheng Tan, Yu-Quan Wei, Wei Wang","doi":"10.1089/hum.2023.144","DOIUrl":"10.1089/hum.2023.144","url":null,"abstract":"<p><p>Distant metastasis and primary tumor relapse are the two main hurdles to the success of surgical treatment for cancer patients. Circulating tumor cells (CTCs) and incomplete surgical resection are the primary cause of distant metastasis and local recurrence of tumors, respectively. Chimeric antigen receptor (CAR)-modified T cells target residual carcinomas and CTCs hold the potential to inhibit primary recurrence and reduce tumor metastasis, but the experimental evidence is lacking. Here, we developed a surgery-induced tumor metastasis model in immunocompetent mice to investigate the efficacy of CAR-T cells therapy in preventing metastasis and local recurrence. We observed that subcutaneous tumor resection has induced a large number of CTCs intravasated into circulation. EpCAM-specific CAR-T was effective in clearing CTCs following surgical removal of the tumor. This resulted in less pulmonary metastasis and longer survival in mice when compared to mice treated with surgery followed by Mock-T cells infusion. In addition, the local relapse was obviously inhibited at the surgical site followed by EpCAM-CAR-T cell treatment. This study demonstrated that CAR-T cell therapy can be an adjuvant treatment following surgery to prevent tumor metastasis and inhibit primary tumor relapse for cancer patients.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71423254","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-01DOI: 10.1089/hum.2023.29262.trf
Terence R Flotte
{"title":"Lived Experience with Gene Therapy.","authors":"Terence R Flotte","doi":"10.1089/hum.2023.29262.trf","DOIUrl":"https://doi.org/10.1089/hum.2023.29262.trf","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138799021","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-01Epub Date: 2023-12-05DOI: 10.1089/hum.2023.035
Julia Festag, Marvin M Festag, Theresa Asen, Jochen M Wettengel, Martin A Mück-Häusl, Shaheed Abdulhaqq, Christiane Stahl-Hennig, Jonah B Sacha, Benjamin J Burwitz, Ulrike Protzer, Karin Wisskirchen
Adoptive T cell therapy using natural T cell receptor (TCR) redirection is a promising approach to fight solid cancers and viral infections in liver and other organs. However, clinical efficacy of such TCR+-T cells has been limited so far. One reason is that syngeneic preclinical models to evaluate safety and efficacy of TCR+-T cells are missing. We, therefore, developed an efficient viral vector strategy mediating expression of human major histocompatibility complex (MHC)-I in hepatocytes, which allows evaluation of TCR-T cell therapies targeting diseased liver cells. We designed adeno-associated virus (AAV) and adenoviral vectors encoding either the human-mouse chimeric HLA-A*02-like molecule, or fully human HLA-A*02 and human β2 microglobulin (hβ2m). Upon transduction of murine hepatocytes, the HLA-A*02 construct proved superior in terms of expression levels, presentation of endogenously processed peptides and activation of murine TCR+-T cells grafted with HLA-A*02-restricted, hepatitis B virus (HBV)-specific TCRs. In vivo, these T cells elicited effector function, controlled HBV replication, and reduced HBV viral load and antigen expression in livers of those mice that had received AAV-HBV and AAV-HLA-A*02. We then demonstrated the broad utility of this approach by grafting macaque T cells with the HBV-specific TCRs and enabling them to recognize HBV-infected primary macaque hepatocytes expressing HLA-A*02 upon adenoviral transduction. In conclusion, AAV and adenovirus vectors are suitable for delivery of HLA-A*02 and hβ2m into mouse and macaque hepatocytes. When recognizing their cognate antigen in HLA-A*02-transduced mouse livers or on isolated macaque hepatocytes, HLA-A*02-restricted, HBV-specific TCR+-T cells become activated and exert antiviral effector functions. This approach is applicable to any MHC restriction and target disease, paving the way for safety and efficacy studies of human TCR-based therapies in physiologically relevant preclinical animal models.
{"title":"Vector-Mediated Delivery of Human Major Histocompatibility Complex-I into Hepatocytes Enables Investigation of T Cell Receptor-Redirected Hepatitis B Virus-Specific T Cells in Mice, and in Macaque Cell Cultures.","authors":"Julia Festag, Marvin M Festag, Theresa Asen, Jochen M Wettengel, Martin A Mück-Häusl, Shaheed Abdulhaqq, Christiane Stahl-Hennig, Jonah B Sacha, Benjamin J Burwitz, Ulrike Protzer, Karin Wisskirchen","doi":"10.1089/hum.2023.035","DOIUrl":"10.1089/hum.2023.035","url":null,"abstract":"<p><p>Adoptive T cell therapy using natural T cell receptor (TCR) redirection is a promising approach to fight solid cancers and viral infections in liver and other organs. However, clinical efficacy of such TCR<sup>+</sup>-T cells has been limited so far. One reason is that syngeneic preclinical models to evaluate safety and efficacy of TCR<sup>+</sup>-T cells are missing. We, therefore, developed an efficient viral vector strategy mediating expression of human major histocompatibility complex (MHC)-I in hepatocytes, which allows evaluation of TCR-T cell therapies targeting diseased liver cells. We designed adeno-associated virus (AAV) and adenoviral vectors encoding either the human-mouse chimeric HLA-A*02-like molecule, or fully human HLA-A*02 and human β2 microglobulin (hβ2m). Upon transduction of murine hepatocytes, the HLA-A*02 construct proved superior in terms of expression levels, presentation of endogenously processed peptides and activation of murine TCR<sup>+</sup>-T cells grafted with HLA-A*02-restricted, hepatitis B virus (HBV)-specific TCRs. <i>In vivo</i>, these T cells elicited effector function, controlled HBV replication, and reduced HBV viral load and antigen expression in livers of those mice that had received AAV-HBV and AAV-HLA-A*02. We then demonstrated the broad utility of this approach by grafting macaque T cells with the HBV-specific TCRs and enabling them to recognize HBV-infected primary macaque hepatocytes expressing HLA-A*02 upon adenoviral transduction. In conclusion, AAV and adenovirus vectors are suitable for delivery of HLA-A*02 and hβ2m into mouse and macaque hepatocytes. When recognizing their cognate antigen in HLA-A*02-transduced mouse livers or on isolated macaque hepatocytes, HLA-A*02-restricted, HBV-specific TCR<sup>+</sup>-T cells become activated and exert antiviral effector functions. This approach is applicable to any MHC restriction and target disease, paving the way for safety and efficacy studies of human TCR-based therapies in physiologically relevant preclinical animal models.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10825313/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41116894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-01Epub Date: 2023-11-06DOI: 10.1089/hum.2023.29255.pwl
Phillip W L Tai
{"title":"Integration of Gene Therapy Vectors: A Risk Factor for Tumorigenesis or Another Commensal Property of Adeno-Associated Viruses That Benefits Long-Term Transgene Expression?","authors":"Phillip W L Tai","doi":"10.1089/hum.2023.29255.pwl","DOIUrl":"10.1089/hum.2023.29255.pwl","url":null,"abstract":"","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71480983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-01Epub Date: 2023-10-26DOI: 10.1089/hum.2023.030
Yan Lu, Chao-Xuan Zhang, Patrick Rodrigues, Lei Yang, Aaron Shafer, Zoran Rankovic, Frank Fazio
Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.
基于慢病毒载体的基因治疗在治疗各种疾病方面越来越受欢迎。圣犹达正在开发各种LV载体,用于在细胞基因治疗中转导细胞。一些LV载体是使用稳定的293T包装细胞系生产的,其中包括gag-pol-rev-tat和病毒糖蛋白。反式激活因子(transactuator of transcription[Tat])是一种能显著提高慢病毒转录效率的调节蛋白。Tat的残留分析对基因载体的质量和安全性至关重要。在这项工作中,我们开发了一种高灵敏度的液相色谱-串联质谱法来分析慢病毒中残留的Tat,作为酶联免疫吸附测定的替代方法。左心室中残留的Tat可以高特异性准确定量,检测限为0.3 ng/mL。
{"title":"Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Sensitive Analysis of Residual Protein Tat Bh1-101 in Lentiviral Vectors for Gene Therapy.","authors":"Yan Lu, Chao-Xuan Zhang, Patrick Rodrigues, Lei Yang, Aaron Shafer, Zoran Rankovic, Frank Fazio","doi":"10.1089/hum.2023.030","DOIUrl":"10.1089/hum.2023.030","url":null,"abstract":"<p><p>Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10170222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}