Human Gene Therapy Methods最新文献_第10页

A Rapid Cell Expansion Process for Production of Engineered Autologous CAR-T Cell Therapies. 用于生产工程化自体CAR-T细胞疗法的快速细胞扩增过程。

Human Gene Therapy Methods Pub Date : 2016-12-01 DOI: 10.1089/HGTB.2016.120

Tangying Lu, O. Pugach, R. Somerville, S. Rosenberg, J. Kochenderfer, M. Better, S. Feldman

{"title":"A Rapid Cell Expansion Process for Production of Engineered Autologous CAR-T Cell Therapies.","authors":"Tangying Lu, O. Pugach, R. Somerville, S. Rosenberg, J. Kochenderfer, M. Better, S. Feldman","doi":"10.1089/HGTB.2016.120","DOIUrl":"https://doi.org/10.1089/HGTB.2016.120","url":null,"abstract":"The treatment of B-cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials.1-6 The anti-CD19 CAR-T cell production method used to support initial trials relied on numerous manual, open process steps, human serum, and 10 days of cell culture to achieve a clinical dose.7 This approach limited the ability to support large multicenter clinical trials, as well as scale up for commercial cell production. Therefore, studies were completed to streamline and optimize the original National Cancer Institute production process by removing human serum from the process in order to minimize the risk of viral contamination, moving process steps from an open system to functionally closed system operations in order to minimize the risk of microbial contamination, and standardizing additional process steps in order to maximize process consistency. This study reports a procedure for generating CD19 CAR-T cells in 6 days, using a functionally closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 6 1","pages":"209-218"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/HGTB.2016.120","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"61230196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 42

Adeno-Associated Virus 5 Transduces Adipose-Derived Stem Cells with Greater Efficacy Than Other Adeno-Associated Viral Serotypes. 腺相关病毒5比其他腺相关病毒血清型更有效地转导脂肪来源的干细胞。

Human Gene Therapy Methods Pub Date : 2016-12-01 DOI: 10.1089/HGTB.2016.123

Priyanka Sharma, S. Wimalawansa, Gregory C. Gould, R. Johnson, K. Excoffon

{"title":"Adeno-Associated Virus 5 Transduces Adipose-Derived Stem Cells with Greater Efficacy Than Other Adeno-Associated Viral Serotypes.","authors":"Priyanka Sharma, S. Wimalawansa, Gregory C. Gould, R. Johnson, K. Excoffon","doi":"10.1089/HGTB.2016.123","DOIUrl":"https://doi.org/10.1089/HGTB.2016.123","url":null,"abstract":"Adipose-derived stem cells (ASCs) have shown potential in the treatment of a myriad of diseases; however, infusion of cells alone is unlikely to provide the full range of potential therapeutic applications. Transient genetic manipulation of ASCs could increase their repair and regeneration characteristics in a disease-specific context, essentially transforming them into drug-eluting depots. The goal of this study was to determine the optimal parameters necessary to transduce ASCs with recombinant adeno-associated virus (rAAV), an approved gene therapy vector that has never been associated with disease. Transduction and duration of gene expression of the most common recombinant AAV vectors were tested in this study. Among all tested serotypes, rAAV5 resulted in both the highest and longest term expression. Furthermore, we determined the glycosylation profile of ASCs before and after neuraminidase treatment and demonstrate that rAAV5 transduction requires plasma membrane-associated sialic acid. Future studies will focus on the optimization of gene delivery to ASCs, using rAAV5 as the vector of choice, to drive biological drug delivery, engraftment, and disease correction.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 6 1","pages":"219-227"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/HGTB.2016.123","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"61230323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products. 复制缺陷腺病毒载体的部分E3缺失允许潜在毒性转基因产物的稳定表达。

Human Gene Therapy Methods Pub Date : 2016-10-01 Epub Date: 2016-09-07 DOI: 10.1089/hgtb.2016.044

Larissa H Haut, Amanda L Gill, Raj K Kurupati, Ang Bian, Yan Li, Wynetta Giles-Davis, Zhiquan Xiang, Xiang Yang Zhou, Hildegund C J Ertl

{"title":"A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.","authors":"Larissa H Haut, Amanda L Gill, Raj K Kurupati, Ang Bian, Yan Li, Wynetta Giles-Davis, Zhiquan Xiang, Xiang Yang Zhou, Hildegund C J Ertl","doi":"10.1089/hgtb.2016.044","DOIUrl":"https://doi.org/10.1089/hgtb.2016.044","url":null,"abstract":"Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 5","pages":"187-196"},"PeriodicalIF":0.0,"publicationDate":"2016-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.044","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34424882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Optimization of Internally Deleted Dystrophin Constructs. 内部缺失肌营养不良蛋白结构的优化。

Human Gene Therapy Methods Pub Date : 2016-10-01 Epub Date: 2016-07-31 DOI: 10.1089/hgtb.2016.026

Mojgan Reza, Steve H Laval, Andreas Roos, Stephanie Carr, Hanns Lochmüller

{"title":"Optimization of Internally Deleted Dystrophin Constructs.","authors":"Mojgan Reza, Steve H Laval, Andreas Roos, Stephanie Carr, Hanns Lochmüller","doi":"10.1089/hgtb.2016.026","DOIUrl":"https://doi.org/10.1089/hgtb.2016.026","url":null,"abstract":"Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterized in relation to their structure and function in vivo. To optimize a dystrophin cDNA construct for therapeutic application we designed and produced four human minidystrophins within the packaging capacity of lentiviral vectors. Two novel minidystrophins retained the centrally located neuronal nitric oxide synthase (nNOS)-anchoring domain in order to achieve sarcolemmal nNOS restoration, which is lost in most internally deleted dystrophin constructs. Functionality of the resulting truncated dystrophin proteins was investigated in muscle of adult dystrophin-deficient mdx mice followed by a battery of detailed immunohistochemical and morphometric tests. This initial assessment aimed to determine the overall suitability of various constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 5","pages":"174-186"},"PeriodicalIF":0.0,"publicationDate":"2016-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.026","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34719721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice Cultures. 腺相关病毒载体在三维切片培养中转导成熟的人脂肪细胞。

Human Gene Therapy Methods Pub Date : 2016-10-01 DOI: 10.1089/HGTB.2016.137

KallendruschSonja, SchopowNikolas, C. StadlerSonja, BüningHildegard, T. HackerUlrich

引用次数: 1

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy 液滴数字PCR在干细胞基因治疗中估计载体拷贝数状态的应用

Human Gene Therapy Methods Pub Date : 2016-10-01 DOI: 10.1089/hgtb.2016.059

Huan-Ting Lin, T. Okumura, Yukino Yatsuda, S. Ito, H. Nakauchi, M. Otsu

{"title":"Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy","authors":"Huan-Ting Lin, T. Okumura, Yukino Yatsuda, S. Ito, H. Nakauchi, M. Otsu","doi":"10.1089/hgtb.2016.059","DOIUrl":"https://doi.org/10.1089/hgtb.2016.059","url":null,"abstract":"Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 1","pages":"197 - 208"},"PeriodicalIF":0.0,"publicationDate":"2016-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"61230048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 15

Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy. 优化一种用于人类基因治疗的质粒载体中宿主细胞DNA实时定量聚合酶链反应方法。

Human Gene Therapy Methods Pub Date : 2016-08-01 DOI: 10.1089/hgtb.2015.155

Serge Ferro, Isabelle Fabre, Xavier Chenivesse

{"title":"Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.","authors":"Serge Ferro, Isabelle Fabre, Xavier Chenivesse","doi":"10.1089/hgtb.2015.155","DOIUrl":"https://doi.org/10.1089/hgtb.2015.155","url":null,"abstract":"Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of \"contaminating\" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 4","pages":"159-70"},"PeriodicalIF":0.0,"publicationDate":"2016-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2015.155","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34689282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Debate on Germline Gene Editing. 关于生殖系基因编辑的争论。

Human Gene Therapy Methods Pub Date : 2016-08-01 DOI: 10.1089/hgtb.2016.28999.deb

Nathalie Cartier-Lacave, Robin Ali, Seppo Ylä-Herttuala, Kazuto Kato, Bernard Baetschi, Robin Lovell-Badge, Luigi Naldini, Adrian Thrasher

引用次数: 5

Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses. 高效低剂量AAV血清型载体的研制

Human Gene Therapy Methods Pub Date : 2016-08-01 DOI: 10.1089/hgtb.2016.054

Chen Ling, Baozheng Li, Wenqin Ma, Arun Srivastava

引用次数: 20

Evaluation of an Optimized Injection System for Retinal Gene Therapy in Human Patients. 一种用于人类视网膜基因治疗的优化注射系统的评价。

Human Gene Therapy Methods Pub Date : 2016-08-01 DOI: 10.1089/hgtb.2016.086

M Dominik Fischer, Doron G Hickey, Mandeep S Singh, Robert E MacLaren

{"title":"Evaluation of an Optimized Injection System for Retinal Gene Therapy in Human Patients.","authors":"M Dominik Fischer, Doron G Hickey, Mandeep S Singh, Robert E MacLaren","doi":"10.1089/hgtb.2016.086","DOIUrl":"https://doi.org/10.1089/hgtb.2016.086","url":null,"abstract":"Many retinal gene therapy clinical trials require subretinal injections of small volumes of adeno-associated viral (AAV) vector solutions in patients with retinal dystrophies, using equipment not specifically designed for this purpose. We therefore evaluated an optimized injection system in order to identify variables that might influence the rate of injection and final dose of vector delivered. An optimized injection system was assembled with a 41G polytetrafluoroethylene tip for retinal gene therapy. Flow rate was recorded at relevant infusion pressures (2-22 psi [14-152 kPa]), different target pressures (0.02-30 mm Hg [0.003-4 kPa]) and temperatures (18°C vs. 36°C) using a semiautomated Accurus(®) Surgical System. Retention of AAV2/8 and AAV2/8(Y733F) vector was quantified after simulating loading/injection with or without 0.001% Pluronic(®) F-68 (PF-68). The optimized injection system provided a linear flow rate (μl/s)-to-infusion pressure (psi) relationship (y = 0.62x; r(2) = 0.99), independent of temperature and pressure changes relevant for intraocular surgery (18-36°C, 0.02-30 mm Hg). Differences in length of 41G polytetrafluoroethylene tips caused significant variation in flow rate (p < 0.001). Use of PF-68 significantly (p < 0.001) reduced loss of vector genomes in the injection system by 55% (AAV2/8) and 52% (AAV2/8(Y733F)). A customized subretinal injection system assembled using equipment currently available in the operating room can deliver a controlled volume of vector at a fixed rate across a range of possible clinical parameters encountered in vitreoretinal surgery. The inclusion of 0.001% PF-68 had a significant effect on the final dose of vector genomes delivered. The described technique is currently used successfully in a clinical trial.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"27 4","pages":"150-8"},"PeriodicalIF":0.0,"publicationDate":"2016-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.086","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34721701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 49