Human Gene Therapy Methods最新文献_第8页

Suppression of Choroidal Neovascularization in Mice by Subretinal Delivery of Multigenic Lentiviral Vectors Encoding Anti-Angiogenic MicroRNAs. 通过视网膜下递送编码抗血管生成microrna的多基因慢病毒载体抑制小鼠脉络膜新生血管。

Human Gene Therapy Methods Pub Date : 2017-08-01 DOI: 10.1089/hgtb.2017.079

Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, Lars Aagaard, Toke Bek, Jacob Giehm Mikkelsen, Thomas Juhl Corydon

{"title":"Suppression of Choroidal Neovascularization in Mice by Subretinal Delivery of Multigenic Lentiviral Vectors Encoding Anti-Angiogenic MicroRNAs.","authors":"Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, Lars Aagaard, Toke Bek, Jacob Giehm Mikkelsen, Thomas Juhl Corydon","doi":"10.1089/hgtb.2017.079","DOIUrl":"https://doi.org/10.1089/hgtb.2017.079","url":null,"abstract":"Lentivirus-based vectors have been used for the development of potent gene therapies. Here, application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model is presented. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells. Six days post injection (PI), robust and widespread fluorescent signals of eGFP are already observed in the retina by funduscopy. The eGFP expression peaks at day 21 PI and persists with stable expression for at least 9 months. In parallel, prominent AsRED co-expression, encoded from the VMD2-driven microRNA expression cassette, is evident in retinal sections and flat-mounts, revealing RPE-specific expression of microRNAs. Furthermore, LV-delivered microRNAs targeting the Vegfa gene in RPE cells reduced the size of laser-induced CNV in mice 28 days PI, as a consequence of diminished VEGF levels, suggesting that LVs delivered locally are powerful tools in the development of gene therapy-based strategies for treatment of age-related macular degeneration.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 4","pages":"222-233"},"PeriodicalIF":0.0,"publicationDate":"2017-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2017.079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35329241","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: 19

Suppression of choroidal neovascularization in mice by subretinal delivery of multigenic lentiviral vectors encoding anti-angiogenic microRNAs. 通过视网膜下递送编码抗血管生成小rna的多基因慢病毒载体抑制小鼠脉络膜新生血管。

Human Gene Therapy Methods Pub Date : 2017-07-27 DOI: 10.1089/hum.2017.079

Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, L. Aagaard, T. Bek, J. Mikkelsen, T. Corydon

{"title":"Suppression of choroidal neovascularization in mice by subretinal delivery of multigenic lentiviral vectors encoding anti-angiogenic microRNAs.","authors":"Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, L. Aagaard, T. Bek, J. Mikkelsen, T. Corydon","doi":"10.1089/hum.2017.079","DOIUrl":"https://doi.org/10.1089/hum.2017.079","url":null,"abstract":"Lentivirus-based vectors have been used for the development of potent gene therapies. Here, we present application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA, and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells. Already 6 days post injection (PI) robust and widespread fluorescent signals of eGFP are observed in the retina by fundoscopy. The eGFP expression peaks at day 21 PI and persists with stable expression for at least 9 months. In parallel, prominent AsRED co-expression, encoded from the VMD2-driven microRNA expression cassette, is evident in retinal sections and flat-mounts, revealing RPE-specific expression of microRNAs. Furthermore, LV-delivered microRNAs targeting the Vegfa gene in RPE cells reduced the size of laser-induced CNV in mice 28 days PI, as a consequence of diminished VEGF levels, suggesting that LVs delivered locally are powerful tools in the development of gene therapy-based strategies for treatment of age-related macular degeneration (AMD).","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hum.2017.079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42762720","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: 6

(RT)-qPCR for detection of and differentiation between RNA and DNA of HIV-1-based lentiviral vectors. (RT)-qPCR用于hiv -1慢病毒载体RNA和DNA的检测和分化。

Human Gene Therapy Methods Pub Date : 2017-07-27 DOI: 10.1089/hgtb.2017.081

M. Pavlovic, Nina Koehler, M. Anton, Anna Dinkelmeier, Maren Haase, Thorsten Stellberger, U. Busch, A. Baiker

引用次数: 2

Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities. 提高腺相关病毒载体制备的质量:产品相关杂质的挑战。

Human Gene Therapy Methods Pub Date : 2017-06-01 Epub Date: 2017-04-17 DOI: 10.1089/hgtb.2016.188

Maria Schnödt, Hildegard Büning

{"title":"Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities.","authors":"Maria Schnödt, Hildegard Büning","doi":"10.1089/hgtb.2016.188","DOIUrl":"https://doi.org/10.1089/hgtb.2016.188","url":null,"abstract":"Adeno-associated viral (AAV) vectors have emerged as one of the most popular gene transfer systems in both research and clinical gene therapy. As AAV vectors are derived from a stealth, nonpathogenic virus and lack active integrase activity, these vectors are frequently applied for in vivo gene therapy of liver, muscle, and other postmitotic tissues. Although long-term transgene expression from AAV vector episomes is reported from these tissues, the episomal nature of AAV-once regarded as disadvantage-has become an attractive feature for gene-editing approaches targeting proliferating cells. In response to the high demand, AAV vector production is receiving special attention. Besides particle yields and biological activity, the most important concern is improving vector purity. The most difficult task in this regard is removal of defective particles, that is, capsids that are either empty or contain DNA other than the full-length vector genomes. Herein, we characterize and discuss these so-called product-related impurities, methods for their detection, as well as strategies to avoid or reduce their formation.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 3","pages":"101-108"},"PeriodicalIF":0.0,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.188","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34837848","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

A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate. 对粗裂解物中重组腺相关病毒的载体基因组进行定量的可扩展的精确方法。

Human Gene Therapy Methods Pub Date : 2017-06-01 Epub Date: 2017-04-13 DOI: 10.1089/hgtb.2016.173

Jianzhong Ai, Raed Ibraheim, Phillip W L Tai, Guangping Gao

{"title":"A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate.","authors":"Jianzhong Ai, Raed Ibraheim, Phillip W L Tai, Guangping Gao","doi":"10.1089/hgtb.2016.173","DOIUrl":"10.1089/hgtb.2016.173","url":null,"abstract":"Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in crude lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to crude lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 3","pages":"139-147"},"PeriodicalIF":0.0,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488319/pdf/hgtb.2016.173.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34982653","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: 0

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines. aavs1靶质粒在AAV产生细胞系中的整合

Human Gene Therapy Methods Pub Date : 2017-06-01 Epub Date: 2017-04-14 DOI: 10.1089/hgtb.2016.158

Yuxia Luo, Amy Frederick, John M Martin, Abraham Scaria, Seng H Cheng, Donna Armentano, Samuel C Wadsworth, Karen A Vincent

{"title":"AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.","authors":"Yuxia Luo, Amy Frederick, John M Martin, Abraham Scaria, Seng H Cheng, Donna Armentano, Samuel C Wadsworth, Karen A Vincent","doi":"10.1089/hgtb.2016.158","DOIUrl":"https://doi.org/10.1089/hgtb.2016.158","url":null,"abstract":"Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 3","pages":"124-138"},"PeriodicalIF":0.0,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.158","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34996911","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

Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells. 利用新一代测序技术对昆虫细胞中腺相关病毒载体的DNA种类进行准确鉴定和定量。

Human Gene Therapy Methods Pub Date : 2017-06-01 Epub Date: 2017-04-21 DOI: 10.1089/hgtb.2016.185

Magalie Penaud-Budloo, Emilie Lecomte, Aurélien Guy-Duché, Sylvie Saleun, Alain Roulet, Céline Lopez-Roques, Benoît Tournaire, Benjamin Cogné, Adrien Léger, Véronique Blouin, Pierre Lindenbaum, Philippe Moullier, Eduard Ayuso

{"title":"Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells.","authors":"Magalie Penaud-Budloo, Emilie Lecomte, Aurélien Guy-Duché, Sylvie Saleun, Alain Roulet, Céline Lopez-Roques, Benoît Tournaire, Benjamin Cogné, Adrien Léger, Véronique Blouin, Pierre Lindenbaum, Philippe Moullier, Eduard Ayuso","doi":"10.1089/hgtb.2016.185","DOIUrl":"https://doi.org/10.1089/hgtb.2016.185","url":null,"abstract":"Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (≤0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 3","pages":"148-162"},"PeriodicalIF":0.0,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.185","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34959339","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: 37

CAR T-Cell Therapy: Progress and Prospects. CAR - t细胞治疗:进展和前景。

Human Gene Therapy Methods Pub Date : 2017-04-01 DOI: 10.1089/hgtb.2016.153

Olivia Wilkins, Allison M Keeler, Terence R Flotte

{"title":"CAR T-Cell Therapy: Progress and Prospects.","authors":"Olivia Wilkins, Allison M Keeler, Terence R Flotte","doi":"10.1089/hgtb.2016.153","DOIUrl":"https://doi.org/10.1089/hgtb.2016.153","url":null,"abstract":"Lentivirus-mediated transduction of autologous T cells with a chimeric antigen receptor (CAR) to confer a desired epitope specificity as a targeted immunotherapy for cancer has been among the first human gene therapy techniques to demonstrate widespread therapeutic efficacy. Other approaches to using gene therapy to enhance antitumor immunity have been less specific and less effective. These have included amplification, marking, and cytokine transduction of tumor infiltrating lymphocytes, recombinant virus-based expression of tumor antigens as a tumor vaccine, and transduction of antigen-presenting cells with tumor antigens. Unlike any of those methods, the engineering of CAR T cells combine specific monoclonal antibody gene sequences to confer epitope specificity and other T-cell receptor and activation domains to create a self-contained single vector approach to produce a very specific antitumor response, as is seen with CD19-directed CAR T cells used to treat CD19-expressing B-cell malignancies. Recent success with these therapies is the culmination of a long step-wise iterative process of improvement in the design of CAR vectors. This review aims to summarize this long series of advances in the development of effective CAR vector since their initial development in the 1990s, and to describe emerging approaches to design that promise to enhance and widen the human gene therapy relevance of CAR T-cell therapy in the future.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 2","pages":"61-66"},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.153","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34844439","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: 55

RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells. 靶向内源性β-珠蛋白前信使RNA的RNA反式剪接

Human Gene Therapy Methods Pub Date : 2017-04-01 Epub Date: 2017-02-14 DOI: 10.1089/hgtb.2016.077

Naoya Uchida, Kareem N Washington, Brian Mozer, Charlotte Platner, Josiah Ballantine, Luke P Skala, Lydia Raines, Anna Shvygin, Matthew M Hsieh, Lloyd G Mitchell, John F Tisdale

{"title":"RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.","authors":"Naoya Uchida, Kareem N Washington, Brian Mozer, Charlotte Platner, Josiah Ballantine, Luke P Skala, Lydia Raines, Anna Shvygin, Matthew M Hsieh, Lloyd G Mitchell, John F Tisdale","doi":"10.1089/hgtb.2016.077","DOIUrl":"https://doi.org/10.1089/hgtb.2016.077","url":null,"abstract":"Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 2","pages":"91-99"},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.077","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34789119","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: 4

Improvement of De Novo Cholesterol Biosynthesis Efficiently Promotes the Production of Human Immunodeficiency Virus Type 1-Derived Lentiviral Vectors. 新生胆固醇生物合成的改善有效促进人类免疫缺陷病毒1型衍生慢病毒载体的产生。

Human Gene Therapy Methods Pub Date : 2017-04-01 DOI: 10.1089/hgtb.2016.150

N. Holic, Sophie Frin, A. Seye, Anne Galy, D. Fenard

{"title":"Improvement of De Novo Cholesterol Biosynthesis Efficiently Promotes the Production of Human Immunodeficiency Virus Type 1-Derived Lentiviral Vectors.","authors":"N. Holic, Sophie Frin, A. Seye, Anne Galy, D. Fenard","doi":"10.1089/hgtb.2016.150","DOIUrl":"https://doi.org/10.1089/hgtb.2016.150","url":null,"abstract":"The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+ hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.","PeriodicalId":13126,"journal":{"name":"Human Gene Therapy Methods","volume":"28 2 1","pages":"67-77"},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/hgtb.2016.150","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"61230120","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