Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors
Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov...
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Format: | Article |
Language: | English |
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Elsevier
2020-06-01
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Series: | Molecular Therapy: Methods & Clinical Development |
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Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050120300371 |
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doaj-b38a05093c5a4598a4a393131cc871b5 |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Gai Ran Xiao Chen Yilin Xie Qingyun Zheng Jinyan Xie Chenghui Yu Nikea Pittman Sixian Qi Fa-Xing Yu Mavis Agbandje-McKenna Arun Srivastava Chen Ling |
spellingShingle |
Gai Ran Xiao Chen Yilin Xie Qingyun Zheng Jinyan Xie Chenghui Yu Nikea Pittman Sixian Qi Fa-Xing Yu Mavis Agbandje-McKenna Arun Srivastava Chen Ling Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors Molecular Therapy: Methods & Clinical Development gene therapy rAAV vector library selection site-directed mutagenesis transduction efficiency vector distribution |
author_facet |
Gai Ran Xiao Chen Yilin Xie Qingyun Zheng Jinyan Xie Chenghui Yu Nikea Pittman Sixian Qi Fa-Xing Yu Mavis Agbandje-McKenna Arun Srivastava Chen Ling |
author_sort |
Gai Ran |
title |
Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors |
title_short |
Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors |
title_full |
Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors |
title_fullStr |
Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors |
title_full_unstemmed |
Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors |
title_sort |
site-directed mutagenesis improves the transduction efficiency of capsid library-derived recombinant aav vectors |
publisher |
Elsevier |
series |
Molecular Therapy: Methods & Clinical Development |
issn |
2329-0501 |
publishDate |
2020-06-01 |
description |
Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy. |
topic |
gene therapy rAAV vector library selection site-directed mutagenesis transduction efficiency vector distribution |
url |
http://www.sciencedirect.com/science/article/pii/S2329050120300371 |
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spelling |
doaj-b38a05093c5a4598a4a393131cc871b52020-11-25T03:26:01ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-06-0117545555Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV VectorsGai Ran0Xiao Chen1Yilin Xie2Qingyun Zheng3Jinyan Xie4Chenghui Yu5Nikea Pittman6Sixian Qi7Fa-Xing Yu8Mavis Agbandje-McKenna9Arun Srivastava10Chen Ling11State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaDepartment of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32611, USA; Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL 32611, USAInstitute of Pediatrics, Children’s Hospital of Fudan University, Shanghai 201102, ChinaInstitute of Pediatrics, Children’s Hospital of Fudan University, Shanghai 201102, China; Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, ChinaDepartment of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32611, USA; Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL 32611, USA; Genetics Institute, University of Florida College of Medicine, Gainesville, FL 32611, USAPowell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL 32611, USA; Genetics Institute, University of Florida College of Medicine, Gainesville, FL 32611, USA; Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32611, USA; Department of Molecular Genetics and Microbiology; Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32611, USA; Corresponding author: Arun Srivastava, Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, 2033 Mowry Road, Room 492-A, Gainesville, FL 32610-3633, USA.State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China; Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32611, USA; Corresponding author: Chen Ling, State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Room C419, 2005 Songhu Road, Shanghai 200438, China.Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.http://www.sciencedirect.com/science/article/pii/S2329050120300371gene therapyrAAV vectorlibrary selectionsite-directed mutagenesistransduction efficiencyvector distribution |