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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Main Authors: 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
Format: Article
Language:English
Published: Elsevier 2020-06-01
Series:Molecular Therapy: Methods & Clinical Development
Subjects:
Online Access:http://www.sciencedirect.com/science/article/pii/S2329050120300371
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language English
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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