Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras

Molecular Therapy. Methods & Clinical Development
Phillip W L TaiGuangping Gao

Abstract

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-conta...Continue Reading

Citations

Jul 2, 2019·Human Molecular Genetics·Claire Domenger, Dirk Grimm
Nov 23, 2019·Human Gene Therapy Methods·Patrick WilmottGrant Logan
Feb 3, 2019·Nature Reviews. Drug Discovery·Dan WangGuangping Gao
Feb 10, 2021·Signal Transduction and Targeted Therapy·Jote T BulchaGuangping Gao
Aug 11, 2020·Molecular Therapy. Methods & Clinical Development·Ngoc Tam TranPhillip W L Tai
Mar 20, 2021·Molecular Therapy. Methods & Clinical Development·Andreas L GimpelRichard D Braatz
Aug 31, 2021·Molecular Therapy : the Journal of the American Society of Gene Therapy·Dhwanil A DalwadiMarkus Grompe
Oct 7, 2021·Gene Therapy·Xiufang PanDongsheng Duan

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Datasets Mentioned

BETA
PRJNA383145

Methods Mentioned

BETA
transfection
electrophoresis
AAV-GPseq
ChIP-seq
PCR

Software Mentioned

bam2fastq
Galaxy
plot
Integrative Genomes Viewer ( IGV )
AAV
GPseq
SMRT Analysis
SMRT
SMRTbell
R package

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