Title |
Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras
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Published in |
Molecular Therapy - Methods & Clinical Development, February 2018
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DOI | 10.1016/j.omtm.2018.02.002 |
Pubmed ID | |
Authors |
Phillip W.L. Tai, Jun Xie, Kaiyuen Fong, Matthew Seetin, Cheryl Heiner, Qin Su, Michael Weiand, Daniella Wilmot, Maria L. Zapp, Guangping 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)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors. |
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Geographical breakdown
Country | Count | As % |
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United States | 4 | 24% |
Japan | 1 | 6% |
Austria | 1 | 6% |
Russia | 1 | 6% |
Switzerland | 1 | 6% |
United Kingdom | 1 | 6% |
Unknown | 8 | 47% |
Demographic breakdown
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Members of the public | 9 | 53% |
Scientists | 8 | 47% |
Mendeley readers
Geographical breakdown
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Unknown | 177 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 54 | 31% |
Student > Ph. D. Student | 27 | 15% |
Other | 11 | 6% |
Student > Bachelor | 9 | 5% |
Student > Master | 8 | 5% |
Other | 9 | 5% |
Unknown | 59 | 33% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 51 | 29% |
Agricultural and Biological Sciences | 31 | 18% |
Immunology and Microbiology | 7 | 4% |
Engineering | 7 | 4% |
Chemical Engineering | 7 | 4% |
Other | 17 | 10% |
Unknown | 57 | 32% |