Horae Gene Therapy Center; Li Weibo Institute for Rare Diseases Research; Department of Microbiology and Physiological Systems; Viral Vector Core; Program in Molecular Medicine; Center for AIDS Research
Bioinformatics | Genetics and Genomics | Therapeutics
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.
AAV-GPseq, gene therapy vector QC, rAAV-ITR, recombinant adeno-associated virus, single molecule real-time sequencing
Rights and Permissions
Copyright © 2018 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
DOI of Published Version
Mol Ther Methods Clin Dev. 2018 June 15;9:130-141. doi: 10.1016/j.omtm.2018.02.002. eCollection 2018 Jun 15. Link to article on publisher's site
Molecular therapy. Methods and clinical development
Tai, Phillip W. L.; Xie, Jun; Fong, Kaiyuen; Seetin, Matthew; Heiner, Cheryl; Su, Qin; Weiand, Michael; Wilmot, Daniella; Zapp, Maria L.; and Gao, Guangping, "Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras" (2018). Open Access Articles. 3497.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.