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
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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 PW, Xie J, Fong K, Seetin M, Heiner C, Su Q, Weiand M, Wilmot D, Zapp ML, Gao G. (2018). Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras. Open Access Articles. https://doi.org/10.1016/j.omtm.2018.02.002. Retrieved from https://escholarship.umassmed.edu/oapubs/3497
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.