UMMS Affiliation

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

Publication Date

2018-06-15

Document Type

Article

Disciplines

Bioinformatics | Genetics and Genomics | Therapeutics

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.

Keywords

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

10.1016/j.omtm.2018.02.002

Source

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

Journal/Book/Conference Title

Molecular therapy. Methods and clinical development

Related Resources

Link to Article in PubMed

PubMed ID

29766023

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.