| Autor: |
Phillip W.L. Tai, Jun Xie, Kaiyuen Fong, Matthew Seetin, Cheryl Heiner, Qin Su, Michael Weiand, Daniella Wilmot, Maria L. Zapp, Guangping Gao |
| Jazyk: |
angličtina |
| Rok vydání: |
2018 |
| Předmět: |
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| Zdroj: |
Molecular Therapy: Methods & Clinical Development, Vol 9, Iss , Pp 130-141 (2018) |
| Druh dokumentu: |
article |
| ISSN: |
2329-0501 |
| DOI: |
10.1016/j.omtm.2018.02.002 |
| Popis: |
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, recombinant adeno-associated virus, single molecule real-time sequencing, rAAV-ITR, gene therapy vector QC |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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