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Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras.


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.

SUBMITTER: Tai PWL 

PROVIDER: S-EPMC5948225 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras.

Tai Phillip W L PWL   Xie Jun J   Fong Kaiyuen K   Seetin Matthew M   Heiner Cheryl C   Su Qin Q   Weiand Michael M   Wilmot Daniella D   Zapp Maria L ML   Gao Guangping G  

Molecular therapy. Methods & clinical development 20180213


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 t  ...[more]

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