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Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras
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 mo...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948225/ https://www.ncbi.nlm.nih.gov/pubmed/29766023 http://dx.doi.org/10.1016/j.omtm.2018.02.002 |
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author | Tai, Phillip W.L. Xie, Jun Fong, Kaiyuen Seetin, Matthew Heiner, Cheryl Su, Qin Weiand, Michael Wilmot, Daniella Zapp, Maria L. Gao, Guangping |
author_facet | Tai, Phillip W.L. Xie, Jun Fong, Kaiyuen Seetin, Matthew Heiner, Cheryl Su, Qin Weiand, Michael Wilmot, Daniella Zapp, Maria L. Gao, Guangping |
author_sort | Tai, Phillip W.L. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5948225 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
spelling | pubmed-59482252018-05-14 Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras Tai, Phillip W.L. Xie, Jun Fong, Kaiyuen Seetin, Matthew Heiner, Cheryl Su, Qin Weiand, Michael Wilmot, Daniella Zapp, Maria L. Gao, Guangping Mol Ther Methods Clin Dev Article 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. American Society of Gene & Cell Therapy 2018-02-13 /pmc/articles/PMC5948225/ /pubmed/29766023 http://dx.doi.org/10.1016/j.omtm.2018.02.002 Text en © 2018 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Article Tai, Phillip W.L. Xie, Jun Fong, Kaiyuen Seetin, Matthew Heiner, Cheryl Su, Qin Weiand, Michael Wilmot, Daniella Zapp, Maria L. Gao, Guangping Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras |
title | Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras |
title_full | Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras |
title_fullStr | Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras |
title_full_unstemmed | Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras |
title_short | Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras |
title_sort | adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948225/ https://www.ncbi.nlm.nih.gov/pubmed/29766023 http://dx.doi.org/10.1016/j.omtm.2018.02.002 |
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