Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing
Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAV...
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MDPI AG
2023-05-01
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Online Access: | https://www.mdpi.com/1999-4915/15/6/1228 |
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author | Mitchell Yip Jing Chen Yan Zhi Ngoc Tam Tran Suk Namkung Eric Pastor Guangping Gao Phillip W. L. Tai |
author_facet | Mitchell Yip Jing Chen Yan Zhi Ngoc Tam Tran Suk Namkung Eric Pastor Guangping Gao Phillip W. L. Tai |
author_sort | Mitchell Yip |
collection | DOAJ |
description | Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the <i>rep</i> gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the <i>rep</i>-<i>cap</i> genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the <i>rep</i>/<i>cap</i> plasmid occurs under several events and rcAAVs spawn from diverse clones. |
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issn | 1999-4915 |
language | English |
last_indexed | 2024-03-11T01:50:29Z |
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series | Viruses |
spelling | doaj.art-e0893a9bb4f240999ab3a9fbe1ce30852023-11-18T13:00:48ZengMDPI AGViruses1999-49152023-05-01156122810.3390/v15061228Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time SequencingMitchell Yip0Jing Chen1Yan Zhi2Ngoc Tam Tran3Suk Namkung4Eric Pastor5Guangping Gao6Phillip W. L. Tai7Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA 01605, USASpirovant Sciences, Inc., Philadelphia, PA 19104, USASpirovant Sciences, Inc., Philadelphia, PA 19104, USAHorae Gene Therapy Center, UMass Chan Medical School, Worcester, MA 01605, USAHorae Gene Therapy Center, UMass Chan Medical School, Worcester, MA 01605, USASpirovant Sciences, Inc., Philadelphia, PA 19104, USAHorae Gene Therapy Center, UMass Chan Medical School, Worcester, MA 01605, USAHorae Gene Therapy Center, UMass Chan Medical School, Worcester, MA 01605, USAClinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the <i>rep</i> gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the <i>rep</i>-<i>cap</i> genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the <i>rep</i>/<i>cap</i> plasmid occurs under several events and rcAAVs spawn from diverse clones.https://www.mdpi.com/1999-4915/15/6/1228adeno-associated virusreplication-competent AAVsingle moleculereal-time sequencingAAV-genome population sequencing |
spellingShingle | Mitchell Yip Jing Chen Yan Zhi Ngoc Tam Tran Suk Namkung Eric Pastor Guangping Gao Phillip W. L. Tai Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing Viruses adeno-associated virus replication-competent AAV single molecule real-time sequencing AAV-genome population sequencing |
title | Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing |
title_full | Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing |
title_fullStr | Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing |
title_full_unstemmed | Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing |
title_short | Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing |
title_sort | querying recombination junctions of replication competent adeno associated viruses in gene therapy vector preparations with single molecule real time sequencing |
topic | adeno-associated virus replication-competent AAV single molecule real-time sequencing AAV-genome population sequencing |
url | https://www.mdpi.com/1999-4915/15/6/1228 |
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