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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Main Authors: Mitchell Yip, Jing Chen, Yan Zhi, Ngoc Tam Tran, Suk Namkung, Eric Pastor, Guangping Gao, Phillip W. L. Tai
Format: Article
Language:English
Published: MDPI AG 2023-05-01
Series:Viruses
Subjects:
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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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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