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Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing

Yip, Mitchell
Chen, Jing
Zhi, Yan
Tran, Ngoc Tam
Namkung, Suk
Pastor, Eric
Gao, Guangping
Tai, Phillip W L
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Abstract

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 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 - 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 / plasmid occurs under several events and rcAAVs spawn from diverse clones.

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Yip M, Chen J, Zhi Y, Tran NT, Namkung S, Pastor E, Gao G, Tai PWL. Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing. Viruses. 2023 May 24;15(6):1228. doi: 10.3390/v15061228. PMID: 37376529; PMCID: PMC10305535.

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10.3390/v15061228
PubMed ID
37376529
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Attribution 4.0 International