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

Automated summary

Clinical-grade AAV vectors for gene therapy undergo diagnostic tests to determine their quality, including the detection of DNA contaminants. One type of contaminant, rcAAVs, are replicative and potentially infectious viral particles formed through recombination events. A commonly used method to detect rcAAVs involves passaging transduced cells and using qPCR to detect the rep gene. However, this method is unable to determine the diversity of recombination events or provide insights into rcAAV formation. Through SMRT sequencing, we analyzed expanded virus-like genomes from rcAAV-positive preparations and found evidence of sequence-independent and non-homologous recombination events leading to the formation of diverse rcAAV clones.