Quantification of high-capacity helper dependent adenoviral vector genomes in vitro and in vivo using quantitative TaqMan real time polymerase chain reaction (qPCR)

PUNTEL; CURTIN; ZIRGER; MUHAMMAD; XIONG; LIU; HU; KROEGER; CZER; SCIASCIA; MONDKAR; LOWENSTEIN; CASTRO

HUMAN GENE THERAPY

MARY ANN LIEBERT INC

Año: 2006 p. 531 - 544

1043-0342

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehiclesfor transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviralvectors in preclinical trials have been limited by immune-related adverse side effects. This has been, inpart, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untowardimmune adverse side effects elicited when high doses of these vectors were used. HC-Ads have severaladvantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makesthem invisible to the host’s immune system. Another advantage is their large cloning capacity (up to 35 kb).However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus(RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently,the development of accurate, simple, and reproducible methods to standardize and validate adenoviralpreparations for the presence of contaminant genomes is required. By using a molecular method thatallows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, andRCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of thesethree genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro,and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistributionof HC-Ad and improve the safety and efficacy of clinical trials.