Molecular Determination of High-Capacity Helper Dependent Adenoviral Vector Genomes In Vitro and In Vivo

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

Baltimore, Mariland, USA

Congreso; American Society of Gene Therapy; 2006

Molecular Determination of High-CapacityHelper Dependent Adenoviral Vector Genomes InVitro and In VivoMariana Puntel,1,2 James F. Curtin,1,2 Jeffrey M. Zirger,1,2 A .K.M. Muhammad,1,2 Xiong Weidong,1,2 Chunyan Liu,1,2 JinweiHu,1,2 Kurt M. Kroeger,1,2 Peter Czer,1,2 Sandra Sciascia,1,2 PedroR. Lowenstein,1,2 Maria G. Castro.1,21Gene Therapeutics Research Institute, Cedars-Sinai MedicalCenter, Los Angeles, CA; 2Department of Medicine andDepartment of Medical and Molecular Pharmacology, Universityof California Los Angeles, Los Angeles, CA.First generation (RAd) and high capacity (HC-Ad) adenoviralvectors are efficient delivery vehicles for transferring therapeutictransgenes in vivo into tissues/organs. The initial successes reportedusing adenoviral vectors in preclinical trials have been limited byimmune related adverse side effects. This has been, in part, attributedto the use of poorly characterized preparations of adenoviral vectorsand also to the untoward immune adverse side effects elicited whenhigh doses of these vectors were used. HC-Ads have severaladvantages over RAds, including the lack of viral coding sequences,which after infection and uncoating, makes them invisible to thehost’s immune system. Another advantage is their large cloningcapacity (up to ∼35Kb). However, accurate characterization of HCAdvectors, and contaminating replication competent adenovirus(RCA) or helper virus is necessary before these preparations can beused safely in clinical trials. Consequently, the development ofaccurate, simple and reproducible methods to standardize andvalidate adenoviral preparations for the presence of contaminantgenomes is required. Using a molecular method that allows accurate,reproducible and simultaneous determination of HC-Ad,contaminating helper virus and RCA genome copy numbers basedon real time quantitative PCR (qPCR), we demonstrate accuratedetection of these three genomic entities, within CsCl purified vectorstocks, total DNA isolated from cells transduced in vitro, and frombrain tissue infected in vivo. This approach will allow accurateassessment of the levels and biodistribution of HC-Ad and improvethe safety and efficacy of clinical trials.