title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.
abstract: Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.
authors.affiliation: Center of Excellence for Regenerative Health Biotechnology, University of Florida , Alachua, Florida, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: D'Costa
authors.affiliation: Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes , Nantes, France.
authors.forename: Veronique
authors.initials: V
authors.lastname: Blouin
authors.affiliation: Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes , Nantes, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Broucque
authors.affiliation: Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes , Nantes, France.
authors.forename: Magalie
authors.initials: M
authors.lastname: Penaud-Budloo
authors.affiliation: Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes , Nantes, France.
authors.forename: Achille
authors.initials: A
authors.lastname: François
authors.affiliation: Center of Excellence for Regenerative Health Biotechnology, University of Florida , Alachua, Florida, USA.
authors.forename: Irene C
authors.initials: IC
authors.lastname: Perez
authors.affiliation: Genethon , Evry, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Le Bec
authors.affiliation: Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes, Nantes, France; Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, Florida, USA.
authors.forename: Philippe
authors.initials: P
authors.lastname: Moullier
authors.affiliation: Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, Florida, USA; Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes, Nantes, France; Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, Florida, USA.
authors.forename: Richard O
authors.initials: RO
authors.lastname: Snyder
authors.affiliation: Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes , Nantes, France.
authors.forename: Eduard
authors.initials: E
authors.lastname: Ayuso