Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR - Inserm - Institut national de la santé et de la recherche médicale Access content directly
Journal Articles Molecular Therapy - Methods and Clinical Development Year : 2016

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.
Fichier principal
Vignette du fichier
DCosta 2016.pdf (757.9 Ko) Télécharger le fichier
Origin : Publication funded by an institution
Loading...

Dates and versions

inserm-01799010 , version 1 (24-05-2018)

Identifiers

Cite

Susan d'Costa, Véronique Blouin, Frederic Broucque, Magalie Penaud-Budloo, Achille François, et al.. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Molecular Therapy - Methods and Clinical Development, 2016, 5, pp.16019. ⟨10.1038/mtm.2016.19⟩. ⟨inserm-01799010⟩
115 View
208 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More