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.

Distinct in vivo roles of secreted APP ectodomain variants APP sα and APP sβ in regulation of spine density, synaptic plasticity, and cognition

“…72 h following transfection, virions were purified and concentrated from cell lysate and supernatant by ultracentrifugation on a iodixanol density gradient followed by buffer exchange to 0.01% pluronic/phosphate-buffered saline (PBS) via a 100 kDa Amicon centrifugal filter unit. Genome copies in the vector stocks were determined by free inverted terminal repeat (ITR)-specific quantitative TaqMan PCR and expressed as genomic copies per ll of concentrated stocks (gc/ll) as described (D'Costa et al, 2016).…”

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

“…We decided to use AAV8RSM for validation because its titer is higher and also because it has been extensively characterized. Many details were included in a series of publications 26, 32, 38, 42 , making it possible to compare our data with the literature values.…”

“…It is highly sensitive to experimental conditions, making it susceptible to errors. Factors such as PCR primers, reagents, equipment and DNA standards etc., can all significantly influence the test results 25, 26 . Because of that, significant inter- and intra- laboratory variations were often reported 27 .…”

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

“…Vector genomes were quantified in crude cell lysates by free-ITR quantitative PCR (qPCR) (48) and baculovirus genomes by a qPCR targeting the baculoviral DNA polymerase gene using primers Bac-F and Bac-R (Data Set S2) and probe FAM-CAA ACA CGC GCA TTA ACG AGA GCA CC-TAMRA. Assembled AAV2 capsid titers were determined in crude cell lysates using the A20 ELISA kit (Pratv; Progen Biotechnik) according to the manufacturer's manual.…”