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.

Figure 1

Generation of free ends for the plasmid inverted terminal repeats (ITRs). (a) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. (b) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. (c) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. (d) Plasmid DNA purity and concentration measured by spectrophotometry.

Figure 2

ITR2 quantitative polymerase chain reaction (qPCR) using psub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats (ITRs) with free ends. (a) The differences between mean quantification cycles (C_q) obtained in each experimental condition with an equal amount of plasmid quantified by spectrophotometry are shown. (b) r-square, slope, and intersection values of the qPCR tests shown in a).

Figure 3

Quantitative polymerase chain reaction (qPCR) targeting the rep sequence using pSub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats with free ends. (a) The minimal differences observed between mean quantification cycles (C_q) obtained in each experimental condition indicate that the DNA concentrations determined by spectrophotometry are similar. (b) r-square, slope, and intersection values of the qPCR tests shown in a.

Figure 4

Design of AAV5 inverted terminal repeats (ITR) quantitative polymerase chain reaction (qPCR). (a) Top: 5’ ITR secondary hairpin structure of wild-type AAV5 and localization of the AAV5 ITR qPCR-specific primers and probe. Bottom: Corresponding 71-bp PCR product. (b) ITR5 qPCR using pAAVPGK plasmid linearized with ScaI, or digested with KpnI to create ITRs with free ends. The differences between mean quantification cycles (C_q) obtained in each experimental condition with an equal amount of plasmid quantified by spectrophotometry are shown. (c) r-square, slope, and intersection values of the qPCR tests shown in a. (d) Titration of an rAAV5 vector sample using the two different linearized plasmids (ITR5 versus free-ITR5). The mean and standard deviations listed for rAAV5 vectors are from four different dilutions of the samples. *P < 0.05.