Quantitative and Digital Droplet-Based AAV Genome Titration

Abstract

The adeno-associated viral vector (AAV) platform has developed into a primary modality for efficient in vivo, and in more limited settings, in vitro or ex vivo gene transfer. Its applications range from a tool for experimental purposes to preclinical and clinical gene therapy. The ability to accurately and reproducibly quantify vector concentration is critical for any of these applications. While several quantification assays are available, here we outline a detailed protocol for the quantification of DNase-I protected vector genomes reliant on the polymerase chain reaction (PCR) as a measure of the active component of the vector, namely its transgene cargo. With the emergence of droplet digital PCR (ddPCR), we provide side-by-side protocols for traditional TaqMan™ real-time, quantitative PCR (qPCR) and ddPCR, as well as comparative data generated with both methods. Lastly, we discuss the importance of the use of surfactant (here, Pluronic^® F-68) in the execution of the assay to limit DNA and AAV adherence to various carriers during the titration, particularly at low concentrations. We believe these protocols can lead to reduced variability and increased comparability between AAV studies.

Keywords: AAV; Adeno-associated virus; Droplet digital PCR; Genome; Quantitative PCR; Real-time PCR; Titration; Vector; ddPCR; qPCR.

Fig. 1

Protocol schematic and workflow

Fig. 2

Protect the LED screens of electronic pipettes from UV light damage

Fig. 3

Workflow for determining linear plasmid copy number and dilution. Calculate the copy number concentration of the linearized plasmid and the volume needed for a dilution of 2E+9 copies per μL (1E+10 copies per 5 μL), based on the known length of the plasmid, “Plasmid Length (bp),” and the mass concentration of the linearized plasmid as determined in Subheading 3.2, steps 6 and 7, “measured mass concentration (g).” Choose pipettes that maximize the accuracy and precision of reagent delivery. *See Subheading 3.2, steps 6 and 7 and Notes 19 and . **The final concentration of the stock linear plasmid dilution may be adjusted according to the linear plasmid yields obtained. For example, this value may be reduced from 2E+09 to 2E+08 copies/μL to create a stock of 1E+09 copies/5 μL, rather than 1E+10 copies/ 5 μL, if the yield of linear plasmid is low. ***See Subheading 2 for exact formulation

Fig. 4

Serial dilution for MV quantification. Follow the same protocol whether diluting virus or plasmid

Fig. 5

Schematic view of a 96-well plate layout for the Bio-Rad QX200® Quanta Soft™ ddPCR software. Sample and TaqMan™ assay locations are mapped in advance to assist in the calculation of reagent volumes and determination of loading strategy

Fig. 6

ddPCR titration is improved by inclusion of Pluronic® F-68 according to AAV serotype. The addition of F-68 during AAV guantification confers to a significant increase in signal that improves vector guantification. The degree of gain ranges from modest (two- to threefold) for AAV2/9 and AAV2/8, to large (five- to tenfold) for AAV2/Anc80 and AAV2/2. The same TagMan™ assay was used for these measurements, and all five serotypes carried the same single-stranded genome. Based on these observations, it appears that AAV capsids differentially attach to plastic

Fig. 7

Pluronic® F-68 optimization for ddPCR. Increasing the amount of F-68 does not significantly improve AAV titration of several serotypes. The same TagMan™ assay was used for all measurements, and all vectors carried the same single-stranded genome. In light of these results, it was decided to abide by the manufacturer’s recommendations for future measurements, and to use a final F-68 concentration of 0.1% (1×) for untested AAV capsid variants

Fig. 8

Quantification of a linearized qPCR standard plasmid. Fluorometry (Qubit™) was compared against Spectrophotometry (BioTek® Take3™). When the DNA being measured is clonally pure and clean, these two types of measurements closely agree. Fluorometry measurements may be lower than spectrophotometer O.D. measurements, because the former is highly specific for double-stranded DNA

Fig. 9

qPCR amplification plot showing the effect of 0.1% Pluronic® F-68 on qPCR standard performance. Standards of the same copy number concentration, serially diluted from the same source material, demonstrate lower Ct values (a “shift to the left”) in the presence of F-68, indicating their higher abundance in solution due to reduced attachment to plastic. This shift translates to higher titers for AAV quantification

Fig. 10

ddPCR vs. qPCR comparison across AAV serotypes carrying the same genome. The cis-plasmid used to manufacture these vectors was used for the qPCR standard. The same TaqMan™ assay was used for all measurements and exactly the same sample dilutions were assayed by both technologies. Pluronic® F-68 improves qPCR-based AAV genome quantification to a level comparable to ddPCR by making more target available for quantification and correcting qPCR standard performance

Fig. 11

ddPCR intra-laboratory assessment of quantification variability. Trainees should first observe a run set up by a more experienced user. They should then set up their own run under supervision before taking up the assay independently. Samples of known concentration should be used to ensure accurate quantification by new users