Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

Abstract

Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.

A novel method for quantitation of AAV genome integrity using duplex digital PCR

“…Recently, a formula for genome integrity utilizing the calculation of percent “linkage” has been suggested, where genes contained on the same template are considered linked, and genes that are physically separated are considered unlinked [ 22 ]. Linkage is defined as the number of double-positive droplets in excess of what is expected due to chance co-localization of two unlinked targets [ 23 ].…”

“…In instances where the concentration of each target is similar, Regan et al suggest that percent linkage (i.e., genome integrity) can be calculated by dividing the linkage concentration by the average concentrations of the 5’ and 3’ target (CMV and polyA respectively, Formula 3 ) [ 23 ]. The authors note that if the concentrations of the two targets are unequal due to amplification bias resulting from experimental conditions (method-induced genome fragmentation, differences in genome accessibility, or differences in amplicon size) that a compensated version of the equation can be used, which involves adjustment of the linkage value by addition of the absolute difference in concentrations of the 5’ and 3’ target, followed by division by the maximum value of either the concentration the 5’ or the 3’ target ( Formula 4 ) [ 22 ]. When either Formula 3 or Formula 4 was used to calculate the percent genome integrity of the plasmid samples, the results were relatively stable across the tested concentration range (8–5000 copies/μL), as demonstrated by the assessment of the RSD (≤17.1%, ≤27.4% respectively, Table 2 ).…”

Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR

“…Surfactants such as Pluronic F-68 and blocking agents like sheared salmon sperm DNA are commonly added to the dilution buffer to facilitate the dissolution and dispersion of viral particles. Capsid lysis and genome DNA release are usually performed after serial dilution [4][5][6][7] or could be carried out before dilution. Viral genome extraction before dilution and quantification has also been seen in previous studies [8][9][10].…”

“…Finally, dPCR reaction and copy number analysis can be run on various instruments based on different principles. Despite the overall similarities in vector genome titration protocols, subtle differences exist [4][5][6][7][8][9][10], potentially contributing to variations in vector genome titer determination. Such discrepancies pose challenges in comparing and standardizing dosing between studies or laboratories, potentially impacting the safety and efficacy profiles of rAAV in clinical trials.…”

Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification

“…Recently, it has been demonstrated that ddPCR can be employed to detect salmonid alphavirus from seawater [ 7 ] and accurately quantify adeno-associated viral vectors [ 8 , 9 ]. During SARS-CoV-2 RNA detection in wastewater with ddPCR, Semliki Forest virus (SFV) vectors were employed as a standard to evaluate the effectiveness and success rate of SARS-CoV-2 extraction techniques [ 10 ].…”