title: Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.
abstract: Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.
authors: Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania , Philadelphia, PA 19104.
authors: Martin
authors: M
authors: Lock
authors: Mauricio R
authors: MR
authors: Alvira
authors: Shu-Jen
authors: SJ
authors: Chen
authors: James M
authors: JM
authors: Wilson
mesh_terms: D017931:DNA Primers / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000032:analysis; D000229:Dependovirus / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors / Q000032:analysis*; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded