APR January/February 2022 - 73

« INSTRUMENTATION
PCR-Based Methodologies to Characterize
AAV Gene Therapy Vectors
Pete Clarner,1*
Katelyn M. Mansfield2*
1 Gene Therapy Accelerator Unit, Biotherapeutics and Medicinal Sciences, Biogen Inc.
2Analytical Development, Product and Technology Development, Biogen Inc.
*Both authors contributed equally
Abstract
Gene therapy is a modality that requires a
complex analytical control strategy to ensure
safety and efficacy of the drug product. Adenoassociated
virus (AAV) is widely used as a
vector for preclinical research and clinical gene
therapy development. AAV gene therapies
require characterization of many product
quality attributes such as the quantitation of
genome titer and aspects of AAV potency such
as internalization, infectivity, expression, and
functional potency (Figure 1). PCR is one of the
most well-known and widely used methods
to quantitatively analyze AAV gene therapy
candidates at all stages of development.
Here, we discuss the many uses of PCRbased
technologies as an analytical tool for
characterizing AAV gene therapies.
Detection Technologies
The two key detection technologies used for
PCR-based quantitative methods are SYBR
and TaqMan (Figure 2). Each requires the
development of a sequence-specific forward
and reverse primer. SYBR is a chelating dye
that significantly increases in fluorescence
when bound to double-stranded DNA
(dsDNA), including newly formed PCR
product dsDNA.1
TaqMan technology uses a third DNA oligo,
the sequence-specific hydrolysis probe. The
probe has a fluorescent reporter dye on the
5' end and a quencher on the 3' end. When
the probe is bound to the target sequence, 5'
exonuclease activity of the DNA polymerase
cleaves the reporter dye from the probe,
resulting in separation from the quencher
Figure 1. PCR can be used to characterize physical quantity and multiple stages of the AAV
transduction pathway. A) Genome titer. B) Cellular internalization. C) Transgene mRNA and
small RNA expression. D) Target mRNA knockdown. Figured created with Biorender.com
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