APR January/February 2022 - 76

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INSTRUMENTATION
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mRNA demonstrates the mechanism of action of the therapy and can
demonstrate functional potency. This type of assay can either utilize
PCR or immunoassays to quantitate the levels of the target mRNA
or protein, respectively. Sometimes both assays may be developed
during phase-appropriate development and may be used in parallel
to show consistency between the orthogonal methods.
Process Related Analyses
PCR methods can be used to detect process-related impurities
such as residual plasmid DNA, residual host cell DNA, and residual
helper viruses. As these impurities may pose a risk to product
quality and safety, their accurate detection and quantitation is
required for all gene therapy products. These impurities arise during
vector production and may remain in the product even after the
purification process. The concentration of impurities in a product can
be determined by PCR amplifying a sequence found in the impurity
(e.g., antibiotic resistance sequence of a DNA plasmid). For processrelated
DNA impurities, the FDA recommends having less than 10ng
of DNA per dose and that the size of the DNA is less than 200 base
pairs to reduce potential oncogenicity and infectivity.10
In addition
to quantifying the concentration of residual DNA in gene therapy
products, 2-dimensional ddPCR (2D-ddPCR) can be used to determine
the approximate fragment size by using primer sets to multiple areas
along the sequence.
PCR is also a commonly used technique for assessing genome
integrity of AAV. As AAV packaging may result in incomplete genomes,
accurately measuring what has been packaged in the AAV is vital
to ensuring product safety and efficacy. As previously stated, qPCR
results can be affected by the standard curve material selected for the
study, and therefore ddPCR is often preferred for genome integrity
analyses as it results in absolute quantification of the target. 2D-ddPCR
for genome integrity can quantify two separate parts of the same viral
genome, therefore increasing the accuracy of detection of incomplete
AAV genomes as compared to 1D-ddPCR.11
Additional Considerations
When measuring the expression level of endogenous mRNA, whether
this is the reference gene or target gene within the cell, detection of
contaminating genomic DNA (gDNA) from the cell can be mitigated
through reagent design. By designing primer/probe sets that span two
exons of the coding sequence of the endogenous gene, this will allow
only quantitation of cDNA reverse transcribed from properly spliced
mRNA (Figure 3). For example, two primer/probe sets, one annealing
within a single exon and the other spanning adjacent exons, were
compared for specificity, demonstrating that primer/probe sets within
a single exon detected contaminating gDNA. In some cases, this can
skew mRNA quantitation leading to less accurate expression results.
(Figure 3, Table 3).
Depending on the intended application of a PCR method, enzymatic
treatment of samples can be a crucial sample processing step. In
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| January/February 2022
Figure 3. Exon-spanning primer/probe set design. Primer/Probe
Set 1 anneals within a single exon whereas Primer/Probe Set 2
spans adjacent exons and will only amplify cDNA from properly
spliced mRNA.
Table 3. Comparison of primer/probe sets for mRNA specific
detection. Both primer/probe sets detect RNA a high level. Primer/
Probe Set 1 also detects contaminating gDNA, whereas Primer/
Probe Set 2 only detects cDNA from properly spliced mRNA. LLOQ:
lower limit of quantitation.
Primer/Probe Set
Set 1
Set 2
Exon-Spanning
No
Yes
gDNA
(Copies/µL)
136.1



APR January/February 2022

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