APR Sept/Oct 2023 - 68

CGT CIRCUIT
Navigating the Complex Testing Strategies
for Viral Vector-based Gene Therapies
Robert Cartee, PhD
Senior Director, Biopharmaceutical Services
SGS North America, Inc.
The safe and effective introduction of
a transgene into a cell to cure or reduce
disease severity is one of th key challenges
facing the field of gene therapy.
Viral
vectors have proven to be one of the
most efficient means to achieve this goal
by taking advantage of a virus's natural
ability to invade and introduce their
genetic material into human cells. As a
result, viral vectors are being developed
to treat a variety of diseases including
monogenic syndromes and cancers and
can be delivered both in vivo, in situ, and
ex vivo (cell therapy).
Several different viruses have been used
as the basis for viral vectors including
adenoviruses
(AV),
adeno-associated
viruses (AAV), retroviruses, lentiviruses
(LV), and herpes simplex viruses (HSV),
with each virus having its advantages and
limitations.
Similar to the actual virus,
viral vectors consist of genetic material
encased in a protective protein shell or
capsid and may be infectious. However,
unlike the virus
from which they are
derived, viral vectors are engineered to
be replication defective through deleting
genetic sequences from the viral genome
necessary for viral replication and/or
production of a capsid. This deletion also
serves to make room in the viral vector
genome for the transgene.
The development and manufacture of
viral vector-based gene therapy products
is an intricate process with rigorous
quality standards. Shown in Figure 1,
the manufacture of a viral vector for
gene therapy begins with plasmids that
are usually produced and purified from
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| September/October 2023
bacteria. One of these plasmids contains
the therapeutic transgene with any
necessary capsid packaging sequences.
The other plasmids, often referred to
as helper plasmids, contain the genes
that encode the capsid proteins and any
replication machinery. These plasmids are
transfected into a host cell, where through
the encoded sequences, the transgene is
packaged into the viral capsid to produce
the viral vector. Following purification of
the viral vector drug substance particles,
they are formulated and filled into
containers for either in vivo delivery or
for transducing allogenic or autologous
cells from subjects or patients for use as a
cell therapy.
Throughout the development and
manufacture of a viral vector, rigorous
testing of the raw materials, any ancillary
materials, plasmids, cell lines, and viral
vector drug substance and drug product
is performed to determine identity,
content/viability/potency, quality, safety,
and stability. These are all critical steps
in meeting regulatory requirements and
getting viral vector-based drug products
to market safely.
This intricate testing
requires specialized scientific expertise
and state-of-the-art instrumentation.
Identity Testing is performed to confirm
that the purified viral vector drug
substance or drug product contains
the correct nucleic acid sequence and
viral capsid proteins.
The nucleic acid
component can be identified though
sequencing (Sanger or Next Generation
[NGS]) or by a quantitative polymerase
chain reaction (qPCR).
The identity of
the viral vector capsid protein can be
determined using either Western blots
or liquid chromatography with detection
by absorbance or mass spectrometry.
Content/Potency Testing is performed
to determine the amount of infectious
viral vector particles present in the drug
substance or drug product. This can be
achieved by quantitating the number
of viral genomes contained in capsid
particles using qPCR. While this method
will indicate the number of genomes it
may not accurately reflect the potency of
Figure 1.
APR Sept/Oct 2023

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