Human Gene Therapy - April 2023 - 326

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TROXELL ET AL.
INTRODUCTION
ADENO-ASSOCIATED VIRUSES (AAV) ARE promising vehicles
for the treatment and potential cure of numerous human
genetic diseases. These viruses are nonpathogenic, exhibit
lower immunogenicity than most viruses, and are capable
of transducing a variety of cell types.1,2 In addition, a viral
vector can package*4.7 kb of DNA, be produced at scale
to meet clinical trial demands with evolved capsids and
tissue-specific cassette design that allow for targeted delivery
and expression.3-6 The icosahedral AAV capsid is
composed of viral proteins (VP) VP1, VP2, and VP3 in an
*1:1:10 ratio.7-10
Recent evidence suggests that the VP ratios comprising
an AAV capsid population may be more divergent and
variable than earlier data indicated.11 The design and directed
evolution ofVPs provide a unique platform to test the
impact of amino acid changes on the immune evasion and
tissue tropism of AAV. There will likely be a continued
need to improve the manufacturability and in vivo efficacy
as more genetic indications are targeted for treatment.
Using the search term ''adeno associated virus,'' there
are >100 clinical trials ongoing in the United States that
utilize recombinant AAV (rAAV) to deliver genetic cargo
to correct genetic diseases (ClinicalTrials.gov). As technology
improves and demand increases, the number of
approved rAAV gene therapies will likely increase dramatically.
Manufacturing of rAAV typically relies on the
following genetic components provided in trans: Rep and
Cap genes, target DNAflanked by inverted terminal repeats
(ITRs), and adenovirus helper genes. These are located on
separate DNAmolecules to minimize the packaging of offtarget
DNA sequences. Despite these efforts, residual host
and plasmid DNA, partially packaged target DNA, and
empty capsids are often purified with rAAV capsids that
contain the therapeutic gene of interest.12-14
These product-related impurities may cause unwanted
results during administration to humans; however, the
contribution of empty capsids toward in vivo efficacy is
not clear. Animal studies with AAV8 demonstrate a reduced
efficacy when partially-packaged or empty AAV
capsids were present.12 Alternatively, other studies have
shown that these impurities may act as decoys for the host
immune system, thereby improving full vector efficacy.15,16
Regardless of the in vivo impact, it is clear that for
a gene therapy program to advance, analytical capabilities
must be able to quantify the levels of empty and full
capsids in manufactured AAV.
Previous studies have demonstrated the capability of
analytical methods to quantify full, partial, and empty capsids.17-22
Both cryogenic electron microscopy (cryo-EM)
and sedimentation velocity analytical ultracentrifugation
(SV-AUC) have been used to gain fundamental knowledge
and to quantify full and empty particles of AAV.18,23-27
Mass photometry as well as orbitrap-based charge-detection
mass spectrometry have demonstrated utility for quantification
of full, partial, and empty capsids.28,29 In addition,
size exclusion chromatography with UV and multiangle
light scattering (SEC-MALS) has been applied to quantify
numerous critical quality attributes (CQAs) of AAV.30,31
Additional approaches that quantify the nucleic acid content
and capsid concentrations have been used to obtain a ratio of
genome to capsid content. The absorbance profile ofpurified
rAAV vectors without separation by chromatography has
also been used to quantifyDNAand protein content, thereby
obtaining a full-to-empty ratio.17
Arrhythmogenic right ventricular cardiomyopathy
(ARVC) is a cardiac condition that is caused by several
factors.32 Several genetic loci have been associated with
manifestation of ARVC,33 which suggests that gene
therapy may be a viable approach to treatment. To this end,
a capsid variant derived from AAV9 was isolated from
directed evolution studies, named STRV84, and used for
program advancement. In addition, a DNA cassette with
tissue-specific regulatory control of a gene believed to
improve the ARVC condition was developed. This vector,
named STRX-330, was produced within StrideBio as part
of preclinical studies. As part of the program advancement,
analytical methods were developed. Because full-toempty
quantification is a known CQA for gene therapies,
multiple approaches to assess this CQA were performed.
Specifically, SV-AUC, cryo-EM, SEC-MALS, droplet
digital PCR (ddPCR)/enzyme-linked immunosorbent assay
(ELISA), and bulk absorbance measurements (via the
Stunner instrument from Unchained Labs) were used to
quantify full and empty particles. A test article from a 200L
process confirmation production was used to develop and
optimize a SEC-MALS method. For comparison of all
methods, material from a 500L production was used. This
material was also used in a non-Good Laboratory Practices
(GLP) animal study for a gene therapy program. Of the
tested methods, SEC-MALS displayed the strongest agreement
with SV-AUC and required minimal development and
optimization. In addition, the concentration of vector genomes
(VG) obtained by SEC-MALS was in agreementwith
DNase-resistant ddPCR and data from the Stunner instrument.
Because of the impact of dosing on preclinical studies,
the use of multiple methods to quantify VG is beneficial.
Furthermore, forced degradation studies with pH conditions
demonstrated that SEC-MALS was similar to
ddPCR, capsid ELISA, and a cell-based assay (50% tissue
culture infectivity [TCID50]) in the ability to measure
product instability. The data support that SEC-MALS is a
robust analytical technique for advancement of gene
therapy programs.
MATERIALS AND METHODS
Reagents, chemicals, and supplies
The HeLaRC32 cell line (catalog CRL-2972) and
Adenovirus serotype 5 (catalog VR-1516) were purchased
http://www.ClinicalTrials.gov
Human Gene Therapy - April 2023

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