MAIN TEXT
Adeno-associated viruses (AAV) have proven to be safe and efficacious
gene transfer vectors, with 149 completed or ongoing clinical trials and
5 approved therapies for various forms of cell and gene therapy (Kuzmin
et al., 2021). The bulk of AAV vectors used for research, clinical
trials, and approved therapies are currently produced by multi-plasmid
transfection of mammalian cells (Clement & Grieger, 2016; Wang, Tai, &
Gao, 2019). Though variations on the process exist, production of AAV by
transfection typically uses three plasmids: a transfer plasmid encoding
the gene of interest flanked by viral inverted terminal repeat elements;
a helper plasmid encoding the minimal helper virus genes necessary for
the AAV lifecycle; and a packaging plasmid containing the AAV REPand CAP genes (D. Sharon & Kamen, 2018; Wang et al., 2019).
In this study HEK-293SF cells producing an AAV2-GFP vector were used as
a model to determine what proportion of transfected cells generate fully
assembled capsids during vector production. Commonly used cationic
transfection reagents (polyethyleneimine, calcium phosphate,
lipofectamine, etc.) coprecipitate heterogeneous plasmid mixtures into
larger complexes for transit across the cell membrane, and so in theory
all successfully transfected cells should contain the genetic elements
necessary to produce AAV (Cardarelli et al., 2016; Erbacher et al.,
2004; Fus-Kujawa et al., 2021). Reported transfection efficiencies
(measured by the expression of a fluorescent marker) for well optimized
processes range from 40-60% (Chahal, Schulze, Tran, Montes, & Kamen,
2014; Nguyen et al., 2021). However, the degree to which transfection
efficiency corresponds with the proportion of productive cells in the
culture remains largely untested. Clarifying this will give an
indication of how much of the cell biomass is being utilized with
current AAV manufacturing protocols, and in turn how much those
protocols might be improved.
To establish a model for subsequent experiments, a plasmid transfection
to produce AAV2-GFP was carried out on HEK-293SF cells in suspension,
based on an optimized process developed by Chahal et al. (Chahal
et al., 2014). As seen in Figure 1a, cell density was relatively stable
up to the harvest point at 48 hours post-transfection (hpt), the
previously determined optimal harvest point for this process (Chahal et
al., 2014). Volumetric vector yields at 48 hpt were in the range of
109 VG/ml (Figure 1b). Overall, the measurements
obtained in Figure 1 are in line with previous studies where AAV vectors
were produced by transfection in HEK-293 or derivative cell lines.
To determine what proportion of cells in our transfection model produce
fully assembled vector capsids, transfected cells were stained with a
conformation-specific antibody that binds only to assembled viral
capsids, allowing the measurement of this subpopulation by flow
cytometry (Wobus et al., 2000; Xiao, Warrington, Hearing, Hughes, &
Muzyczka, 2002). The presence of a GFP expression cassette on the
transfer plasmid allowed simultaneous assessment of transfection
efficiency. Cells infected with replication competent (RC) AAV2 and a
human adenovirus type 5 (hAd5) helper virus were used as a positive
control for capsid assembly.
As shown in Figure 2a, mean transfection efficiency at 48 hpt was
roughly 60%. However, only a small fraction
(~7% ) of transfected cells produced measurable
amounts of assembled AAV capsid. This contrasts with the RC AAV2 and
helper virus infected control where, as expected, nearly all cells are
positive for assembled AAV capsid. Note that AAV capsids will
spontaneously assemble without necessarily encapsulating a viral genome,
and so these measurements may be an overestimate of the proportion
productive cells in the culture (Hajba & Guttman, 2020). These results
were independently replicated by separate teams at McGill University and
the National Research Council of Canada
The observation that only ~7% of transfected cells
appeared to produce assembled AAV capsids raised the question as to what
proportion of cells were expressing the necessary factors for AAV vector
production. The three transfected plasmids collectively encode
~17 protein and RNA elements, making measurement of
every factor impractical (D. Sharon & Kamen, 2018; Wang et al., 2019).
Instead, a subset of proteins from each of the three transfected
plasmids were visualized in transfected cells via immunofluorescence.
As shown in Figure 3, GFP was broadly expressed in imaged cells.
Interestingly though, GFP expression does not seem to reliably indicate
coexpression of AAV capsid monomers or the hAd5 E2A helper factor, both
of which were expressed at detectable levels in a much smaller subset of
cells. This may partially explain the observation in Figure 2b that only
~7% transfected cells contain assembled AAV capsids,
despite an apparent transfection efficiency nearly tenfold higher.
Low yields in transfection-based AAV vector production platforms are a
long-standing issue, and have spurred the development of numerous
alternatives. Plasmid-free systems such as baculovirus expression
vectors, herpesvirus vectors, and more recent self-silencing adenoviral
systems boast significantly increased yields and are far more amenable
to scaleup (Cawood, 2020; D. Sharon & Kamen, 2018). There have also
been attempts both within academia and industry to develop a stable
producer cell line, though little of the latter work is published
(Clement & Grieger, 2016). While these platforms may supplant
transfection for late-stage and approved therapies, the unparalleled
speed and simplicity of transfection-based manufacturing means it is
likely to remain a mainstay of AAV vector production for early clinical
and research applications in the foreseeable future.
The results presented here indicate that current transfection-based AAV
vector production protocols utilize only a fraction of the available
cell biomass, and that significant increases in yield may yet be
realized. To this end, the innovation and optimization of this process
remains an active area of research. Computational work modeling plasmid
uptake, expression, and vector assembly kinetics in HEK-293 has
demonstrated utility in identifying molecular bottlenecks to improve
vector yield and quality (Nguyen et al., 2021). Design of Experiments
approaches to optimize process parameters have also recently been shown
to be effective in increasing AAV vector yields across a wide range of
serotypes (Zhao et al., 2020).
The results of Figure 2 also demonstrate that the expression of a
transfection marker does not necessarily imply a cell is producing AAV
vector particles. While transfection efficiency remains an important
process development metric, the confirmation-specific antibody staining
against assembled AAV capsids that was used here may also prove useful
in the future development and optimization of transfection-based AAV
vector platforms.