INTRODUCTION
Gene therapy gives rise to hopes for a large spectrum of genetic
diseases, that are mainly untreatable using conventional pharmacology.
Over the past decade, recombinant Adeno-Associated viruses (rAAV) have
been the most studied viral vectors and were successfully used in
clinical trials for in-vivo gene transfer. In the case of
Duchenne Muscular Dystrophy (DMD) which is the most common lethal muscle
genetic disease, animal-based studies suggest that a whole-body
treatment to achieve efficient systemic gene delivery would require
infusion of high doses of rAAV vector within the range of
1014 vector genomes/kg (Guiner et al., 2013; Le Guiner
et al., 2017). Although several strategies have been developed to
optimize vector transduction in targeted muscle cells, the large-scale
production of high-quality rAAV vectors is still a limiting step to move
forward for clinical trials into large patient population.
Most commonly, rAAVs are produced in HEK293 adherent mammalian cells by
co-transfection of two or three plasmids containing (i) the AAV genes,
(ii) the essential adenoviral helper genes that are supplemented intrans , and (iii) the viral genome with a maximal size of 4.7kb
which is framed by inverted terminal repeats (ITRs) required for genome
replication and encapsidation (Galibert and Merten, 2011). This method
has been used worldwide, including the manufacturing of AAV2 and AAV8
reference-standard materials (RSM) to be exploited by the scientific
community and regulatory agencies (Lock et al., 2010; Ayuso et al.,
2014) in order to provide common quantification methods for the particle
and genome. However, the expansion and transfection of adherent cells
may limit manufacturing of rAAV biotherapeutic products. To scale-up the
procedure, suspension systems have been developed and optimized during
the last decade, using suspension mammalian cells and serum-free medium
(Grieger et al., 2016) or the insect cells/baculovirus system (Cecchini
et al., 2011; Kotin and Snyder, 2017).
The baculovirus expression vector (BEV) platform has become an
established manufacturing platform for the production of viral vaccines
and gene therapy vectors and it offers many advantages over plasmid
transfection of mammalian adherent cells such as manufacturing speed,
cost efficient and scalability. Of note, the insect cell-based system
has shown to be adapted and efficient for industrial application with
examples in the flu vaccine industry (Cox and Hashimoto, 2011; Milián
and Kamen, 2015) using bioreactors of 20,000 liters (Flublock, Protein
Sciences).
The standard method to generate a recombinant baculovirus is based on
site-specific Tn7 transposition of an exogenous DNA cassette carrying
the gene of interest (GOI) from a plasmid donor to the Autographa
californica multicapsid nucleopolyhedrovirus (AcMNPV) baculovirus DNA
called “bacmid” (Kubo and Craig, 1990; Luckow et al., 1993).
Recombinant baculoviruses are produced in Sf9 insect cell line
(Spodoptera frugiperda ) which has been adopted as workhorse in
many laboratories for baculoviral vectors amplification because of its
ability to combine both a high production yield of infectious particles,
and growth in suspension at 27°C-31°C without CO2 in
serum-free media, an advantage for large-scale cGMP manufacturing.
Originally, BEV system has been adapted for rAAV production by
co-infecting insect cells with three recombinant baculoviruses
(rep, cap and GOI) where AAV promoters were replaced by
baculovirus ones to allow expression of AAV genes into insect cells
(Urabe et al., 2002). Later, R. Kotin and collaborators developed a
simplified dual baculoviruses system by combining the rep andcap helper functions into one unique construct (Smith et al.,
2009).
The baculovirus/Sf9 platform has proven its efficiency for the
manufacturing of rAAV viral vectors in particular through the market
authorization of Glybera, a gene therapy product indicated for the
treatment of Lipoprotein Lipase Deficiency. However, the regulatory
agencies requested to the company uniQure for residual baculovirus DNA
assessment due to a potential side effects (Bryant et al., 2013). For
this purpose, we have developed a protocol based on high-throughput
sequencing to identify and quantify residual DNAs, called thesingle-stranded virus sequencing (SSV-Seq), and we demonstrated
that baculoviral DNA is the major source of DNA contamination in the
final rAAV product with up to 2.1% of total NGS reads (Penaud-Budloo et
al., 2017). Worryingly, the gentamycin resistance gene coding sequence,
that has been used to select recombinant baculovirus after Tn7
transposition, was detected by qPCR in research-grade rAAV lots.
Finally, it has also been reported in the literature that BEV genome
derived from the Tn7-bacmid system is subjected to instability and
spontaneously deletion, leading to the generation of defective
interfering viruses (DIs) which accumulate at the expense of the intact
ones and limiting scale-up of batch production (Pijlman et al., 2004,
2003). Altogether, these technical concerns prompted us to reconsider
other options to overcome these barriers and to define new standards to
produce baculoviral reagents for rAAV vector manufacturing.