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.