Gene therapy is one of the most innovative and fastest growing fields in the pharmaceutical industry. The first approved gene therapy utilized a recombinant AAV vector (rAAV), and dozens of additional rAAVs for gene therapy are presently in clinical trials. rAAV gene therapy drugs have been priced in the region of € 500’000 and above, which is partially a result of them being manufactured by highly complex processes combining multiple components, requiring 5-7 separate GMP production runs.
In the present ERC Proof-of-concept project AAV Factory, we set out to introduce a scalable, single-baculovector rAAV production platform to resolve this bottleneck. Our objective was to investigate whether rAAV particles could be produced in high quality and quantity using an innovative all-in-one baculovector strategy we had pioneered in DNA-DOCK (the parent ERC Advanced grant of PoC AAV Factory). In AAV Factory, we addressed the question whether the baculovirus platform we had engineered could produce rAAVs superior to the state-of-the-art in terms of quality, efficacy and associated costs, potentially resolving one of the foremost bottlenecks currently impeding rAAV production.
Towards this goal, we produced rAAV particles using our innovative baculoviral platform comprising an engineered, partly synthetic baculovector system (SynBac). We compared our production setup with rAAV production in a classical multiplasmid transfection approach carried out in parallel. Purification of the particles produced, either by classical multiplasmid co-transfection or by our innovative one-in-all baculovector approach, was carried out side-by-side following established standard operation protocols (SOPs) for rAAVs. We contrasted the the rAAVs we produced, in terms of quality, quantity and functionality, with the current industry norm for batch acceptance/rejection.
The AAV Factory PoC study resulted in a resounding success.
Our innovative baculoviral platform produced rAAV in high quality and quantity, as evidenced by a range of quality control criteria (CBB-stained SDS-PAGE, nucleic acid gel electrophoresis (NAGE), transmission electron microscopy (TEM), target cell transduction), with much less effort and lower costs, requiring a single production run. The QC data clearly showed that our innovative approach is superior to classical multiplasmid co-transfection in terms of productive rAAV yield, while fully maintaining the efficacy in target cell transduction.
SDS-PAGE and electron microscopy evidenced that the particles we produced were homogenous and largely devoid of contaminants. Inspection of the TEM images and NAGE supported high-level nucleic acid occupancy of the rAAV particles analysed. Of note, the particles we produced with our system, even without further optimizing the purification procedures, fulfilled the industrial batch acceptance criterion based on transmission electron microscopy as a quality control (QC) method. In fact, the high quality and uniformity of the rAAV particles we produced, potentially allows high-resolution structure determination by single particle electron cryo-microscopy (cryo-EM), which we aim to pursue in the near future as an ultimate quality control.
The work we carried out in the framework of ERC PoC AAV-Factory thus established our innovative all-in-one baculovector manufacturing approach as a powerful rAAV production platform. We are hopeful that our manufacturing innovation in due course can contribute to reducing the costs of gene therapy interventions, thus reduce strain on health care systems and make these type of therapies accessible to patients who currently cannot afford them.
