Adeno-associated virus (AAV) gene therapies for ultra-rare disorders utilize complex and expensive raw materi- als and procedures that complicate clinical preparation. The high cost of existing AAV therapies (e.g., $850,000 for Luxterna) reflects the high costs of goods (e.g. DNA) and procedures used in the vector manufacturing pro- cess. Solutions are needed that provide near-personalized scale-out, and agile AAV production for one to a few patients at a time, at significantly reduced cost. Our objectives to reduce complexity and cost are three-fold: integrating (1) rapidly-produced synthetic DNA as raw material for cell transfection, (2) a single-use static bio- reactor that enables high cell density filterless perfusion and (3) continuous virus collection in which AAV is dis- placed from the bioreactor during perfusion, and concentrated using affinity capture resins that bind AAV sur- face proteins. First, we will leverage rolling circle amplification (RCA DNA) for synthetic DNA production to replace cur- rent reliance on cGMP plasmids, accelerating DNA production time from >4 months to <4 days and reducing DNA production costs ~10-fold by removing bacterial fermentation and downstream purification processes. RCA DNA is produced cell-free and endotoxin-free, can produce gram-quantities of tandemly repeated DNA in a day. We have demonstrated proof-of-concept for AAV production using triple transfection of RCA DNA into HEK293 cells, resulting in titers comparable to standard plasmid transfection. Second, we will leverage a low shear filterless static perfusion bioreactor that supports up to 30x106 cells per mL to develop high density transfection protocols that reduce both transfection reagent usage and cell culture medium consumption during virus production for a further ~3 to 10-fold relative cost reduction. Third, we will design a continuous AAV production/collection protocol using the low shear filterless static perfusion bioreactor. We will establish methods for high titer recovery of secreted AAV (post-transfection with synthetic RCA DNA) via a continuous perfusion method that collects secreted virus while maintaining producer cells in the bioreactor. The secreted AAV will be collected by affinity capture resins that bind AAV capsid pro- teins. Our approach will enable continuous media harvest of AAV from high-density transfected cells by exploiting the simplicity of RCA-based DNA production, minimizing media costs through high density cell culture, and uti- lizing a continuous virus collection procedure that provides a higher cumulative AAV yield than obtained in cur- rent batch-based lytic processes.
Adeno-associated virus (AAV) gene therapies for ultra-rare disorders utilize complex and expensive raw materi- als and procedures that complicate clinical preparation. The high cost of existing AAV therapies (e.g., $850,000 for Luxterna) reflects the high costs of goods (e.g., DNA) and procedures used in the vector manufacturing pro- cess. Solutions are proposed that provide near-personalized scale-out, and agile AAV production for one to a few patients at a time, at significantly reduced cost.
