Microfluidic production of nanogels as alternative triple transfection reagents for the manufacture of adeno-associated virus vectors.

Adeno-associated viral vectors (AAVs) have proved a mainstay in gene therapy, owing to their remarkable transduction efficiency and safety profile. Their production, however, remains challenging in terms of yield, the cost-effectiveness of manufacturing procedures and large-scale production. In this work, we present nanogels produced by microfluidics as a novel alternative to standard transfection reagents such as polyethylenimine-MAX (PEI-MAX) for the production of AAV vectors with comparable yields. Nanogels were formed at pDNA weight ratios of 1 : 1 : 2 and 1 : 1 : 3, of pAAV cis -plasmid, pDG9 capsid trans -plasmid and pHGTI helper plasmid respectively, where vector yields at a small scale showed no significant difference to those of PEI-MAX. Weight ratios of 1 : 1 : 2 showed overall higher titers than 1 : 1 : 3, where nanogels with nitrogen/phosphate ratios of 5 and 10 produced yields of ≈8.8 × 10 8 vg mL -1 and ≈8.1 × 10 8 vg mL -1 respectively compared to ≈1.1 × 10 9 vg mL -1 for PEI-MAX. In larger scale production, optimised nanogels produced AAV at a titer of ≈7.4 × 10 11 vg mL -1 , showing no statistical difference from that of PEI-MAX at ≈1.2 × 10 12 vg mL -1 , indicating that equivalent titers can be achieved with easy-to-implement microfluidic technology at comparably lower costs than traditional reagents.