# An autonucleolytic suspension HEK293F host cell line for high-titer serum-free AAV5 and AAV9 production with reduced levels of DNA impurity

## TL;DR

Scientists engineered HEK293F cells to secrete a nuclease, reducing DNA impurities in AAV5 and AAV9 gene therapy production without affecting viral yield or effectiveness.

## Contribution

Development of a HEK293F cell line that secretes nuclease to reduce DNA impurities in AAV production.

## Key findings

- NuPro-1S cells produced 3.06 × 10¹⁰ AAV5 genomes/mL, significantly higher than HEK293F cells.
- AAV9 production from NuPro-1S cells reached 1.8 × 10¹³ genomes/mL, with no loss in transduction ability.
- DNA impurities in AAV process streams from NuPro-1S cells were reduced by ~60% compared to HEK293F.

## Abstract

We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, “NuPro-1S.” When cultivated in serum-free media, NuPro-1S cells yielded 3.06 × 1010 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85 × 109 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8 × 1013 vg/mL from NuPro-1S cells compared with 7.35 × 1012 vg/mL from HEK293F cells. AAV9 from both HEK293F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue in vivo. Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared with HEK293F cells. A similar reduction in HEK293F cells was only achievable with a 50 U/mL Benzonase treatment.

Nesbeth and colleagues engineered HEK293F cells with a transgene encoding a secreted staphylococcal nuclease. This engineering step did not compromise AAV5 or AVV9 yield or infectiousness when the cells were used as hosts for transient transfection, but did effect reduced levels of DNA impurity in process streams.