AAVR disruption as a producer-cell engineering strategy for enhanced supernatant-based recovery of selected AAV...

Recovery of adeno-associated virus (AAV) vectors from culture supernatants has attracted increasing interest as a strategy to simplify downstream processing and improve product purity. However, several AAV serotypes can be re-internalized by producer cells, raising the possibility that newly released particles are recaptured during production and thereby limiting extracellular yield. Because AAV entry is mediated by the universal receptor KIAA0319L (AAVR), receptor-dependent re-uptake may contribute to this limitation, yet its role in vector manufacturing remains unexplored. Here, we investigated whether disruption of AAVR enhances supernatant-based AAV recovery. Using CRISPR-Cas9-mediated genome editing, we generated AAVR-knockout (AAVR-KO) HEK293-EB producer cells by deleting exon 2 containing the translational start codon. AAVR ablation abolished susceptibility to multiple AAV serotypes and markedly reduced cellular uptake of extracellular particles. In supernatant AAV vector recovery assays, AAVR knockout increased supernatant vector genome recovery for selected serotypes in a representative clone, although this effect varied among independently isolated clones. Complementary AAVR overexpression reduced accumulation of extracellular AAV1 vector genomes, supporting a role for AAVR expression levels in supernatant recovery while also suggesting additional effects on production-related cellular processes. These findings identify AAVR-dependent cellular uptake or retention as a modifiable post-release process that can influence supernatant-based AAV vector recovery.