# Heterogeneity in an adeno-associated virus transfection-based production process limits the production efficiency

## TL;DR

This study finds that only a small fraction of cells produce rAAV vectors during transfection, suggesting ways to improve production efficiency for gene therapy.

## Contribution

The study identifies cell heterogeneity and specific biological processes limiting rAAV production efficiency through single-cell transcriptomics.

## Key findings

- Only ~3% of cells contain assembled rAAV9 capsids, indicating low production efficiency.
- High plasmid gene expression is observed in 8% of cells, but only 3% produce rAAV9.
- Genes related to cell cycle, immune response, and protein unfolding differ between high and low expression cells.

## Abstract

The popularity of rAAV vectors in gene therapy are placing a burden on current production systems. To improve the accessibility of these life changing treatments, increases in production yields and a reduction in the cost-of-goods are needed. Transient transfection is the most common way to introduce rAAV-encoding plasmids to producer cells but it suffers from significant drawbacks such as low and inconsistent yields as well as high cost due to its need for plasmid DNA. This study aims to address the low yield of transient transfection-based rAAV production through advanced methods in process characterization. Adherent and suspension cultures of a HEK293T cell line were triple-transfected for rAAV9 production using polyethylenimine (PEI). Samples were taken at various times post-transfection for analysis with bulk and single-cell transcriptomics. It was revealed that 46% of the cells lacked transcripts of genes from at least one plasmid, indicating that a significant proportion of the cells did not have the genes necessary for rAAV9 production. Among the remaining 54% of the cells expressing genes from all three plasmids, only 8% showed high plasmid gene expression. Flow cytometric analysis of intracellular rAAV9 confirmed these results by showing that only ~ 3% of cells contained assembled rAAV9 capsids. Titre analysis by qPCR of the supernatant and lysate of the producer cells indicated an average culture performance of 1013 vg/L. Analysis of the single-cell transcriptomic data showed that a significant proportion of cells that had high plasmid gene expression were in the S-phase. Trajectory inference highlighted that genes involved in the G2-M phase transition, immune response, and protein unfolding were differentially expressed at the branch point between high and low plasmid expression. This study reveals a significant bottleneck in the transient transfection-based production of rAAV. With less than 5% of cells producing rAAV, significant improvements in titres can be achieved if this fraction can be increased. Moreover, regulation of the cell-cycle, inhibition of the immune response, and alleviating protein misfolding all potentially offer the key to enabling these life changing treatments to reach a wider audience.

The online version contains supplementary material available at 10.1038/s41598-025-26261-0.