Does promoter activity in a plasmid predict AAV performance?

A promoter that performs well in a plasmid assay will not necessarily perform equally well in every gene therapy vector. One of the biggest reasons is assay topology—whether the regulatory element is tested on an episomal DNA molecule or after genomic integration.

Studies comparing massively parallel reporter assays (MPRAs) have shown that episomal and integrated assays can rank the same regulatory elements quite differently (Inoue et al., 2017; Agarwal et al., Nature, 2025). Our own AIM™ platform demonstrates the same principle. The key takeaway is simple: the assay should match the biology of your therapeutic vector.

Episomal and integrated reporter assays measure different aspects of promoter function.

Integrated assays evaluate promoters within chromatin after genomic integration, making them highly reproducible and well suited for integrating vectors. Episomal assays instead measure promoter activity from extrachromosomal DNA, where chromatin organization and genomic position effects differ.

Neither assay is inherently better—they answer different biological questions.

The therapeutic vector determines which assay is most informative.

AAV genomes generally persist as episomal concatemers, whereas lentiviral vectors and many engineered cell therapies rely on genomic integration. As a result, a promoter selected in an integrated assay may not be the optimal choice for AAV, just as an episomal winner may not be ideal for an integrating product.

Matching the screening assay to the intended modality increases the likelihood that the observed activity will translate.

SuRE™ measures promoter activity from plasmid DNA, making it a closer approximation to the episomal biology of AAV than an integrated reporter assay. For integrating products, we instead use integrated readouts that better reflect the final genomic context.

Importantly, we always report how a promoter was tested, because assay topology determines how confidently the results can be translated to a therapeutic vector.

Neither episomal nor integrated reporter assays fully reproduce the complexity of the native genomic environment. Chromatin state, higher-order genome organization, and local regulatory interactions all influence promoter activity in vivo.

For AAV vectors, additional factors such as inverted terminal repeat (ITR)-associated transcriptional activity and concatemer formation can further affect expression. For this reason, promising promoters identified in MPRA screens should always be confirmed in the final therapeutic construct and relevant target cells.

Talk to us. Tell us your delivery modality and we will match the assay topology to it.