RNA-TITER-An ADAR-based transcript detection assay for accelerated functional titer assessment of adeno-associated viruses.

In gene therapy, payload design is a critical process that often requires the comparison of multiple vector architectures. However, current analytical pipelines for assessing transgene expression are time intensive, labor heavy, and costly. Here, we describe an RNA editing-based titratable indirect transgene expression reporter (RNA-TITER), an in cellulo reporter that senses transgene transcript levels. By coupling an RNA-editing strategy to a luminescence-based readout, RNA-TITER eliminates the need to co-deliver an additional reporter protein on the adeno-associated virus (AAV) genome for functional titer assessment. Compared to RNA isolation, reverse transcription, and quantitative PCR (qPCR), RNA-TITER achieves at least 5-fold reduction in processing time and requires substantially fewer pipetting steps, while maintaining exceptional concordance with traditional quantitative reverse-transcription PCR (RT-qPCR) results (R2 = 0.9974). In addition to reducing hands-on effort and the risk of technical errors, this workflow significantly lowers per-experiment costs. Collectively, this methodology enables rapid screening of different constructs and batches.