Abstract Background Adeno-associated virus (AAV) vectors are the leading platform for in vivo gene therapy. The vast majority of gene therapeutic approaches rely on the (over) expression of a single therapeutic transgene. Gene replacement can restore function or alleviate symptoms, but monogenic diseases as well as polygenic or acquired conditions involve dysregulation of multiple processes, rendering overexpression of a single transgene suboptimal or insufficient. Delivering multiple transgenes from one vector offers a more powerful strategy for correcting complex disease mechanisms, achieving stronger therapeutic outcomes. Existing strategies for multigenic therapies rely on 2A peptides, internal ribosome entry sites, or dual-promoter systems. However, these strategies either reduce AAV’s already limited packaging capacity or lack the ability to independently regulate transgene expression. To overcome these limitations, we develop a bidirectional cardiomyocyte-specific promoter (Bi-Card) that drives independent expression of two transgenes. Methods We used cardiac-specific chromatin accessibility and vertebrate conservation to select promoter sequences. Neonatal rat ventricular myocytes (NRVM), human induced pluripotent stem cell derived ventricular myocytes (hiPSC-VM), HEK 293T and HEP G2 cells were transduced with recombinant AAV6 containing mScarlet3 and GFP transgenes at a multiplicity of infection of 10, 000. Cells were processed 4-5 days post-transduction. Expression of the transgenes was measured by qPCR and flow cytometry. Additionally, mice were retro-orbitally injected with 5 x 10¹1 viral genomes recombinant AAV9 containing mScarlet3 and GFP transgenes. Mice were killed 2 weeks after injection and viral and genomic DNA and RNA where isolated from ventricles, atria, skeletal muscle, liver and kidney to confirm delivery and measure biodistribution and expression. Results Our here developed Bi-Card promoter demonstrated strong, specific expression in hiPSC-VMs and NRVMs, but not in HEK 293T or HEP G2 cells, as exemplified by a 9-fold and 15-fold enrichment of mScarlet3 and GFP signal respectively (figure 1), confirming cardiomyocyte specificity. Additionally, we show that transgene levels of transcripts can be individually controlled by the addition of an upstream open reading frame or CW3SL element. Finally, specificity was validated in vivo, where expression was only detected in the heart. Conclusion In conclusion, our cardiomyocyte-specific bidirectional promoter can be used to deliver two individually tuneable therapeutic transgenes from a single recombinant AAV vector. Figure 1. Normalized fluorescence intensity of mScarlet3 and GFP expressed by a Bi-CMV and the Bi-Card promoter in HEK 293T cells and NRVMs. mScarlet3 is expressed in the ‘forward’ orientation (as if by a unidirectional promoter) and GFP is expressed in the ‘reverse’ orientation. Figure 1For image description, please refer to the figure legend and surrounding text.
Visser et al. (Sun,) studied this question.