Improving the precision of AAV lung gene therapy for SP-B deficiency using computationally derived lung-specific promoters

Abstract

Recombinant adeno-associated virus (rAAV) platforms have achieved significant success in clinical gene therapy; however, many still rely on ubiquitous promoters. This robust and widespread transgene expression can cause off-target effects, immune activation, and systemic toxicity, limiting their suitability for diseases requiring tissue-specific expression, such as surfactant protein B (SP-B) deficiency. Here, we aimed to improve the precision of AAV-lung gene therapy by evaluating computationally predicted lung-specific promoters with AAV6.2FF, a capsid with strong lung tropism. Promoter strength and specificity were assessed following administration of AAV6.2FF encoding the human placental alkaline phosphatase (AP) reporter gene in mice. Cross-species activity was evaluated in precision-cut lung slices (PCLS) from ferrets and pigs. We identified promoter 5979 as lung-specific in mice, outperforming the ubiquitous CASI promoter (comprised of the cytomegalovirus enhancer, chicken β-actin promoter, and ubiquitin C regulatory elements) in transgene expression and specificity, independent of AAV capsid or route of administration. In a conditional SP-B knockout model, AAV6.2FF-5979-hSPB extended survival in SP-B-deficient mice and outperformed its CASI-driven counterpart. Promoter 5979 exhibited the highest activity among the four synthetic promoters in ferret PCLS, but demonstrated limited activity in pig PCLS, underscoring species-specific differences. This study demonstrates the therapeutic value of tissue-specific promoters in targeted gene therapy while emphasizing the importance of refining algorithmic prediction platforms to ensure reliable cross-species and clinical performance.