Novel bile acid inducible promoters for adeno-associated viral vector gene therapy

Resumen

Bile acids (BA) homeostasis is mainly regulated by bile salt excretory pump (BSEP), a hepatocyte transporter that transfers BAs to bile. BSEP expression is regulated by BA levels through activation of farnesoid X receptor transcription factor, which binds to the inverted repeat (IR-1) element in BSEP promoter. Gene therapy of cholestatic diseases could benefit from using vectors carrying endogenous promoters physiologically regulated by BAs, however their large size limits this approach, especially when using adeno-associated viral vector (AAV) vectors. We evaluated the functionality and BA-regulation of minimal versions of human and mouse BSEP promoters containing IR-1 using AAV vectors expressing luciferase. Unexpectedly, a minimal mouse BSEP promoter (imPr) showed higher BA-mediated expression and inducibility than a minimal human promoter (ihPr) or than full-length BSEP promoters in human hepatic cells. In addition, in mice receiving an AAV vector carrying imPr promoter luciferase expression was efficiently regulated by administration of a BA-enriched diet. Interestingly, this vector also expressed significantly higher luciferase levels in Abcb4-/- mice, which have high levels of BAs, compared to wild type mice, or to mice receiving a vector containing luciferase gene downstream of constitutive alpha-1 antitrypsin promoter. In contrast, the AAV vector having ihPr showed very low luciferase expression with no inducibility. Interestingly, AAV vectors having genes coding for BSEP or MDR3 downstream of the minimal mouse BSEP promoter were able to control or revert disease symptoms in clinically relevant mouse models of PFIC2 and PFIC3.Finally, we optimized imPr by adding IR-1 repeats at its 5’ end. A new promoter provided higher levels of luciferase than imPr both in vitro and in vivo. In conclusion, these minimal BSEP promoters could represent useful tools for gene therapy approaches in which physiological BA regulation is desired.