Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vectors

The native PSA enhancer and promoter confer prostate-specific expression wh en inserted into adenovirus vectors capable of efficient in vivo gene deliv ery, although the transcriptional activity is low. By exploiting properties of the natural PSA control regions, we have improved the activity and spec ificity of the prostate-specific PSA enhancer for gene therapy and imaging applications. Previous studies have established that androgen receptor (AR) molecules bind cooperatively to AREs in the PSA enhancer core (-4326 to -3 935) and act synergistically with AR bound to the proximal promoter to regu late transcriptional output. To exploit the synergistic nature of AR action we generated chimeric enhancer constructs by (1) insertion of four tandem copies of the proximal AREI element; (2) duplication of enhancer core; or ( 3) removal of intervening sequences (-3744 to -2855) between the enhancer a nd promoter. By comparing to the baseline construct, PSE, containing the PS A enhancer (-5322 to -2855) fused to the proximal promoter (-541 to +12), t he three most efficacious chimeric constructs, PSE-BA (insertion of ARE4), PSE-BC (duplication of core) and PSE-BAC (insertion of core and ARE4), are 7.3-, 18.9-, and 9.4-fold higher, respectively. These chimeric PSA enhancer constructs are highly androgen inducible and retain a high degree of tissu e discriminatory capability. Initial biochemical studies reveal that the au gmented activity of the chimeric constructs in vivo correlates with their a bility to recruit AR and critical co-activators in vitro. The enhanced acti vity, inducibility and specificity of the chimeric constructs are retained in an adenoviral vector (Ad-PSE-BC-luc). Systemic administration of Ad-PSE- BC-luc into SCID mice harboring the LAPC-9 human prostate cancer xenografts shows that this prostate specific vector retained tissue discriminatory ca pability compared with a comparable cytomegalovirus (CMV) promoter driven v ector.