Robust prostate-specific expression for targeted gene therapy based on the human kallikrein 2 promoter.
Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.
Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.
X
X
Xie
X
X
Zhao
Y
Y
Liu
C Y
CY
Young
D J
DJ
Tindall
K M
KM
Slawin
D M
DM
Spencer
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009368:Neoplasm Transplantation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011467:Prostate / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D020840:Tissue Kallikreins / Q000096:biosynthesis* / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D020840:Tissue Kallikreins