In vivo expression of prostate-specific adenoviral vectors in a canine model

Abstract

The activity and expression of transgene ␤-galactosidase (lacZ) by replication-deficient adenoviral vectors (Ad-lacZ) containing prostate-specific promoters were compared using an in vivo canine model. The prostate tissue-specific promoters were prostate-specific antigen, probasin, and mouse mammary tumor virus long-terminal repeat, which were fused separately to an Escherichia coli lacZ gene. Dogs underwent laparotomy, and adenoviral vectors were delivered by direct intraprostatic injection. At 72 hours postinjection, the prostate and various other organs were harvested to evaluate the degree of prostate expression and dissemination of adenoviral vectors. Expression of lacZ in tissues was determined by 5-bromo-4-chloro-3-indolyl ␤-D-galactoside staining, ␤-galactosidase assay, and E. coli lacZ reverse transcriptase-polymerase chain reaction (PCR). The presence of adenoviral DNA sequences in canine tissues was determined by PCR using primers specific for the type 5 adenoviral genome. All three of the prostate-specific adenoviruses tested effectively expressed the lacZ gene in the canine prostate, but expression levels were lower than that of the control viral vector AdRSVlacZ following intraprostatic injection. By PCR, adenoviral vector DNA was detected in other organs and tissues, including the bladder and vas deferens. However, reverse transcriptase-PCR analysis revealed that prostate-specific Ad-lacZ vectors only transcribed lacZ mRNA in the prostate and not in nonprostatic tissues. Thus, these novel prostate-specific adenoviral vectors each have equal in vivo expression exclusively in the prostate and may potentially be used for prostate cancer gene therapy.

p16/MTS1/INK4A suppresses prostate cancer by both pRb dependent and independent pathways

“…To ensure the quality of total RNA samples, the same RT mixture mentioned above was used for PCR of housekeeper gene b-actin. The primers speci®c to b-actin gene were 5'-TCCTGTGGCATCCAC-GAAACT-3' and 5'-GAAGCATTTGCGGTGGACGAT-3' which resulted a 314 bp PCR product (Steiner et al, 1999b). Same conditions for PCR described above were used for PCR of b-actin except the reaction was carried out at 948C for 4 min; then for 30 cycles at 948C for 1 min, 558C for 1 min, and 728C for 1 min; followed by at 728C for 10 min.…”

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

“…Gene Therapy adenovirus vectors (Ads) have the capacity to deliver genes in vivo and intra-prostatically, 20 and also have been utilized in several on-going human prostate cancer clinical trials. 21,22 Two Ads with either the entire 5.8-kb (−5825 to +12) PSA regulatory region or 1.6-kb (−5322 to −3738) enhancer linked to the proximal PSA promoter (−541 to +12), were able to direct either prostate-specific expression of cytotoxic HSVtk 19 or viral E1A 22 to induce lytic viral replication.…”

“…Homogenizations of the tissue were performed by a PowerGen 125 (Fisher Scientific) Protein concentrations of tissue homogenized lysates were measured by Coomassie Plus reagent (Pierce) according to the manufacturer's directions. [10][11][12][13][14][15][16][17][18][19][20] l of tissue lysate were measured in luminometer assays.…”

Regulation of transgene expression in muscles by ultrasound-mediated hyperthermia

“…Most earlier attempts have been focused on developing tissuespecific promoters (TSPs). 8 Indeed Steiner et al 9 recently successfully demonstrated the potential of a prostatespecific promoter in a canine model. However, a lot of TSP-based systems have not been very satisfactory due to low expression level in targeted tissue and/or high background in nontargeted tissues.…”