Patent Application: US-85316497-A

Abstract:
the present invention provides synthetic ribozyme oligonucleotides alone and within constructs . the ribozyme gene provides methods for the treatment of prostate hyperplasia and other androgen dependent pathologies . improved therapies for such diseases are provided without significant hormonal imbalance and without surgical intervention . also provided are techniques for selecting and synthesizing effective and specifically targeted molecular tools for use in inhibiting androgen receptor gene expression .

Description:
the examples presented herein are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . four oligodeoxynucleotides ( wild - type h1 ( seq id no : 13 ), wild - type hr2 ( seq id no : 2 ), mutant hr2 ( seq id no : 14 ), and antisense oligonucleotide hr2 ( seq id no : 15 ); see fig1 ; table 1 ) were synthesized by phosphoramidite method and purified on a 16 % polyacrylamide / 8m urea gel . the oligonucleotides were tailed with recognition sequences for restriction enzymes , sac i at their 5 ′ ends and ecor i at their 3 ′ ends . then , each oligonucleotide was ligated to a bluescript sk plasmid ( stratagene , la jolla , calif .) that had been digested with sac i and ecor1 , to allow expression of the hammerhead ribozymes or antisense oligonucleotides under the control of a t3 rna polymerase promoter . a 144 bp segment of human ar cdna containing a target site of the h1 hammerhead ribozyme , and a 234 bp segment of human ar cdna containing another target site of the hr2 hammerhead ribozyme were also cloned into the bluescript plasmid to generate ar mrna in vitro . for in vivo study , the full - length human ar cdna was cloned into a mammalian expression vector containing the human cytomegalovirus promoter to create pcmv - ar vector as a target . the mouse mammary tumor virus long terminal repeat promoter containing an ar response element was ligated with the chloramphenicol acetyl transferase gene ( cat ) to create a pmmtv - cat plasmid as a reporter . hammerhead ribozyme elements , that is the wild - type h1 and wild - type hr2 , the mutant hr2 , and the antisense hr2 oligo — all have the flanking hind iii site at the 5 ′ end and the xba i site at the 3 ′ end . thus they could be cloned into the hind ii / xba i sites of the pcdna3 mammalian expression vector ( invitrogen , san diego , calif . ), containing the cmv promoter , to create pcmv - h1 , pcmv - hr2 , pcmv - mut - hr2 and pcmb - anti - hr2 vectors . in order to generate pol ii directing expression plasmid with the hr2 wild - type hammerhead ribozyme , mutant hammerhead ribozyme and antisense hr 2 oligo elements , the corresponding double - stranded dna oligos were cloned in the xba i / sac i sites of a plasmid carries the rat u6 small nuclear rna promoter upstream of the cloning sites ( das , et al ., 1988 ). these constructs were designated as : u6 - hr2 , u6 - mut - hr2 and u6 - anti - hr2 , respectively ( fig1 ). the sequences of all constructs were confirmed by dna sequencing . to generate transcripts in vitro , the bluescript plasmids containing the ar cdna and different hammerhead ribozymes were linearized with a restriction enzyme . the transcription reactions were carried out with t3 or t7 rna polymerase as recommended by the supplier ( promega , madison , wis .). the ar gene transcripts were either labeled using [ α - 32 p ] utp , or synthesized with unlabelled ntps . the products were purified by electrophoresis in a 10 % polyacrylamide / 8m urea gel . the ar mrna substrate and the hammerhead ribozyme na were incubated at 37 ° c . in 50 mm tris - hci , ph 7 . 5 , 10 mm mgcl 2 , 2 mm spermine and 1 mm edta . after adding stop buffer and heating at 95 ° c . for 2 min ., the products were resolved y electrophoresis in a 10 % polyacrylamide / 8m urea gel . the products were detected by autoradiography or ethidium bromide staining . for time - course experiments , 100 μl of a mixture containing the hammerhead ribozyme and ar mrna substrate was incubated at a 1 : 1 molar ratio at 37 ° under the conditions described above . the reaction was followed by removing 10 μl of the mixture at different times and the reaction was quenched by adding 5 μl of 10 mm edta / 90 % formamide / 0 . 02 % xylene cyano1 / 0 . 01 % bromphenol blue . the labeled reaction products were separated on a 10 % polyacrylamide / 8m urea gel and quantified using a phosphorlmager ( molecular dynamics , inc ., sunnyvale , calif .). the mixtures of the hr2 hammerhead ribozyme ( 2 nm ) and the ar mrna substrates ranging from 8 nm to 70 nm were incubated at 37 ° c . in 50 mm tris - hci , ph 7 . 5 , 10 mm mgcl 2 , 2 mm spermine and 1 mm edta for 40 min . the reaction products were analyzed on a 10 % polyacrylamide / 8m urea gel and quantitated using a phosphorlmager ( molecular dynamics , inc .). pc - 3 cells which are ar negative and derived from human prostate adenocarcinoma were transfected using the calcium phosphate method ( chan , et al ., 1995 ). briefly , the pc - 3 cells were plated , grown and cotransfected with a reporter construct ( pmmtv - cat ), a target vector ( pcmv - ar ), and pcmv control vector together with hammerhead ribozyme expression vectors such as pcmv - h1 , pcmv - hr2 , pcmv - mut - hr2 , pcmv - anti - hr2 . after four hours , the transfection medium was replaced with normal growth medium with or without 10 − 9 m dht . cells were harvested 48 hours later and cat activity analyzed . for stable transfection , rat ar cdna was subcloned into a vector containing the neomycin gene ( invirogen , san diego , calif .). the vector containing the rat ar cdna was transfected into a monkey kidney carcinoma cell line , cv - 1 . transfected cv - 1 cells were selected in 0 . 5 mg of g418 / ml ( sigma , st . louis , mo .). individual colonies were expanded and screened by rt - pcr . the pmmtv - cat and pcmv control vectors were cotransfected with either pcmv - h1 or pcmv - hr2 into the stably transfected cells ( cv - 1 / ar ). nih 3t3 cells were also used in cotransfection studies . after 12 hours , the medium was replaced with normal growth medium with or without 10 − 9 m dht . the cells were harvested after 24 hours , and cat activity was detected by the cat - elisa assay . total rna from the transfected cells with or without hammerhead ribozymes was isolated according to the protocol provided in the rneasy kit ( qianga , chatsworth , calif .). briefly , 10 6 cells were washed three times with ice - cold pbs without ca ++ and mg ++ , and then were lysed with an rlt buffer containing guanidinium isothiocyanate . the lysate was mixed with an equal volume of 70 % ethanol and centrifuged through an rneasy spin column . impurities were removed from the column by washing it once with an rwi solution containing guanidinium isothiocyanate and twice with an rpe solution . total rna was eluted . the products were treated with rnase - free pancreatic dnassel ( promega ) in 10 mm mgcl2 / 0 . 1 mm dithiothreitol / 10 mm rnase inhibitor for 30 min . at 37 ° c . ( ojwang , et al ., 1992 ). to generate an antisense ar rna probe , rat ar cdna was digested with sst i to release a 179 bp fragment between 1697 and 1865 . the fragment was cloned into the ssti site of the bluescript vector . the vector containing 179 bp fragment of the ar cdna was linearized with xba i and an antisense ar rna probe was synthesized with [ α - 32 p ] utp , ctp , atp and gtp and t7 rna polymerase . the probe was purified through a 5 % polyacrylamide / 8m urea gel . the β - actin antisense rna probe was also synthesized as an internal control . rnase protection assays were performed using a ribonuclease protection assay kit rpaii ( ambion , inc ., austin , tex .). briefly , 1 and 8 μg of total rna were hybridized with 5 × 10 5 cpm of radiolabeled antisense β - actin rna probe and 5 × 105 cpm of radiolabeled antisense ar rna probe . the products were digested with a diluted rnasea / t1 mixture and precipitated with ethanol . the protected ar mrna and β - actin mrna products were separated on 5 % polyacrylamide / 8 m urea gels . the gels were dried , and ar mrna was quantitated using a phosphorlmager ( dynamic molecule , inc .). in order to detect hammerhead ribozyme expression in transfected cells , 200 ng of total na from different treated cells was subjected to rt - pcr using two primers , 5 ′- ttccgaactgatgagtcc - 3 ′ ( seq id no : 4 ) from the hr2 hammerhead ribozyme stem i region ( see fig1 ) and 5 ′- agtgggagtggcaccctt - 3 ′( seq id no : 5 ) from the polylinker sequence in the pcdna3 vector . two primers of β - actin gene , 5 ′- tgcgtgacattaaggagaagc - 3 ′ ( seq id no : 6 ) from position 667 to 687 , and 5 ′- atccacacggagtacttggg - 3 ′ ( seq id no : 7 ) from position 1063 to 1044 , were also synthesized for controls . one oligonucleotide of each primer pair was labeled with [ γ - 32 p ] atp and t4 kinase . the cycling conditions were as follows : 94 ° c . for 1 min , 57 ° c . for 1 min and 72 ° c . for 2 min for 21 cycles . the pcr products were separated in a 5 % polyacrylamide gel , and specific bands were quantitated using a phosphorlmager . the expression of the hammerhead ribozyme in different treated cells was analyzed and normalized to β - actin rna . immunohistochemical studies of pc - 3 cells transfected with or without the hammerhead ribozyme were performed following the experimental conditions described ( doumit , et al ., 1996 ). the cells were washed with pbs three times and fixed in pbs containing 2 % paraformaldehyde and 10 % sucrose , ph 7 . 2 , for 20 min , then permeabilized in pbs containing 10 % mouse serum for 30 min , and cells were incubated with primary ar antibody in blocking reagent overnight at room temperature . then the cells were washed three times in pbs and exposed to biotinylated goat anti - rabbit igg ( 1 : 100 ) as secondary antibody in vectastain elite abc reagent ( vector lab ., burlingame , calif .) for 30 min at room temperature . after three washes in pbs , ar positive staining with the enzyme activity produced a brown reaction product when exposed to 3 , 3 ′- diaminobenzidine ( sigma , st . louis , mo .) containing h 2 o 2 . a vax 8600 and a vax 8800 computer with the sequence analysis software package from genetic computer group ( university of wisconsin , madison , wis .) were used . theminimal fee energy fold of ar mrna was computed using mfold program version 8 . 1 . the mfold program predicts optimal and suboptimal rna secondary structures based on the energy minimum method ( zuker and stieger , 1981 ; zuker , 1989 ). graphic representations were obtained using squiggles ( university of wisconsin , madison , wis .). the following examples are presented only to describe preferred embodiments and utilities of the present invention , and are not meant to limit the scope of the present invention unless specifically indicated otherwise in the claims appended thereto . the present example demonstrates the site specificity of the synthetic ribozyme of the invention . two hammerhead ribozymes , h1 ( seq id no : 13 ) and hr2 ( seq id no : 2 ), cleave human androgen receptor ( ar ) mrna at the guc sequence at positions 1394 and 2375 , respectively . in an in vivo assay , both of these hammerhead ribozymes , h1 and hr2 , cleave the target ar mrna substrate into two products at the expected sites . the extent of cleavage varied with the time of incubation , and the molar ratio of ribozyme to substrate . at 30 seconds of incubation at 37 ° c ., hr2 cleaves 37 % of the target mrna at 1 : 1 molar ratio . complete cleavage of the target ar mrna by the two hammerhead ribozymes at 1 : 1 molar ratio occurs within 30 min . hr2 is more active than h1 . a mutant ribozyme ( mut - hr2 ; seq id no : 14 ) and a oligodeoxynucleotide antisense ( antisense hr2 oligo ; seq id no : 15 ) to the target ar mrna sequence fail to catalyze cleavage of the ar mrna substrate in vitro . mut - hr2 has mutations at two bases in the catalytic part ( stem ii ) and the antisense hr2 oligo lacks the catalytic part of hr2 ( fig1 ). the wild - type hammerhead ribozymes , h1 and hr2 , the mut - hr2 and the antisense hr2 oligo were cloned into a mammalian expression vector ( pcmv ) utilizing the rna polymerase ii promoter to create pcmv - h1 , pcmv - hr2 , pcmv - mut - hr2 , and pcmv - anti - hr2 , respectively . these constructs were tested for their effects on ar gene expression in culture prostatic cells . cotransfection of either the h1 or the hr2 expression construct into mammalian cells along with the ar expression plasmid ( pcmv - ar ) and an ar - responsive reporter plasmid ( pmmtv - cat ) results in the inhibition of cat activity . both the ar mrna level and the ar protein level also decline . the extent of the decrease in ar and mrna is dependent on the level of the expressed hammerhead ribozyme and the decreased ar mrna also correlates with the extent of inhibition in the cat activity . however , the β - actin mrna level is not affected , indicating that the hammerhead ribozymes h1 and hr2 target specifically the ar mrna . similar to the in vitro study , the hr2 ribozyme is more effective than the h1 ribozyme in vivo . the wild - type hr2 ribozyme is much more active in inhibiting ar mrna expression and cat activity than the corresponding mutant ribozyme ( mut - hr2 ) and the antisense hr2 . the present example demonstrates that the described synthetic hammerhead ribozymes are designed to cleave specifically ar mrna within cells , as well as utility therapy in vivo . the hr2 hammerhead ribozyme was cloned downstream of the rat u6 small nuclear rna promoter which is transcribed by rna polymerase iii , to give u6 - hr2 plasmid . compared with pcmv - hr2 , which is transcribed by pol ii , u6 - hr2 is more efficient in inhibiting ar mrna and cat activity in vivo . a 1 : 5 molar ratio of pcmv - ar : u6 - hr2 achieved 90 % reduction of cat activity , whereas 90 % reduction of cat activity will require a 1 : 100 molar ratio of pcmv - ar : pcmv - hr2 . the present example demonstrates the selection of ar mrna regions targeted by hammerhead ribozymes . the predicted primary and secondary structures of the entire ar mrna was searched for open loops that contain the consensus sequence for hammerhead ribozyme cleavage , 5 ′- hux - 3 ′, which is cleaved 3 ′ of x ( x can be a , c or u ). rna substrate with the guc triplet adjacent to cleavage site was reported to yield very high cleavage efficiency compared with rna substrate containing other triplet sequences such as cuc , gua and aua ( rufner , et al ., 1990 ; shimayama , et al ., 1995 ; hendix , et al ., 1996 ). all guc triplets in the open loop regions were tagged as potential cleavage sites for the hammerhead ribozyme . then both sides of sequences surrounding these guc triplets were scanned through the genbank data base to eliminate sequences with substantial homology to other mrnas . in order to provide more discrimination and ensue a high rate of cleavage by the hammerhead ribozyme , a / u - rich regions of the flanking sequences ( stems i and iii ) were chosen , because of their generally lesser stability than g / c - rich regions . the greater stability of a g - c base pair diminishes the probability of dissociation of the cleavage products . the presence of a - rich sequences in the flanking sequences of the hammerhead ribozyme also would avoid the possibility of u - g wobble pair formation that would tend to decrease the specificity of the enzyme ( herschlag , et al ., 1991 ; bertrand , et al ., 1994 ). two potential target sites were selected within the open reading frame of the ar mrna to cleave between residues 1393 - 1394 and between residues 2208 - 2209 . these sites are targeted by ribozymes h1 and hr2 , as shown in fig3 in open stem - loop regions of the mrna . one sequence targeted by h1 consists of 19 residues , 58 % a / u - rich . the sequence targeted by hr2 has 18 residues , 61 % a / u - rich ( fig1 ). in vitro sequence - specific catalytic cleavage of the ar mrna by the hammerhead ribozymes cleavage reactions of the 144 nt and 234 nt fragments of the ar mrna substrate by the h1 and hr2 ribozymes in vitro are shown in fig4 . cleavage is dependent on time and the molar ratio of ribozyme : substrate . using a 1 : 1 molar ratio of ribozyme : substrate , both ribozymes completely cleave the ar mrna substrate in less than 30 min , generating the expected products : for h1 , a 104 nt and a 40 nt product from the 144 nt ar mrna substrate ( fig4 a ), and for hr2 , a 182 nt and a 52 nt fragment from the 234 nt ar mrna substrate ( fig4 b ). when 32 p - labeled 234 nt fragment of the ar mrna and unlabeled hr2 are incubated at a 1 : 1 molar ratio at 37 ° c . for different time intervals , 50 % of cleavage products are observed at about 13 min ( fig4 c ). on the other hand , when different ratios of the ribozyme and substrate are mixed , complete cleavage of the 234 nt fragment of the ar mrna is observed using a 1 : 3 molar ratio of hr2 : ar mrna substrate in 30 min at 37 ° c . ( fig5 a ). by contrast , complete cleavage of the 144 nt fragment requires a 1 : 1 molar ratio of h1 : substrate in the same time ( fig5 b ). the results show that hr2 is more active than is h1 . steady - state cleavage velocities were measured for 2 nm of hr2 with mrna substrate concentrations ranging from 8 to 70 nm . when the different concentrations of 32 p - labeled 234 nt substrate were incubated with cold hr2 , the ribozyme was effectively saturated with substrate at high concentrations . under the reaction conditions at 37 ° c . in 10 mm mgcl2 / 50 mm tris - hci , ph 7 . 5 / 2 mm spermine / 1 mm edta for 40 min , hr2 ribozyme cleaved more products of the rna substrate with increasing the rna substrate concentrations ( fig6 ). in vitro cleaved the 234 nt substrate with high efficiency . that these hammerhead ribozymes specifically recognize their target sequences was demonstrated by the fact that hr2 was totally ineffective in catalyzing the cleavage of the ar mrna substrate for h1 ( fig7 a , lane 3 ), and vice versa ( fig7 a , lane 6 ). point mutations ( ac and gu ) in the catalytic domain of hr2 ( fig1 ) resulted in complete loss of the catalytic activity in vitro ( fig7 b , lanes 7 , 10 ). the specificity elements of hr2 by antisense hr2 deoxyoligo also failed to cleave the 234 nt ar mrna substrate ( fig7 b , lanes 5 , 8 ). the mutant hr2 hammerhead ribozyme construct , pcmv - mut - hr2 , was tested in transfection studies to distinguish the antisense effect from the enzymatic activity on the ar mrna substrate . efficiency and specificity of the hammerhead ribozyme in transient contransfection assays toward the target ar mrna in cell culture in order to demonstrate the efficiency of h1 and hr2 inactivation of ar gene expression at the cellular level , h1 and hr2 ribozymes , mutant ribozyme ( mut - hr2 ) and the antisense oligo hr2 alone ( without the catalytic loop ) were cloned into a mammalian expression vector ( pcmv ) and transfected them into pc - 3 cells with the target vector ( pcmv - ar ) and the reporter vector ( pmmtv - cat ). an analysis of the expression of pmmtv - cat showed that cat activity was inhibited with increasing doses of hammerhead ribozyme transfected in the form of pcmv - h1 and pcmv - hr2 . at 1 : 20 , 1 : 50 and 1 : 100 molar ratios of target : h1 ribozyme , cat activity was reduced by 10 %, 55 % and 80 %, respectively , relative to that transfected with the control vector ( fig8 a ). also , cat activity was reduced by 60 %, 75 % and 95 %, respectively , at the same molar ratios of the target / hr2 ribozyme ( fig8 b ). these results demonstrate the efficacy of the transfected ribozyme in cultured cells . to assess catalytic activity versus antisense effect of the ribozyme on inactivating ar gene expression , wild - type hr2 ( pcmv - hr2 ) with mutant hr2 were compared , which lacks catalytic activity ( fig7 b , lanes 7 , 10 ) and antisense rna hr2 ( pcmv - anti - hr2 ). the mutant ribozyme and antisense rna had inhibitory effects , but much smaller than with wild - type hr2 . at 1 : 50 and 1 : 100 molar ratios of substrate to ribozyme or antisense rna , wild - type hr2 inhibited 6 % and 95 % of the cat activity , respectively , while the mutant ribozyme inhibited 37 % and 60 %, respectively , and the antisense rna only 20 % and 65 %, respectively ( fig9 ). these results showed that inhibition of the cat activity is due mainly to the catalytic property of the hammerhead ribozyme , not to antisense effect . the vector containing the ar cdna was transfected into cv - 1 cells that were derived from monkey kidney tumor cells . positive clones expressing ar mrna were screened by rt - pcr . a cv - 1 clone with stably expressing ar mrna ( cv - 1 / ar ) was transfected with pmmtv - cat in the presence of 10 − 8 mdht or absence of dht . chloramphenicol acetyl transferase activity was induced nine - fold by dht ( fig1 ). when cv - 1 / ar cells were cotransfected with pmmtv - cat and either pcmv control vector , or pcmv - h1 , or pcmv - hr2 and cultured in the presence of dht , cat activity of the cells transfected with the ribozyme was nine - fold lower than that of the cells cotransfected with the control vector ( fig1 ). the ar hammerhead ribozymes can be concluded to inactivate the ar mrna expression with high specificity and efficiency . it has been observed that a hammerhead ribozyme whose expression is under the control of an rna pol iii promoter is more effective in inhibiting target gene expression as compared to one whose expression is under the control of an rna pol ii promoter ( cotton and brinstiel , 1989 ; yu et al ., 1993 ; michienzi et al ., 1996 ; perriman et al ., 1996 ). to determine if this could more efficiently inhibit ar mrna expression , hr2 wild - type , mutant ribozyme and antisense oligo were subcloned downstream of the rat u6 small nuclear gene promoter which is driven by the rna pol iii , to yield u6 - hr2 , u6 - mut - hr2 and u6 - anti - hr2 constructs . the pcmv - ar and pmmtv - cat , along with either u6 - hr2 , or u6 - mut - hr2 , or u6 - anti - hr2 or u6 control vector were transfected into nih3t3 cells by the calcium phosphate method ( chan et al ., 1995 ). for comparison , pcmv - hr2 was used in parallel ( fig8 ). the data showed that u6 - hr2 was more effective in inhibiting cat activity in nih3t3 cells ( fig1 ). at a 1 : 5 molar ratio of pcmv - ar : u6 - hr2 , cat activity was reduced by about 90 % ( fig1 ), whereas the same reduction in cat activity in the case of the pcmv - hr2 required a 1 : 100 molar ratio of the pcmv - ar : pcmv - hr2 ( fig8 ). in agreement with the findings of yu et al . ( 1993 ), the ribozyme under the control of the rna pol iii promoter exhibits stronger inactivation of gene expression than under the control of the rna pol 11 promoter ( yu et al ., 1993 ). the mutant ribozyme and anti sense rna yielded less than 20 % of inhibition of cat activity at the same molar ratio ( fig1 , lanes 4 , 5 ). the present example demonstrates that the decrease in cat activity results directly from a decrease in ar mrna due to the action of the ribozyme . total rnas were isolated from the pc - 3 cells transfected with the ar cdna expression vector and either pcmv - hr2 , or pcmv - mut - hr2 or pcmv - anti - hr2 at 1 : 25 , 1 : 50 or 1 : 100 molar ratios . the rnase protection assays were performed using an antisense ar rna probe that spans the expected cleavage site of the ar mrna and analyzed the ar mrna levels in different treatments of the pc - 3 cells . the results shown in fig1 a , b and c indicate that ar mrna normalized to the amount of the control β - actin mrna in each sample was lower in cells transfected with hammerhead ribozyme . wild - type hr2 more efficiently inhibited ar mrna at different molar ratios of the pcmv - ar : pcmv - hr2 than the control , and was more effective in inhibiting ar mrna expression than the mutant ribozyme and antisense rna . when different concentrations of wild - type and the mutant hr2 were transfected into pc - 3 cells , the levels of ribozyme expression were detected by quantitative rt - pcr , using specific primers from the stem i sequences of the hr2 ribozyme ( fig1 c ). taken together , these results show that the inhibitory activity is dependent on the level of ribozyme expression in cultured cells . in order to further study the effects of the ribozyme transcribed by rna pol iii on inhibition of ar mrna expression , pcmv - ar expression vector was transfected into pc - 3 cells with either u6 - hr , or u6 - mut - hr , or u6 - anti - hr2 , or u6 control vector at 1 : 5 and 1 : 25 molar ratios . ribonuclease protection assays were performed to analyze ar mrna levels in different transfected cells . results as shown in fig1 demonstrate that the wild - type ribozyme is not only more active in inhibiting the target ar mrna than the mutant ribozyme and antisense rna , but also more effective than hr2 which is transcribed by the rna pol 11 ( compare fig1 with fig1 ). the decrease in ar mrna by the ribozyme correlates with the decrease in cat activity . however , the products resulting from the cleavage of the ar mrna by the ribozyme . were not detected . these cleavage products may be degraded too quickly to be detected ( cofton and bimstiel , 1989 ; yuan et al ., 1992 ; sullenger and cech , 1993 ; xing and whitton , 1993 ; lieber and strauss , 1996 ). in this study , it was shown that the proximal 5 ′ flanking promoter region of the ar gene lacks obvious tata or caat boxes , but contains a pur / pyr - rich region . this region is conserved in human , rat and mouse species . in the rat , six copies of the gggga repeat sequence from position − 123 to − 94 are located immediately upstream of the gc - rich box which is bound by sp1 nuclear transcription factor . in recent studies , it has been demonstrated that the pur / pyr - rich region can form a non - b dna conformation and plays an important role in a number of tata - less gene promoters such as the promoter for the epidermal growth factor receptor , human ha - ras , human c - myc genes and others ( cooney et al ., 1988 ; hoffman et al ., 1990 ; grigoriew et al ., 1992 ; roy , 1993 ; mayfield et al ., 1994 ; mouscadat et al ., 1994 ). the present invention provides evidence that the pur / pyr - rich region of the ar gene can form a non - b dna conformation that is sensitive to the single - strand specific s1 nuclease . fine mapping analysis reveals that both dna strands of the pur / pyr - rich region are cleaved by s1 nuclease and form an asymmetric cleavage pattern . further studies show that the pur / pyr - rich region forms a triple helical h - form dna conformation . this pur / pyr - rich region can be bound by antiparallel and parallel purine rich oligonucleotides , but not by pyrimidine - rich tfos or random dna sequences under physiological conditions . gel mobility shift assays and dnase i footprinting studies show that the pur / pyr - rich region binds a novel pyrimidine single - strand dna binding protein and also a double - strand dna binding protein , the nuclear transcription factor sp1 . mutation of the region showed that the pur / pyr - rich region serves as an enhancer element and indicates an important regulatory element of ar gene expression . the novel pyrimidine - single - stranded dna binding protein will be cloned to determine how the protein regulates the ar gene transcription . also , the relationship between the single - stranded dna binding protein and nuclear transcription factor sp1 in the pur / pyr - rich region will be investigated . in addition , tfos will be designed to study its effect on ar gene expression . in the present study , two hammerhead ribozymes were designed to target specific guc specimens in ar mrna . in a cell - free system , both hammerhead ribozymes can cleave ar mrna at the expected sites , but the mutant hammerhead ribozyme and antisense oligonucleotide do not cleave the target mrna sequences . at 1 : 1 molar ratio of substrate : hammerhead ribozyme , both hammerhead ribozymes cleave the mrnas completely within 30 min at 37 ° c . the hammerhead ribozyme recognizes only its target sequences , and catalyzes the cleavage of the mrna substrates , indicating its specificity and efficiency . the hammerhead ribozyme can cleave the ar mrna in cultured cells . this effect is due to the endonuclease activity rather than to the antisense effect . compared to the hi hammerhead ribozyme , the hr2 hammerhead ribozyme is more effective in vitro and in vivo . the hr2 hammerhead ribozyme driven by the rna pol iii promoter is more powerful than the one driven by the rna pol ii promoter . transgenic mice expressing hammerhead ribozyme have been created ( efrat et al ., 1994 ; larsson et al ., 1994 ; l &# 39 ; huillier et al ., 1996 ). the hammerhead ribozyme inhibits target gene expression to different levels in such mice . in order to detect whether ribozyme can inhibit endogenous ar mrna in cells . lncap cells derived from human prostate cancer cells were chosen . it produces endogenous ar mrna and prostate specific antigen ( psa ). psa expression is dependent on androgen and ar . when hr2 ribozyme expression vector was transfected into lncap cells , psa activity was analyzed 48 hour post - transfection . results showed that the ribozyme inhibit 50 % of psa activity compared to the control group . in further studies , transgenic mice expressing hammerhead ribozyme targeting the ar gene will be generated . the hammerhead ribozyme will be cloned downstream of the rat probasin promoter that is specific for prostate tissue ( greenberg et al ., 1994 ). hammerhead ribozyme expression by rt - pcr in transgenic mice will be detected . transgenic mice expressing the hammerhead ribozyme will be selected . to examine ar gene and hammerhead ribozyme expression in prostate glands , histological studies of the prostate gland in the transgenic mice will also be conducted . ar regulates the development of the male reproductive organs . a hammerhead ribozyme contains antisense sequence to the target substrate . for this reason , it is important to establish whether this type of ribozyme truly functions as a catalytic rna endonuclease or whether the “ activity ” is due instead to an antisense effect in cultured cells . a wild - type hammerhead ribozyme and a mutant one with substitutions in the catalytic core were designed . the mutant hammerhead ribozyme ( mut - hr2 ) and the antisense oligo ( antisense hr2 ) do not cleave the target rna substrate in vitro ( fig7 b ). the wild - type , mutant hammerhead ribozymes and the antisense rna oligo in vivo were further examined . it was found that both the mutant hammerhead ribozyme and the antisense rna oligo have inhibitory effects on ar mrna expression and cat activity , but the wild - type hammerhead ribozyme is much more inhibitory than the antisense oligo and / or the mutant hammerhead ribozyme ( fig9 and 11 ). it was further shown that the hammerhead ribozyme action is due to endonuclease activity rather than to an antisense effect ( scanlon et al ., 1991 ; ojwang et al ., 1992 ; lange et al ., 1993 ; inokuchi et al ., 1994 ). the levels of ar mrna and the cat activities in transfected cells suggested that the expression of ar mrna is inversely correlated with the expression of hammerhead ribozyme . with increasing amounts of transfected hammerhead ribozyme , ar gene expression is further suppressed . to achieve a higher effect of hammerhead ribozyme on its substrate , it must be used in excess and properly localized . a promoter driving a hammerhead ribozyme must be much stronger than a promoter generating a target rna substrate . compared with the ribozyme transcribed by the rna pol iii , a higher dose of the ribozyme transcribed by the rna pol ii is required to achieve the same degree of inhibition . it is known that the yield of rna transcribed by the rna pol ii is lower in cell systems than that transcribed by the rna pol iii ( brafty et al ., 1993 ; bertrand et al ., 1994 ; chowrira et al ., 1994 ). the rna pol ii system is usually employed for mrna transcription or expression of a long antisense rna . this system generates the cap and poly a structures that are required for the stability of rna in vivo . however , rna pol ii is not suitable for production of short rna molecules ( sanfacon and hohn , 1990 ). in contrast , rna pol iii produces small rnas at a higher rate of transcription and in various tissues ( coften and brinstiel , 1989 ; perriman et al ., 1996 ). using rna pol iii , it is possible to generate a short hammerhead ribozyme in cells with a stable secondary structure that protects the hammerhead ribozyme from nuclease attack ( perriman et al ., 1996 ). it has been reported that a hammerhead ribozyme driven by the rna pol iii promoter in a cell culture system can reduce target gene expression to a greater extent than a hammerhead ribozyme driven by the rna pol ii promoter ( cotten and brinstiel , 1989 ; yu et al ., 1993 ; thompson et al ., 1996 ). studies were conducted to compare the ability of the hammerhead ribozyme transcribed by rna pol ii and by rna pol iii promoters to inhibit ar mrna expression in cultured cells were undertook . the hr2 ribozyme was cloned into expression vectors with either a rna pol ii promoter ( pcmv - hr2 ) or a rna pol iii promoter ( u6 - hr2 ). it was found that the u6 - hr2 driven by rna pol iii dramatically inhibits ar mrna level and cat activity ( fig1 and 13 ). the cat activity and ar mrna are reduced by about 90 % and 70 %, respectively , at a 1 : 5 molar ratio of the target vector ( pcmv - ar ): ribozyme ( u6 - hr2 ). similar inhibition of ar expression and cat activity by the hr2 ribozyme driven by rna pol ii promoter requires a 1 : 100 molar ratio of target vector ( pcmv - ar ): hammerhead ribozyme ( pcmv - hr2 ) ( fig8 and 12 ). this demonstrates that the rna pol iii promoter . more efficiently promotes transcription of the hammerhead ribozyme gene than does the rna pol ii promoter . moreover , detection of the ar mrna by ribonuclease protection assay and of the ar protein level by immunohistochemistry in cultured cells provide direct proof for the hammerhead ribozyme effect in vivo . the present example defines a method to be used in the treatment of human prostate hyperplasia . the ribozyme construct will include a ribozyme gene sequence as provided in seq id no : 9 and a promoter sequence from prostate specific antigen gene ( psa ). this promoter sequence may be derived from the psa available at gen bank accession no : u37672 . as part of the claimed invention the method for treating prostate hyperplasia comprises administering a pharmacologically active preparation that includes a vector construct of the ribozyme gene as defined at seq id no : 9 and a promoter sequence of psa in an amount effective to reduce antigen receptor gene expression . a pharmacologically active amount of the preparation as used in the description of the present invention as defined as an amount that will provide a reduction of androgen receptor gene expression sufficient to provide a clinically detectable reduction or at least inhibition of prostate gland enlargement . while any variety of carrier vectors may be employed , it is anticipated that adenoviral and retroviral vector constructs may be used in particular applications of the claimed method . the gene therapy methods provided herein may be used alone or in combination with other treatments , such as surgical removal / reduction of prostate gland and / or administration of enzyme inhibitors , such as alpha reductase . cyproterone acetate ( schering ag ) along with proscar ™ ( merck ) may also be employed in combination with the claimed methods to provide improved clinical outcome for the patient . all of the compositions and methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined herein . the above is a detailed description of particular embodiments of the invention . those with skill in the art should , in light of the present disclosure , appreciate that obvious modifications of the embodiments disclosed herein can be made without departing from the spirit and scope of the invention . all of the embodiments disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . the full scope of the invention is set out in the claims that follow and their equivalents . the claims and specification should not be construed to unduly narrow the full scope of protection to which the present invention is entitled . the following references , to the extent they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . adler , a j ., scheller , a , and robins , d . m . 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