Patent Application: US-14277999-A

Abstract:
the present invention provides a method of inhibiting the proliferation of cells . the method comprises inhibiting induction or decreasing expression of egr - 1 or decreasing the nuclear accumulation or activity of the egr - 1 gene product . the present invention also provides a method of reducing the incidence of restenosis in a subject . the method comprises administering to the subject an agent which inhibits induction or decreases expression of egr - 1 or decreases the nuclear accumulation or activity of the egr - 1 gene product .

Description:
in order that the nature of the present invention may be more clearly understood the preferred forms of the present invention will now be described in greater detail . fig1 shows uptake of radiolabeled antisense e11 by smooth muscle cells ( passive ; ▪ “ lipofectamine ”). fig2 shows effect of oligonucleotides ( 1 μm ) on smooth muscle cell proliferation . in a survey of immediate - early genes that could be induced by acute vascular injury in the rat aorta , the expression of the early - growth - response gene product egr - 1 ( krox - 24 ; ngf - ia , zif268 , t1s8 ), a serum - inducible zinc - finger nuclear phosphoprotein and member of a family of related transcription factors ( 3 ) was examined . in this examination aortic endothelium of male sprague - dawley rats ( 400 g ) was partially denuded using an uninflated 2f balloon catheter . deendothelialized regions were identified by intravenous injection of evans blue ( 0 . 3 ml of 5 % solution in pbs ) 10 min prior to sacrifice . animals were perfusion - fixed with phosphate - buffered 4 % paraformaldehyde . vessel segments were treated with 1 μg / ml proteinase at 37 ° c ., prehybridized for 2 h at 55 ° c . in 0 . 3m nacl , 20 mm tris , ph7 . 5 , 5 mm edta , 1 × denhardt &# 39 ; s , 10 % dtt and 50 % formamide , and incubated with the appropriate 35 s - utp - labeled riboprobe for 16 h . after washing , the slides were coated with autoradiographic emulsion and exposed for 3 wk . the images were photographed and digitized . the hybridization signal of the radiolabeled probe appears as white grains . all specimens observed under dark field illumination after nuclear counterstain with hematoxylin . immunostaining for factor - viii - related antigen confirmed that injury was limited to endothelium . in situ hybridization techniques which visualize the endothelium of the vessel wall en face revealed that egr - 1 expression was dramatically induced exclusively at the endothelial wound edge within 30 min of partial denudation . egr - 1 expression was undetectable in endothelium from unmanipulated arteries . induced egr - 1 mrna was apparent after 2 h , and the time - dependent decrease in the specific hybridization signal demonstrates the transient induction of endothelial egr - 1 expression by injury . in contrast , the sense egr - 1 riboprobe failed to hybridize with mrna from normal or injured tissue . pdgf - b - chain transcript levels were also low in unmanipulated vessels , consistent with previous findings using other techniques ( 4 ). partial denudation did not induce pdgf - b gene expression at the endothelial wound edge until 4 h after injury and continued for several weeks during endothelial regeneration ( 5 ). the colocalization of the spatial patterns of egr - 1 and pdgf - b gene expression , and the temporal association between these two genes in injured arterial endothelium , led to a determination of whether egr - 1 could inducibly regulate the expression of pdgf - b . in response to mechanical injury in vitro , confluent endothelial cells initiate movement into the open “ wounded ” area by actively responding to locally - derived signals or autocoids from injured cells . an in vitro model of vascular injury ( 6 ) was used to address the possible link between egr - 1 and injury - induced pdgf - b gene expression . nuclear run - off analysis revealed that egr - 1 gene transcription was induced in cultured bovine aortic endothelial cells ( baec ) within 1 h of injury . 5 ′ deletion analysis of the pdgf - b promoter in endothelial cells previously defined a region necessary for core promoter activity ( d77 ) which contained a binding site for the ubiquitous transcription factor , sp1 ( 7 ). recent in vivo footprint analysis of the promoter demonstrates that the sp1 element is indeed occupied in intact cells ( 8 ). in vitro dnase i footprinting revealed that recombinant egr - 1 protected a region overlapping this site from partial dnase i digestion . when nuclear extracts from endothelial cells 1 h after injury were incubated with a 32 p - labelled oligonucleotide spanning this region ( 32 p - oligo b , 5 ′- gctgtctccacccacctctcgcactct - 3 ′ seq id no : 2 ), a distinct nucleoprotein complex formed . the injury - induced complex was eliminated by antibodies to egr - 1 . nuclear sp1 also bound to the pdgf - b promoter fragment ; however , its levels are unaltered by injury . thus , injury - induced endothelial egr - 1 expression precedes the induction of pdgf - b , and egr - 1 binds to a distinct region in the pdgf - b promoter also bound by sp1 . the functional importance of this interaction for pdgf - b promoter - dependent gene expression was next determined . northern blot and transient transfection analysis using pdgf - b promoter - reporter constructs previously revealed that this gene is basally expressed in vascular endothelial cells ( 7 ). chloramphenicol acetyltransferase ( cat ) expression driven by the pdgf - b promoter ( d77 - cat ) was induced by injury within 36 h . reporter activity also increased in cells exposed to phorbol 12 - myristate 13 - acetate ( pma ) or by cytomegalovirus - mediated overexpression of egr - 1 . when a mutation that abolished the ability of egr - 1 to bind to the pdgf - b promoter was introduced into the d77 - cat construct , basal expression driven by the promoter was attenuated , and expression inducible by injury was abolished . the mutant construct also failed to mediate increased reporter activity when egr - 1 was overexpressed , or when the cells were exposed to pma . the egr - 1 binding site in the proximal pdgf - b promoter is thus required for inducible promoter - dependent expression in vascular endothelial cells . the interaction of egr - 1 and sp1 with overlapping binding elements in the proximal pdgf - b promoter suggests that sp1 , resident on the promoter in unstimulated cells , may be displaced by increasing levels of egr - 1 . running gel shifts ( 9 ) indicate that recombinant egr - 1 bound to the pdgf - b promoter in a stable and reversible manner . the relative efficiency with which egr - 1 was displaced from 32 p - oligo b by its unlabelled counterpart indicates that egr - 1 interacts with the pdgf - b promoter with a faster off - rate than its comparable site in the proximal pdgf - a promoter ( 9 ). sp1 was displaced from the promoter by egr - 1 in a dose - dependent manner . decreasing levels of egr - 1 in the presence of a fixed concentration of sp1 allowed reoccupation of the promoter by sp1 . the absence of a higher order complex when both factors are present indicates that egr - 1 and sp1 do not bind the promoter simultaneously . these findings with recombinant proteins indicate that an interplay involving egr - 1 and sp1 can occur on the pdgf - b promoter . the localized induction of egr - 1 at the endothelial wound edge precluded a direct determination of whether a displacement mechanism was involved in the induction of pdgf - b gene expression by injury . pma is a model agonist of egr - 1 expression in vascular endothelial cells ( 9 ). the dramatic induction of egr - 1 mrna and protein that precedes the increase in pdgf - b levels in endothelial cells exposed to pma is like the temporal pattern with which these genes are expressed at the endothelial wound edge following arterial balloon injury . transcript and protein levels of sp1 are also not affected by pma . nuclear proteins from pma - treated endothelial cells bound to the pdgf - b promoter with a pattern similar to that observed using injury - induced extracts . immunobinding studies determined that nucleoprotein complexes contained either sp1 or egr - 1 . the profound induction in egr - 1 levels by pma demonstrates the ability of this transcription factor to displace sp1 from the pdgf - b promoter in the context of nuclear extracts . accordingly , the pma - inducible endothelial expression of the pdgf - b gene , like pdgf - a ( 9 ), involves an interplay between egr - 1 and sp1 at overlapping binding sites in the proximal promoter . this contrasts with a previous report suggesting that egr - 1 may serve as a negative regulator of gene transcription by blocking the binding of sp1 to its own recognition sequence ( 10 ). these findings suggest that the localized induction of pdgf - b expression at the endothelial wound edge may also involve displacement of promoter - bound sp1 by elevated levels of nuclear egr - 1 . egr - 1 may be involved in interactions with other transcriptional activators and the basal complex to mediate increased gene expression in response to injury . egr - 1 also appears to play a key role in injury - inducible pdgf expression in smooth muscle cells . in the rat arterial injury model in - situ hybridization with en face preparations indicate that egr - 1 expression is induced in smooth muscle cells concurrent with the expression of pdgf - a at the same location . the antisense approach is based on the ability of an oligonucleotide ( synthetic dna ) to recognize its complementary sequence within the cell , in the form of messenger rna ; the bound complex is then able to sterically interfere with ribosome binding and translation into protein ( 11 ). alternatively , the bound complex triggers cleavage of the mrna by the nuclease rnase h , which is widely present in mammalian cells and specifically recognizes dna - rna duplexes ( 12 ). thus , the overall effect of a given antisense oligonucleotide may be to reduce specific mrna and protein levels if mediated by rnase h , or a reduction in specific protein levels in the case of steric interference ( 13 ). advantages offered by the use of antisense oligonucleotides over conventional inhibitors are specificity and synthesis . this is based on the uniqueness of the target mrna and the general availability of oligonucleotide synthesizers . a drawback in this approach is the propensity of these oligonucleotides to be degraded or inactivated by nucleolytic phosphodiesterases . however , chemical modification of the phosphodiester linkages between individual nucleotides has been found to increase nuclease resistance by up to ten - fold and increase potency as a consequence ( 14 , 15 ). as explained above egr - 1 is an immediate - early gene ( 16 , 17 ) expressed at low or undetectable levels in arterial endothelial cells ( 18 ) or smooth muscle cells . it is dramatically induced by a number of ( patho ) physiologically - relevant agonists and conditions such as fluid shear stress , mechanical injury ( 18 ), heparin - binding growth factor - 1 , as well as the protein kinase c - inducer , phorbol 12 - myristate 13 - acetate ( 9 ). egr - 1 mrna is transcribed and processed in the nucleus ; it then enters the cytoplasm where it is translated to protein . since egr - 1 protein contains a nuclear targeting sequence , it reenters the nucleus and interacts with its nucleotide recognition sequence in the promoters of responsive genes . two genes which are induced by egr - 1 are those encoding the platelet - derived growth factor a - chain ( 9 ) and b - chain ( 18 ). this growth factor is a potent mitogen and chemoattractant for smooth muscle cells ( 19 ) and produced by cells involved in the atherosclerotic or restenotic lesion . accordingly , the pdgf ligand / receptor signalling system has been implicated in the pathogenesis of atherosclerosis . elevated levels of pdgf - a transcripts have been observed in human carotid plaques ( 20 ). the coexpression of pdgf - a with smooth muscle α - actin implicated smcs in the plaque as a source of pdgf - a ( 20 ). murry and colleagues ( 21 ) also found pdgf - a mrna in human atherosclerotic plaques using competitive rt - pcr . rekhter and gordon ( 22 ) used a triple immunolabeling approach to localize pdgf - a protein to smooth muscle - like cells and some endothelial cells within human carotid plaques . libby and colleagues showed that smcs cultured from human atherosclerotic plaques could express pdgf - a transcripts and secrete pdgf - like binding and mitogenic activity ( 23 ). barrett and benditt used northern blot and dot blot analysis to show that levels of pdgf - b - chain mrna were approximately five - fold greater in carotid plaques than normal aorta and carotid arteries ( 24 ). in situ hybridization later corroborated these findings demonstrating that the pdgf - b - chain was associated with endothelium at the luminal surface of the plaque and smooth muscle - like cells within the plaque ( 25 ). ross and coworkers used a double immunostaining technique with carotid endarterectomy specimens to detect pdgf - b protein in macrophages ( 26 ). a number of important considerations were undertaken in the design of hybridization - specific antisense oligonucleotides to egr - 1 . first , each oligonucleotide was synthesized with a phosphorothioate backbone for increased stability and potency . second , oligonucleotides were size - matched to 15 bases ; longer sequences were avoided to reduce the possibility of non - specific effects seen with longer oligonucleotides ( stein , c . a . ( 1996 ) trends in biotechnology 14 : 147 - 149 ). third , a sequence of four consecutive guanosines was avoided in light of recent reports indicating that oligonucleotides bearing this sequence , such as those that have targeted c - myb and c - myc , inhibit proliferation by a nonantisense mechanism ( 27 , 28 ). fourth , the efficacy of multiple oligonucleotides directed toward various regions of egr - 1 mrna was assessed . finally , a size - matched , fully phosphorothioated oligonucleotide with no sequence complementarity to any portion of egr - 1 mrna was used as a negative control . a panel of antisense oligonucleotides complementary to rat egr - 1 mrna were designed to identify regions within the mrna that were suitable target sites . putative target sites were chosen on the basis of being encoded within regions of the mrna that had low secondary structure and theoretically had a greater potential for inter molecular hybridisation . such single stranded regions were identified firstly by using the zuker algorithm for determination of free energy of rna molecules ( zuker , m ( 1989 ) science 244 : 48 - 52 ). once a low energy secondary structure was determined , regions of low frequency intramolecular base - pairing were identified by visual examination . the oligo nucleotides used are set out in table 1 one of these oligonucleotides , e11 , was radiolabeled with 32 p , and assessed for its ability to associate with cultured vascular smooth muscle cells . after various times , the cultures were washed with phosphate - buffered saline and removed from the vessel by scraping . after centrifugation , the cells were transferred to eppendorf tubes , solubilized and either counted in a scintillation counter or electrophoresed on a denaturing polyacrylamide gel . radiolabeled e11 associated with the cells in a time - dependent manner ; maximal uptake was observed after 6 h ( fig1 passive ). the oligonucleotide was still associated with the cells after 9 h and 24 h ( fig1 passive ). electrophoretic analysis indicated that the oligonucleotide did not undergo significant degradation during the course of experiment . the panel of oligonucleotides were assessed for their ability to inhibit smooth muscle cell proliferation in an assay of 3 h - thymidine incorporation into dna . oligonucleotides were added to the culture supernate 6 h after the medium was changed to serum - free at a final concentration of 1 μm and incubated for a further 18 h . the cells were washed and exposed again to 1 μm oligonucleotide in medium containing a concentration of serum that would stimulate 3 h - thymidine incorporation into dna submaximally . after a further 24 h incubation , the cells were pulsed for 6 h with 3 h - thymidine prior to the determination of tca - precipitable 3 h - thymidine incorporation into dna . the control oligonucleotide e1 , an oligonucleotide of random sequence bearing no complementarity to egr - 1 mrna , did not alter the rate of serum - inducible 3 h - thymidine incorporation into dna in smooth muscle cells ( n = 10 ) ( fig2 ). in contrast , two egr - 1 antisense oligonucleotides were able to inhibit dna synthesis . a / s2 and e11 , directed against different portions of egr - 1 mrna , inhibited by 63 % ( n = 10 ), and 50 % ( n = 12 ), respectively ( fig2 ). trypan blue exclusion studies and morphologic observations revealed that inhibition was unlikely to be due to non - specific cytotoxic mechanisms . in contrast , egr - 1 antisense oligonucleotides e6 , e7 or e9 failed to inhibit smooth muscle cell proliferation ( fig2 ). that not every egr - 1 antisense oligonucleotide could inhibit is consistent with the notion that naturally occurring mrna has higher order structure and certain sequences may not be as accessible to certain oligonucleotides as others . western blot analysis was used to assess the effect of egr - 1 antisense oligonucleotides on levels of serum - inducible egr - 1 . oligonucleotides at a final concentration of 1 μm , were added to the culture supernates 6 h after changing the medium to serum - free to render the cells quiescent . after 16 h , the cells were washed with phosphate - buffered saline and incubated with 1 μm oligonucleotide for a further 2 h . the cells were then exposed to a concentration of serum able to stimulate 3 h - thymidine incorporation into dna submaximally . after 2 h , the cell lysate was electrophoresed on denaturing polyacrylamide gels prior to transfer and then assessed for the presence of egr - 1 using specific antibodies . serum induced the synthesis of egr - 1 protein within 2 h . incubation of the cells with e1 did not affect the ability of serum to induce egr - 1 . in contrast , e11 and a / s2 profoundly inhibited the induction of egr - 1 protein . cellular levels of the related zinc - finger transcription factor , sp1 , were unaffected by either e11 or a / s2 demonstrating the target specificity of these oligonucleotides . taken together , these findings demonstrate that antisense oligonucleotides directed to selected regions of egr - 1 mrna reduce the accumulation of the egr - 1 protein . inhibition of egr - 1 is not due to non - specific or cytotoxic mechanisms . cells treated with these oligonucleotides do not undergo morphologic changes or take up trypan blue . moreover , whereas egr - 1 protein levels are profoundly attenuated by these oligonucleotide , levels of the related zinc - finger transcription factor , sp1 , are unaffected . these oligonucleotides can selectively inhibit smooth muscle cell proliferation . since egr - 1 is an inducible transcription factor with binding sites in multiple genes ( 9 , 18 ), these findings strongly suggest that the antiproliferative effects of these oligonucleotides may be due to selective inhibition of the expression of egr - 1 - dependent genes required for proliferation , such as pdgf ( 9 , 18 ), bfgf ( 29 ) or cell - cycle regulatory genes ( 30 ). egr - 1 is not basally expressed in smooth muscle cells or endothelial cells of the vessel wall , unless activated by mechanical injury ( 18 ). therefore , egr - 1 is an appropriate target for antiproliferative therapy . the various oligonucleotides used in these studies were delivered to the cells without a carrier . uptake is an energy - dependent process and is maximal at 37 ° c . ( 31 ). phosphorothioate oligonucleotides have been found to associate with an 80 kd protein on the cell surface , consistent with receptor - mediated endocytosis ( 32 , 33 ). passive delivery , however , is largely an inefficient process ( 34 ). the biologic effects of these oligonucleotides can be augmented by methodologies which facilitate more efficient delivery into cells , such as liposomes ( 35 ). indeed , such approaches may be useful in increasing the biological potency of e11 and a / s2 . the results presented herein indicate that “ lipofectamine ”, for example , can increase both the rate and total uptake of radiolabeled e11 ( fig2 ) without affecting the integrity of the oligonucleotide to any significant extent . since post - angioplasty restenosis is usually focal , local delivery of these oligonucleotides may be useful in the treatment of this disease , which in the united states occurred is approximately 30 - 50 % of the over 300 , 000 procedures performed in 1991 alone ( 36 ). it will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described . the present embodiments are , therefore , to be considered in all respects as illustrative and not restrictive . 1 . m . a . gimbrone , jr ., am . j . cardiol . 75 , 67b ( 1995 ); 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