CN1323344A - DNA zyme and method for treating restenosis - Google Patents
DNA zyme and method for treating restenosis Download PDFInfo
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Abstract
This application provides a DNAzyme which specifically cleaves c-myc mRNA, comprising a 15-nucleotide catalytic domain and two binding domains, one binding domain contiguous with the 5' end of the catalytic domain and the other binding domain contiguous with the 3' end of the catalytic domain. This invention also provides a pharmaceutical composition for inhibiting the onset of restenosis, which comprises the instant DNAzyme and a pharmaceutically acceptable carrier suitable for topical administration. This invention further provides an angioplastic stent for inhibiting the onset of restenosis, which comprises an angioplastic stent operably coated with a prophylactically effective dose of the instant pharmaceutical composition. Finally, this invention provides methods for inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering either the instant pharmaceutical composition or angioplastic stent to the subject.
Description
Invention field
The present invention relates to adopt DNAzyme to suppress the onset of restenosis.Described DNAzyme reaches this purpose by the mRNA of cutting coding c-myc, and the expression of c-mcy is that the generation of restenosis is needed in vascular smooth muscle cell.
The background of invention restenosis
Restenosis is a kind of serious internal disease, and it takes place after the angioplasty of being everlasting.This disease has tormented all angioplasty patients' 30%-60%.
It is believed that restenosis be by (to small part by) smooth muscle cell after the blood vessel injury that takes place in the revascularization process (" SMC ' s ") too breeds and causes.Think that several biological regulators help the propagation of this SMC.These conditioning agents comprise Thr6 PDGF BB (" PDGF "), fibroblast growth factor (" FGF ") and rhIGF-1 (" IGF ") (Ross; Banscota; Libby; Gay).(Kindy takes place to the inducing action of SMC propagation via transactivation in the born of the same parents of many important gene in these conditioning agents; Gadeau).These genes comprise c-myc, c-myb, c-fos and PCNA (proliferating cell nuclear antigen), and generally are cell cycle specifics.
Specifically, in blood vessel injury 30 minutes to 2 hours, c-myc is overexpression in SMC ' s, and is expressed in after this 12 hours and is reduced to normal level.That is to say that angioplasty causes the vascular SMC damage, the latter causes c-myc at damage beginning in back 30 minutes to 2 hours overexpression, and finishes in back 12 hours in damage.
Adopt radiotherapy and pharmacological treatments treatment restenosis at present.Radiotherapy comprises radioactive implant or the radioactivity composition is delivered on the site for the treatment of.Although radiotherapy has demonstrated some achievements likely, the long-term side-effects of crown radiation in having produced.About pharmacological treatments, anti--thrombotin and the antiproliferative method of using so far generally all do not have effect (Bennet).
DNAzvme
In the human gene therapy, antisense technology has been to select to be used to one of main method that makes those expression cause disease and therefore undesirable gene inactivation.The method of described antisense is used a kind of nucleic acid molecule, and it is complementary to the mRNA molecule of the undesirable gene of coding, and therefore hybridization with it.This hybridization has caused the inhibition of genetic expression.
Antisense technology has some shortcoming.The result of antisense hybridization is the formation of DNA/ target mRNA heteroduplex.This heteroduplex is as the substrate of the degraded target mRNA composition of RNA enzyme H mediation.At this, described DNA antisense molecule is with the passive mode effect, because it only helps the required cutting of endogenous RNA enzyme H.Thisly form the ability aspect of stablizing heteroduplex, limited the design of antisense molecule to the chemical property that relies on antisense molecule of RNAse H with its target mRNA.The antisense DNA molecule also has and non-specific active associated problem, under higher concentration even have and the toxicity associated problem.
As the surrogate of antisense molecule, catalytic nucleic acid molecule shown and has been hopeful as being used for the therapeutical agent of inhibition of gene expression, and extensive discussions (Haseloff in the literature; Breaker (1994); Koizumi; Otsuka; Kashani-Sabet; Raillard; And Carmi).Like this, be different from conventional antisense molecule, catalytic nucleic acid molecule is by its target mRNA molecule of real cutting rather than only with it in conjunction with working.If target sequence satisfies some subsistence level, then catalytic nucleic acid can only cut the target nucleic acid sequence.Described target sequence must be complementary to the hybridization zone of catalytic nucleic acid, and this target must comprise distinguished sequence at cleavage site.
There are many data to put down in writing catalysis RNA molecule (" ribozyme ") (Haseloff; Symonds; And Sun), showing and that they can either cut RNA molecule (Haseloff) again can cutting DNA molecule (Raillard).In fact, the development in external selection and evolution technology has made the random variants or the stochastic sequence RNA of the known ribozyme of employing obtain to become possible (Pan at the new ribozyme of known substrate as starting point; Tsang; And Breaker (1994)).
Yet ribozyme is extremely sensitive to the enzymic hydrolysis effect in the cell that they act on.This itself has limited their pharmaceutical application again.
Recently, created the new catalytic molecular (Breaker (1995) that is called " DNAzyme " of a class; Santoro).DNAzyme is a strand, cutting RNA (Breaker (1994); Santoro), also cutting DNA (Carmi).Propose the universal model of DNAzyme, be called " 10-23 " model.DNAzyme according to " 10-23 " model also abbreviates " 10-23DNAzyme " as, and it comprises the catalytic domain of 15 deoxyribonucleotides, and flank is two substrate differential thresholds that contain 7-9 deoxyribonucleotide respectively.Analyzed in vitro shows, such DNAzyme can be at purine under physiological condition: the pyrimidine junction is cut its substrate RNA (Santoro) effectively.
DNAzyme shows and promises to be medicine.Yet it is unpredictable that DNAzyme successfully resists the disease that the existence owing to known mRNA causes.This unexpected property part is owing to two factors.At first, some mRNA secondary structure may hinder DNAzyme in conjunction with and the ability of cutting its target mRNA.Secondly, express the suction possibility not enough result that effectively provide treatment meaning of the cell of target mRNA to DNAzyme.Owing to these reasons, the origin cause of formation target mRNA sequence of only knowing disease and it can not allow people reasonably to expect treatment success at the DNAzyme of target mRNA separately, because lack an invention step.
Summary of the invention
The application provides specificity cutting c-myc the DNAzyme of mRNA, it comprises (a) catalytic domain, it has nucleotide sequence GGCTAGCTACAACGA, and at any purine: the cutting of pyrimidine cleavage site towards it, (b) in abutting connection with catalytic domain 5 ' terminal in conjunction with the territory and (c) in abutting connection with terminal another of catalytic domain 3 ' in conjunction with the territory; Wherein saidly be complementary to two zones that flank is right after the purine residue of the cleavage site that needs the DNAzyme catalyze cleavage among the c-myc mRNA respectively in conjunction with the territory, and therefore hybridization with it, and wherein each is at least 6 Nucleotide in conjunction with length of field, and two are at least 14 Nucleotide in conjunction with territory bonded length overall.
The present invention also provides the medicinal compositions that is used to suppress the restenosis onset, and it comprises described DNAzyme and is applicable to the medicinal acceptable carrier of topical.
The present invention also is provided for suppressing the angioplasty stent of restenosis onset, and it comprises the angioplasty stent of the described medicinal compositions of operationally coating the prevention effective dose.
The present invention also provides the method that is used for suppressing standing curee's restenosis onset of angioplasty, generally gives the described medicinal compositions that described curee prevents effective dose before and after when it is included in angioplasty.
At last, the invention provides the method that is used for suppressing standing curee's restenosis onset of angioplasty, when it is included in angioplasty before and after the described angioplasty stent of the described curee of topical administration.
The accompanying drawing summary
Fig. 1 shows the structure of " 10-23 " DNAzyme that describes among the Santoro.Represent cleavage site by the asterisk between X and the Y.Represent substrate-by N ' s in conjunction with the territory.
Fig. 2 shows the design of c-myc RNA-cutting DNA zyme.The cleavage site of c-myc DNAzyme is selected in initial subcipher of AUG of human c-myc mRNA (second exon).As shown in the figure, cutting occurs between A and the U.
Fig. 3 shows optimized DNAzyme brachium and chemically modified.Designed c-myc-cutting DNA zyme with different brachiums based on " 10-23 " type.3 '-3 ' terminal bases that is illustrated in 3 ' end by shade C or G (3 ' INV) is inverted.
Fig. 4 shows many conversion (multiple turnover) kinetics.Figure A shows the photo densitometry image of 16% polyacrylamide gel, and it is to adopt phosphorescence imager (PhosphorImager) (Molecular Dynamics) and obtain, and is presented under many switch conditions the cutting to synthetic c-mycmRNA.All reactions all use the substrate mRNA (as shown) of 200pM DNAzyme and 2nM, 4nM, 8nM, 16nM and 32nM to carry out.The incubation time of each reaction indicated on the top of per pass between 0-60 minute.Figure B shows DNAzyme cutting process (nM) curve of each concentration of substrate.These data are to obtain from the spectrodensitometry of the cutting band shown in the figure A.
The cutting of the outer c-myc mRNA of Fig. 5 display body.Having
32From the PGEM carrier, transcribe 1.5kb c-myc mRNA substrate under the situation of P-UTP.Cleavage reaction is in 10mMMgCl
2, carried out 60 minutes under the 50mM Tris.HCl pH7.5,37 ℃.
Fig. 6 shows the stability analysis of 3 '-inversion DNAzyme in the human serum.DNAzyme is with human serum (Sigma) incubation of AB type.Shown in different time points collect sample, and use
32The P mark.The DNAzyme of this mark analyzes in 16% polyacrylamide gel.Here the typical gel banding pattern (going up right corner) of the DNAzyme of unmodified and the typical gel banding pattern (following right corner) of 3 '-inverted DNAzyme have been shown.
Fig. 7 is presented among the SV-LT-SMC ' s test to c-myc mRNA cutting DNA zyme.SMC ' the s of cessation of growth cessation stimulates having under the situation of following material with 10%FBS-DME (the improved Eagle substratum of Dulbecco that comprises 0.5% N of tire serum): 10mM is called anti--c-myc mRNA DNAzyme (following), 10mM control oligonucleotide (the arm sequence identical with Rs-6 contains and be inverted catalytic core sequence) or the independent liposome (DOTAP of Rs-6; Be N-[1-(2,3-two oily acyloxy)]-N, N, N-TMA (TriMethylAmine)-Methylsulfate).Data are represented with mean value SD.
Fig. 8 shows the dose-response experiment of Rs-6 DNAzyme among the SMC ' s.This experiment is described in detail according to Fig. 7.Data are represented with the percentage ratio of contrast.
Fig. 9 is presented at the expression of c-myc among the SMC ' s that DNAzyme handles.As usefulness as described in the embodiment 7
35S-methionine(Met) labeled cell, and carry out immuno-precipitation with the proteic expression level of c-myc among the SMC ' s that determines the DNAzyme processing.
Figure 10 shows the genomic dna sequence (exons 1 and 2) of human c-myc gene.
Detailed Description Of The Invention
The object of the invention is to adopt the DNAzyme technology to suppress the ISR onset. The onset of described disease, the physical injury by arterial smooth muscle in the angioplasty process triggers, and it is characterized in that soon after the c-myc overexpression several hours. This c-myc overexpression causes the SMC hyper-proliferative, itself suppresses the ISR onset and suppress this overexpression. The present invention uses c-myc mRNA specific DNA mye by the front and back when the angioplasty and utilizes " window on opportunity " of this c-myc overexpression in damage zone, therefore cuts mRNA and suppresses the ISR onset.
In particular, the application provides the DNAzyme of specificity cutting c-myc mRNA, it comprises (a) catalytic domain, it has nucleotide sequence GGCTAGCTACAACGA, and at any purine towards it: the cutting of pyrimidine cleavage site, (b) in abutting connection with terminal one of catalytic domain 5 ' in conjunction with the territory, and (c) in abutting connection with terminal another of catalytic domain 3 ' in conjunction with the territory; Wherein saidly be complementary to respectively two zones that flank is right after the purine residue of the cleavage site that needs the DNAzyme catalyze cleavage among the c-myc mRNA in conjunction with the territory, and therefore with it hybridization, and wherein each is at least 6 nucleotides in conjunction with length of field, and two overall lengths in conjunction with the territory combination are at least 14 nucleotides.
Here used " DNAzyme " refers to specific recognition and cuts the dna molecular of special target nucleic acid sequence, and it can be DNA or RNA. Described DNAzyme cutting RNA molecule, it is " 10-23 " type as shown in Figure 1, so name is because historical reasons. Such DNAzyme has been described in Santoro. The RNA target sequence that 10-23 DNAzyme needs is by NNNNNNNR*YNNNNNN、
NNNNNNNNR
*YNNNNN or NNNNNNR*Any RNA sequence that YNNNNNNN consists of, wherein R*Y is cleavage site, and R is A or G, and Y is U or C, and N is arbitrarily G, U, C or A.
In parameter of the present invention, can change arbitrarily in conjunction with the length (being also referred to as " brachium " here) in territory, can be identical or different. Envisioned various changes such as 7+7,8+8 and 9+9, and in the embodiment of back, more fully enumerated. Confirmed well in conjunction with the length in territory longer, so it with complementary mRNA sequence just in conjunction with tighter. Therefore, in preferred embodiments, each length in conjunction with the territory is 9 nucleotides. In one embodiment, described DNAzyme has TGAGGGGCAGGCTAGCTACAACGACGTCGTGAC sequence (being also referred to as " Rs-6 " at this).
In the treatment of using based on DNAzyme, DNAzyme in born of the same parents under the environment anti-degraded and stable as much as possible be important. A method that reaches this purpose be by shown in one or more of DNAzyme terminal add one 3 '-3 '-be inverted. More particularly, 3 '-3 '-be inverted (at this also referred to as " inversion ") refer to terminal nucleotide and 3 ' carbon atom of the nucleotides that is adjacent between covalency phosphoric acid bonding. General phosphoric acid bonding between 3 ' and 5 ' carbon atom of such bonding and adjacent nucleotide is reverse, thereby " inversion " word is arranged. Therefore, in preferred embodiments, 3 ' terminal nucleotide residue adjacency catalytic domain 3 ' terminal in conjunction with the territory in be inverted. Except being inverted, described DNAzyme can also comprise the nucleotides of modification. The nucleotides of modifying comprises such as N3 '-P5 ' phosphamide (phosphoramidate) key and peptide-nucleic acid key. These are well known in the art (Wagner).
In the present invention, the purine of any adjacency in c-myc mRNA: pyrimidine nucleotide is to can be as cleavage site.In preferred embodiments, purine: uridylic is the purine of wishing: the pyrimidine cleavage site.
The c-myc mRNA zone that comprises described cleavage site can be any zone.For example, needing the position of DNAzyme-catalyze cleavage among the c-myc mRNA can be translation initiation site, montage recognition site, 5 ' non-translational region and 3 ' non-translational region.In one embodiment, cleavage site is positioned at translation initiation site.
The DNA of the human c-myc mRNA sequence and/or the described sequence of encoding is well-known (Bernard)." c-myc mRNA " as used herein is meant by human c-myc dna sequence dna shown in Figure 10 or by its any any mRNA sequence of the polymorphic form coding of existence naturally.C-myc mRNA comprises ripe mRNA and immature mRNA.In parameter of the present invention, determine c-myc mRNA cleavage site, each is in conjunction with the required sequence in territory and determine that therefore global DNA zyme sequence can carry out according to well-known method.
The present invention also provides the medicinal compositions that is used to suppress the restenosis onset, and it comprises described DNAzyme and is applicable to the medicinal carrier of accepting of topical.
In the present invention, the described medicinal compositions of topical administration can adopt any different methods well known by persons skilled in the art and transfer system to realize or finish.For example, topical administration can be finished via conduit and local injection with via the coating stent of following discussion.
The pharmaceutical carrier that is used for topical administration is widely known by the people in the art, and described carrier and promoting agent blended method to be passed also are widely known by the people.Below using the transfer system of many conventional carriers that use, is the representative that many anticipations are used to give the embodiment of described composition.
The localized delivery system comprises as gel and solution, and can comprise vehicle such as solubilizing agent, penetration enhancers (as lipid acid, fatty acid ester, fatty alcohol and amino acid), and hydrophilic polymer (as polycarbophil and polyvinylpyrrolidone).In preferred embodiments, described pharmaceutically acceptable carrier is liposome or Biodegradable polymeric.The example that can be used for liposome of the present invention comprises as follows: (1) CellFectm, cation lipoid N, N
I, N
II, N
III-tetramethyl--N, N
I, N
II, N
III1: 1.5 (M/M) Liposomal formulation of-four palmityls (palmityl) spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2: 1 (M/M) Liposomal formulations (GlenResearch) of a kind of cation lipoid and DOPE; (3) DOTAP (N-[1-(2,3-two oily acyloxy)-N, N, N-trimethyl ammonium-Methylsulfate] (Boehringer Manheim); (4) Lipofectamine, 3: 1 (M/M) Liposomal formulations (GIBCO BRL) of polycation lipoid DOSPA and neutral lipoid DOPE.
The present invention also is provided for suppressing the angioplasty stent of restenosis onset, and it comprises the angioplasty stent that can operate the described medicinal compositions of coating the prevention effective dose.
The angioplasty stent is also named other title as " stent in the blood vessel " or simple " stent ", and they are well known in the art.They are through being usually used in stoping the vessel sealing that is caused unusually by physical property, and are ingrown as the undesirable vascular tissue that surgery damage causes.They often contain the piped, the expansible grid type structure that match with their function, and selectively biodegradable.
In the present invention, described stent can adopt any suitable method known in the art operationally to be coated with the above medicinal compositions.At this, " operationally coating " stent is meant coating by this way: in case give the stent of described coating, then allow to discharge described medicinal compositions in surrounding tissue to be treated in good time.This coating process for example, can adopt polymer poly pyrroles (polypyrrole).Stent, the method and composition that is used to coat above-mentioned substance go through in United States serial 60/091,217.
The prevention effective dose of determining described medicinal compositions can adopt conventional method of calculation to finish based on animal data.In one embodiment, described prevention effective dose is included in the described DNAzyme between about 0.1mg and the about 1g.In another embodiment, the prevention effective dose is included in the described DNAzyme between about 1mg and the 100mg.In an embodiment again, described prevention effective dose is included in the described DNAzyme between about 10mg and the 50mg.In yet another embodiment, described prevention effective dose comprises the described DNAzyme of about 25mg.
The present invention also provides the method for restenosis onset among the curee who suppresses to stand angioplasty, the described medicinal compositions of the front and back topical administration curee effective dose when described method is included in angioplasty.As used herein, " front and back " when angioplasty give described medicinal compositions, carry out before or after can or following closely at this intra-operative.Give and to carry out in accordance with known methods, as the conduit transmission." inhibition " restenosis onset is meant and weakens the seriousness that betides angioplasty restenosis afterwards, or stops the restenosis onset fully.In preferred embodiments, inhibition restenosis onset is meant and stops the restenosis onset fully.
At last, the invention provides the method for restenosis onset among the curee who suppresses to stand angioplasty, the described angioplasty stent of topical administration curee when described method is included in angioplasty.
By understanding the present invention better, person skilled in the art will readily appreciate that still they just illustrate the present invention who describes more comprehensively as in the following claim book with reference to following embodiment.In addition, enumerated different documents among the application.The content of these documents is attached among the application by reference, more fully to describe the technology status in field under the present invention.Embodiment
The vitro characteristics of embodiment 1 anti--c-myc DNAzyme is identified
The vitro efficacy of DNAzyme is definite by the speed of RNA cutting under the many switch conditions of measurement.About these experiments, the concentration range that adopts substrate is so that [S] 〉=10 times [E], and [E] is fixed on 200pM.Described DNAzyme and
32The synthetic RNA substrate of P-mark respectively at 37 ℃ at 50mM Tris.HCl pH 7.5,10mM MgCl
2With pre-equilibration among the 0.01%SDS 10 minutes.When time zero, DNAzyme and substrate mixed begin reaction.Get in order and the aliquots containig of quencher in 90% methane amide, 20mM EDTA and application of sample dyestuff in different time points at the reaction process post analysis then.Product fragment in these samples and responseless substrate are by electrophoretic separation in 16% denaturing polyacrylamide gel.The level of response of each time point is passed through to be determined by the spectrodensitometry of the gel images of phosphorescence imager (MolecularDynamics) generation.Adopt k
ObsValue (coming) by the slope of these time-histories experiment be created in best-fit in the improved Eadie-Hofstee curve line (k
ObsTo k
Obs/ [S]).In this way, K
mAnd k
CatValue divide other negative slope and y intercept to provide with the tropic.
Adopt the effectiveness (Fig. 4) of the external DNAzyme catalyze cleavage of the short c-myc RNA of many conversion kinetic measurement synthetic sequence.Contain DNAzyme and their unmodifieds contrast and excessive of three kinds of modifications of symmetric 7,8 and 9 base pair substrate brachium conjunctivums
32The synthetic c-myc RNA of P-mark is incubation together.According to k
ObsValue is determined kinetics parameter K
mAnd k
Cat(table 1).
Pass through k
At/ K
mTotal catalytic efficiency of each DNAzyme of measuring of ratio obviously different between the kind of kind of modifying and unmodified.In galianconism DNAzyme (7+7bp), comprise a modification of being inverted base and produce k
Cat/ K
m3 demultiplications little.With the negative of this nicking activity should be opposite, long-armed kind (9+9bp) is owing to have inversion base modification, relative efficiency to strengthen 10 times.The brachium conjunctivum DNAzyme of intermediate length (8+8bp) is subjected to the minimum that influences of modification, shows k
Cat/ K
mValue increases by 2 times.3 ' therefore ,-influence of being inverted terminal bases is according to the length of substrate brachium conjunctivum and difference.In galianconism (7+7bp) DNAzyme, find that modification is harmful to catalytic efficiency.Yet in long-armed (9+9bp) molecule, it improves catalytic activity really.The activity of the DNAzyme of unmodified is with 8bp substrate brachium conjunctivum the best.In galianconism (7bp) DNAzyme, its total efficiency is mainly due to higher K
m(3.4-23nM) and lower.Surpass among the DNAzyme of 8bp (as 9bp) at brachium, because K
mRelative rising (3.4-7nM) and k
CatReduce (0.11-0.06min
-1), gross activity reduces.
Like this, for c-myc mRNA cutting DNA zyme, find that the optimum Cutting efficient of unmodified pattern is the pattern that contains the 8bp arm.According to k separately
Cat/ K
mValue, the 7bp of the c-myc DNAzyme of unmodified and the pattern total efficiency of 9bp are lower.
The kinetics pattern of the c-myc cutting molecule of these three kinds different sizes is owing to comprising the inversion of 3 '-terminal nucleotide and changing significantly.The influence that this dna modification effect is learned c-myc RNA cutting power is obvious especially in short 7bp arm DNAzyme.With the contrast of unmodified type, this molecule is according to its k
Cat/ K
mBe worth inefficent basically.Yet weakening by two other Nucleotide of interpolation in 8bp modification type of this catalytic efficiency recovers even strengthens.This show short dna zyme active weaken owing to DNA/RNA more interactional disturb (be inverted by Nucleotide and cause) to cause, it can be restored to 8bp by increasing arm lengths.Length by further increase modification type DNAzyme is found another slight improvement of catalytic efficiency to 9bp.The situation of this and unmodified type DNAzyme forms contrast, and the situation proof of unmodified type DNAzyme is found active sharply decline during to 9bp when the length that increases arm from 8bp.
These results prove that 8bp is the optimum length of unmodified type DNAzyme to c-myc RNA cutting.The arm lengths of 9bp seems to provide best catalyze cleavage activity in 3 '-inversion type DNAzyme.Contain the weakening of sloping portion reflection enzyme turnover ratio of the catalytic efficiency seen in the unmodified type DNAzyme of 9bp arm, show as lower k
CatValue.This lower turnover ratio may be since described DNAzyme to reactant avidity enhanced results, avidity the dissociating of product of slowing down again itself.Avoided this active weakening in the DNA that is inverted modification by terminal bases, this may be the result to the interactional destabilization of enzyme-product.
Table 1
The kinetics of c-myc-cutting DNA zyme
| ??DNAzyme | Brachium | Modify | k cat(min -1) | ?K m(nM) | ????k cat/K m?(pM -1·min -1) |
| ???Rs-1 | ??7+7 | Do not have | ????0.25 | ???23 | ????10.8 |
| ???Rs-2 | ??7+7 | 3 '-be inverted | ????0.16 | ???50 | ?????3.2 |
| ???Rs-3 | ??8+8 | Do not have | ????0.11 | ???3.4 | ??????32 |
| ???Rs-4 | ??8+8 | 3 '-be inverted | ????0.24 | ????4 | ??????60 |
| ???Rs-5 | ??9+9 | Do not have | ????0.06 | ????7 | ?????8.6 |
| ???Rs-6 | ??9+9 | 3 '-be inverted | ????0.26 | ????4 | ??????65 |
Analyze three kinds of different lengthss, all at the kinetics of the c-myc RNA cutting of the DNAzyme of initial son (modification with unmodified).Be reflected at 10mM MgCl
2Under the situation of 50mMTrisHCl pH7.5, carry out containing under many switch conditions of at least 10 times of excessive substrates.
The external cutting of embodiment 2 total length c-myc mRNA
Total length c-myc mRNA is used for further test dna zyme (10mM MgCl under the physiological condition that stimulates
2, pH7.5,37 ℃) and the ability of c-myc mRNA of cutting different types.Cleavage reaction carries out under single switch condition, adopts long substrate of 10nM (c-myc mRNA) and 50nMDNAzyme.
Fig. 5 has shown that all DNAzyme cut c-myc mRNA effectively with the cutting speed of 20-50%.According to expectation, the DNAzyme of longer arm more effectively cuts substrate.The modification of 3 '-inversion base has reduced the cutting efficiency of 7+7 arm DNAzyme, but has strengthened the cutting efficiency of 9+9 arm DNAzyme.What is interesting is that the DNAzyme cutting efficiency between the DNAzyme of preheating and not preheating does not have difference.This result shows that the accessibility of the cleavage site among the c-mycmRNA is not subjected to the influence of mRNA secondary structure.The chemically modified of embodiment 3DNAzyme and stability
The stability of following methods analyst DNAzyme in 100% human AB serum.Briefly, with the unlabelled DNAzyme of 150 μ M incubation in 37 ℃ of human serum that are incubated at 100 μ l 100%, took out repeat samples 5 μ l in 0,2,4,8,24,48 and 72 hours at time point.During sampling, (10mM TrisHCl, pH7.5 1mlEDTA) join in the aliquot sample of 5 μ l, and carry out phenol/chloroform extraction with 295 μ l TE immediately.The all samples of each time point γ-
32The P-ATP end mark, and be not further purified or precipitate just directly leakage of electricity swimming in the 16%PAGE gel, DNAzyme and degraded product that all are complete shown like this.The result shows that 3 '-3 ' inversion at 3 '-end has improved the stability (t of DNAzyme in human serum widely
1/2=20 hours), and the DNAzyme of unmodified shows transformation period＜2 hour (Fig. 6).
The propagation of the inhibition SMC of embodiment 4DNAzyme mediation
In blood vessel SV40LT (simian virus 40 large T antigen) smooth muscle cell (Simons), test the activity of anti--c-myc DNAzyme.In 0.5%FBS-DMEM after the cessation of growth cessation, by add 10% FBS-DMEM with SMC ' s from G
0Middle release.Simultaneously with cellular exposure in the DNAzyme or control oligonucleotide (promptly containing the 9/9 arm DNAzyme that is inverted catalytic core sequence) that transmit by DOTAO.Growth-inhibition ability of 72 hours detection DNAzyme after transmitting.The scope that the data presentation SMC quantity of different DNAzyme shown in Figure 7 reduces is between 30%-80%, and the not discovery of employing contrast reduces.Based on these analytical resultss, the activity of the most effective molecule Rs-6 (9/9 arm that contains 3 '-inversion base) further detects in dose-response analysis (Fig. 8).Compared with the control, Rs-6 is being low to moderate the growth that obviously suppresses SMC under the concentration of 50nM.Embodiment 5 anti--c-myc DNAzyme are to the influence of SMC cell cycle
DNAzyme to SMC outgrowth influence also adopt two independently technical evaluations, promptly DNA cell cycle analysis and mitotic index determines.The DNA histogram is generation in 72 hours behind serum stimulation.Behind this interval of 72 hours, remain on G with the irritation cell that has only 65%
0/ G
1Compare, 74% not irritation cell remains on G
0/ G
1Yet,, remain on G owing to add DNAzyme Rs-6
0/ G
1The ratio of the irritation cell of phase increases to 71%.Under the contrast, unactivated DNAzyme contrast (Rs-8) is to the not influence of SMC cycle.Prove these results by the mitotic index (be mitotic numerical value in per 1000 cells, determine) of determining to stimulate back 72 hours SMC colonies with microscope.
Table 2
Anti--c-myc DNAzyme is to the influence of the smooth muscle cell proliferation of serum-stimulation
The proteic expression of c-myc among the SMC ' s of embodiment 6DNAzyme transfection
| ???G0/G1(％) | ???S(％) | ???G2/M(％) | Mitotic index (%) | |
| Do not stimulate DOTAP Rs-6 Rs-8 (contrast) | ????73.66 ????65.24 ????70.81 ????67.81 | ????8.56 ???12.59 ????9.93 ???12.33 | ????13.39 ????16.62 ????14.12 ????15.19 | ?????0.5 ?????1.9 ?????0.3 ?????2.2 |
For the effectiveness of anti--c-myc DNAzyme is described, the proteic expression of c-myc among the SMC ' s that employing immuno-precipitation analyzing DNA zyme handles on molecular level.Briefly, SMC ' s did not stagnate 72 hours in having the substratum of serum, cultivated 1 hour in 37 ℃ in no met-free substratum (comprising 5% dialysis foetal calf serum) then.After removing substratum, for the cell replacing contains 5% dialysis foetal calf serum, 100mCi/ml
35The met-free substratum of S-Met and 5mMDNAzyme, and cultivated in addition 2 hours.Adopt as described that method prepares cellular lysate, and adopt the agarose bonded anti--c-myc antibody test c-myc albumen.As shown in Figure 9, determine that it is proteic synthetic to suppress c-myc significantly with anti--c-myc DNAzyme treatment S MC ' s as immuno-precipitation by the metabolic marker material.SMC is with the expression not influence of control oligonucleotide (Rs-8) incubation to c-myc.
Reference Ausubel, people such as F.M.., Analysis of proteins.Cuttent Protocols in MolecularBiology (1995) the 2nd volume, 10.18.3, John Wiley﹠amp; Sons, Inc.Banscota, people such as N., (1989) Mol.Endocrinol. (3): 1183-1190.Bennet, M.R. and Schwartz, S.M. (1995) Circulation 92:1981-1993.Bernard, O., Deng the people., (1983) EMBO J 2:2375-2383.Breaker, R.R. and Joyce, G. (1994) Chemistry and Biology 1:223-229.Breaker, R.R., Joyce, G.F (1995) Chem.﹠amp; Biol. (2): 655-600.Carmi, people such as N, (1996) Chemistry and Biology 3:1039-1046.Gadeau, A. wait the people., (1991) J.Cell Physiol. (146): 356-361.Gay, G., Winkles, J. (1991) Proc.Natl.Acad.Sci.USA (88): 296-300.Haseloff, J., Genach, W.L. (1988) Nature (334): 585-591.Kashani-Sabet, people such as M., (1992) Antisense Research and Development 2:3-15.Kindy, M, Sonenshein, G. (1986) J Biol.Chem.261:12865-12868Koizumi, M. wait the people., (1989) Nucleic Acids Research 17:7059-7069.Libby, P (1992) J.Vasc.Surg. (15): 916-917.Otsuka, E. and Koizumi, M., Japanese Patent the 4th, 235, No. 919.Pan, T. and Uhlenbeck, O.C. (1992) Biochemistry 31:3887-3895.Raillard, S.A. and Joyce, G.F. (1996) Biochemistry 35:11693-11701.Ross, R. wait the people., (1986) Cell 46:155-169.Santoro, S.W., Joyce, G.F. (1997) Proc.Natl.Acad Sci USA 94:4262-4266.Simons, people such as M.. (1994) J.Clin.Invest.93:2351-2356Sun, L.Q. wait the people. (1997) Mol.Biotechnology 7:241-251Symonds, R.H. (1992) Annu.Rev.Biochem.61:641-671.Tsang, J. and Joyce, G.F. (1994) Biochemistry 33:5966-5973. U.S. sequence number 60/091, application on June 30th, 217,1998.Wagner,R.W.(1995)Nature?Medicine?1:1116-1118.
Claims (11)
1. specificity is cut the DNAzyme of c-myc mRNA, and it comprises
(a) catalytic domain, it has nucleotide sequence GGCTAGCTACAACGA,
And at any purine towards it: the cutting of pyrimidine cleavage site,
(b) in abutting connection with described catalytic domain 5 ' terminal in conjunction with the territory and
(c) another of the described catalytic domain 3 ' end of adjacency is in conjunction with the territory; Wherein saidly be complementary to two zones that flank is right after the purine residue of the cleavage site that needs the DNAzyme catalyze cleavage among the c-myc mRNA respectively in conjunction with the territory, and therefore hybridization with it, and wherein each is at least 6 Nucleotide in conjunction with length of field, and two are at least 14 Nucleotide in conjunction with territory bonded length overall.
2. the described DNAzyme of claim 1, wherein each length in conjunction with the territory is 9 Nucleotide.
3. the described DNAzyme of claim 1 is inverted in abutting connection with described catalytic domain 3 ' the described of end in conjunction with the 3 '-terminal nucleotide residue in the territory wherein.
4. the described DNAzyme of claim 1, it has the TGAGGGGCAGGCTAGCTACAACGACGTCGTGAC sequence.
5. the described DNAzyme of claim 1, wherein the cleavage site in c-myc mRNA is a purine: uridylic.
6. the described DNAzyme of claim 1, wherein the cleavage site among the c-myc mRNA is positioned at and selects following zone: translation initiation site, montage recognition site, 5 ' non-translational region and 3 ' non-translational region.
7. be used to suppress the medicinal compositions of restenosis onset, it comprises the described DNAzyme of claim 1 and is applicable to the pharmaceutically acceptable carrier of topical.
8. the described medicinal compositions of claim 7, wherein said pharmaceutically acceptable carrier is selected from: liposome and Biodegradable polymeric.
9. be used to suppress the angioplasty stent of restenosis onset, it comprises the angioplasty stent of the medicinal compositions of the claim 7 of operationally coating effective preventive dose.
10. be used for suppressing standing the method for curee's restenosis onset of angioplasty, described method is included in the medicinal compositions of claim 7 of the effective preventive dose of the described curee of front and back topical administration of described angioplasty.
11. be used for suppressing standing the method for curee's restenosis onset of angioplasty, described method is included in the angioplasty stent of the described curee of the front and back topical administration claim 9 of described angioplasty.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9637498P | 1998-08-13 | 1998-08-13 | |
| US60/096374 | 1998-08-13 |
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| CN1323344A true CN1323344A (en) | 2001-11-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99811966A Pending CN1323344A (en) | 1998-08-13 | 1999-08-12 | DNA zyme and method for treating restenosis |
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|---|---|
| EP (1) | EP1117768A4 (en) |
| JP (1) | JP2002525037A (en) |
| KR (1) | KR20010072475A (en) |
| CN (1) | CN1323344A (en) |
| AU (1) | AU5298499A (en) |
| CA (1) | CA2340322A1 (en) |
| WO (1) | WO2000009672A1 (en) |
| ZA (1) | ZA200101188B (en) |
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|---|---|---|---|---|
| AUPQ201499A0 (en) * | 1999-08-04 | 1999-08-26 | Unisearch Limited | Treatment of inflammatory and malignant diseases |
| JP2002348235A (en) * | 2001-03-23 | 2002-12-04 | Clinical Supply:Kk | Preventive against restenosis |
| DE10346487A1 (en) * | 2003-10-02 | 2005-05-12 | Transmit Technologietransfer | Process for the preparation of a cell and / or tissue and / or disease phase specific drug |
| EP1909803A4 (en) * | 2005-06-28 | 2010-09-29 | Johnson & Johnson Res Pty Ltd | Guanosine-rich oligonucleotides as agents for inducing cell death in eukaryotic cells |
| EP3093022B1 (en) | 2015-05-15 | 2019-08-07 | Sterna Biologicals GmbH & Co. KG | Gata-3 inhibitors for use in the treatment of th2-driven asthma |
| AU2021414089A1 (en) * | 2020-12-30 | 2023-08-17 | The Regents Of The University Of California | A biologically stable xnazyme that efficiently silences gene expression in cells |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IE911115A1 (en) * | 1990-04-10 | 1991-10-23 | Canji Inc | Gene therapy for cell proliferative diseases |
| US5646042A (en) * | 1992-08-26 | 1997-07-08 | Ribozyme Pharmaceuticals, Inc. | C-myb targeted ribozymes |
| US6159946A (en) * | 1993-01-07 | 2000-12-12 | Thomas Jefferson University | Antisense inhibition of c-myc to modulate the proliferation of smooth muscle cells |
| US5807718A (en) * | 1994-12-02 | 1998-09-15 | The Scripps Research Institute | Enzymatic DNA molecules |
| BRPI9809433B1 (en) * | 1997-04-29 | 2015-09-08 | Scripps Research Inst | enzymatic DNA molecules |
| US6361941B1 (en) * | 1998-03-27 | 2002-03-26 | Johnson & Johnson Research Pty Limited | Catalytic nucleic acid-based diagnostic methods |
-
1999
- 1999-08-12 CN CN99811966A patent/CN1323344A/en active Pending
- 1999-08-12 WO PCT/IB1999/001484 patent/WO2000009672A1/en not_active Application Discontinuation
- 1999-08-12 KR KR1020017001893A patent/KR20010072475A/en not_active Application Discontinuation
- 1999-08-12 CA CA002340322A patent/CA2340322A1/en not_active Abandoned
- 1999-08-12 EP EP99938479A patent/EP1117768A4/en not_active Withdrawn
- 1999-08-12 JP JP2000565109A patent/JP2002525037A/en active Pending
- 1999-08-12 AU AU52984/99A patent/AU5298499A/en not_active Abandoned
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| KR20010072475A (en) | 2001-07-31 |
| AU5298499A (en) | 2000-03-06 |
| WO2000009672A1 (en) | 2000-02-24 |
| WO2000009672A9 (en) | 2000-05-18 |
| EP1117768A1 (en) | 2001-07-25 |
| EP1117768A4 (en) | 2003-09-03 |
| JP2002525037A (en) | 2002-08-13 |
| ZA200101188B (en) | 2002-05-13 |
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