CN101501193A - Methods and means for treating DNA repeat instability associated genetic disorders - Google Patents

Abstract

The current invention provides for methods and medicaments that apply oligonucleotide molecules complementary only to a repetitive sequence in a human gene transcript, for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders. The invention also pertains to modified oligonucleotides which can be applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.

Description

The ways and means that is used for the treatment of the inherited disease relevant with the DNA repeat instability

Technical field

The present invention relates to field of medicaments, particularly relate to the inherited disease for the treatment of and in its coding or non-coding sequence, having unstable multiple gene-correlation, the most especially, relate to and causing the choreoid HD gene of human Huntington or causing that the instability in the DMPK gene of I type myotonia atrophica repeats.

Background technology

Little satellite of gene specific and moonlet tumor-necrosis factor glycoproteins cause tumor-necrosis factor glycoproteins length increase in the satellite, and this unstable is relevant with about 35 kinds of people's inherited diseases.For example, trinucleotide repetition instability is found in and causes the chain spinobulbar muscular atrophy of X (spinal and bulbar muscular atrophy, SBMA), I type myotonia atrophica (myotonic dystrophy type 1, DM1), in the gene of fragile X mental retardation (FRAX gene A, E, F), Huntington chorea (HD) and some spinocerebellar ataxia diseases (SCA gene family).Instability repeats to be also shown in the coding region of gene (for example Huntington chorea gene), causes disease phenotype by changing protein function and/or protein folding thus.Unstable repeating unit is also shown in non-translational region, for example I type myotonia atrophica (DM1) in 3 ' UTR, perhaps in intron sequences, II type myotonia atrophica (DM2) for example.For DMPK, normal repeat number is about 5 to 37, but is increased to 2 to 10 times or more with complete morbid state before the sudden change, and as many as 50,100 is 1000 or more multiple multiple unit sometimes.For DM2/ZNF9, existing reporting is increased to 10000 or more multiple answer (Cleary and Pearson, Cytogenet.Genome Res.100:25-55,2003).

Cause the gene HD of Huntington chorea to be positioned at karyomit(e) No. 4.Huntington chorea is with the heredity of autosomal dominant mode.When the trinucleotide that surpasses 35 CAG that has one section polyglutamyl amine of coding when this gene repeated, repeat number can enlarge in the follow-up generation.Because this multiple length increases gradually, the severity of disease increases usually, and occurs at the littler age in the follow-up generation, and this process is called antedating.The product of HD gene is the cytoplasmic protein Huntington protein (huntingtin) of 348kDa.In normal form, Huntington protein has the characteristic sequence that is less than 40 glutamine amino-acid residues; Morbific sudden change Huntington protein has 40 residues of surpassing.The continuous expression of mutant Huntington protein molecule in neuronal cell causes forming big protein deposit, finally causes necrocytosis, especially in frontal lobe and basal ganglion (mainly in caudatum).The severity of disease generally is directly proportional with the number of extra residue.

DM1 is a modal muscular dystrophy among the grownup, and it is a kind of carrying out property, degeneration, multisystem inherited disease that mainly occurs in skeletal muscle, heart and the brain.DM1 is (Brook etc., Cell, 1992) of being caused by unstable trinucleotide (CTG) n multiple amplification in the 3 ' non-translational region of DMPK gene (myotonic dystrophy protein kinase) on the people 19q karyomit(e).II type myotonic dystrophy (DM2) is (Liquori etc., Science 2001) that the amplification by CCTG in the introne 1 of ZNF9 gene causes.Under the situation of I type myotonic dystrophy, the nuclear of DMPK transcript-kytoplasm output is blocked by the increase of this tumor-necrosis factor glycoproteins length, and it forms hair clip sample secondary structure, accumulates in nuclear speckle (nuclear foci).The DMPK transcript that has a segment length (CUG) n tumor-necrosis factor glycoproteins can form the hair clip spline structure, and the protein bound of itself and the blind albumen of flesh (muslceblind) family is assembled in the ribose nuclear speckle in nuclear (ribonuclear foci) then.These nuclear inclusion are considered to and the blind albumen chelating of flesh, and might with other factor chelating, pair cell produces restricted subsequently.In DM2, have increased the similar point of formation in the nuclear that is accumulated in of multiple ZNF9 RNA of (CCUG) n.Because the blind albumen of flesh is splicing factor, so their shortage causes the remarkable rearrangement of other transcript montage.Many gene transcription things begin aberrant splicing thereupon, for example comprise the fetus exon, or get rid of exon, and it is impaired to cause producing non-functional protein and cell function.

Argumentation above and neodoxy have brought such understanding, and the promptly unstable disease (for example I type myotonic dystrophy, Huntington chorea etc.) that repeats can cause the unusual transcript of this disease to be treated by removing fully or to small part.For DM1, the unusual transcript of accumulation can be reduced or the part removal in nuclear.With regard to DM, even the unusual relatively little reduction of transcript also can discharge the cytokine that (might be capacity) in a large number be chelated, and helps to recover normal ribonucleic acid thus and processes and cellular metabolism (Kanadia etc., PNAS 2006).In the situation of HD, the minimizing of Huntington protein accumulation of deposits can improve disease symptoms in HD patient's cell.

More existing use antisense nucleic acides, RNA interference or ribozyme are designed for unstable the repeat methods of treatment of disease I type myotonia atrophica and the trial of medicament.(i) Langlois etc. (Molecular Therapy, Vol.7 No.5,2003) has designed the ribozyme that can cut DMPK mRNA.Provide and had and the hammerhead ribozyme that is close to CUG repetition one section RNA of DMPK 3 ' UTR complementary before.In vivo, the carrier ribozyme of transcribing can will comprise in transfectional cell and increased CUG multiple mRNA and normal mRNA cutting and reduced 63% and 50% respectively.Therefore, the normal transcription thing also is subjected to having a strong impact on of this method, and containing of being influenced increased multiple mRNA not by target specifically.

(ii) Langlois etc. (Journal Biological Chemistry, vol 280, no.17,2005) uses RNA to disturb to have carried out another kind of method.The short hairpin RNA (shRNA) that slow virus is sent is introduced in the DM1 sarcoplast, proved the mutant DMPK mRNA that keeps in its downward modulation nuclear.Tested 4 kinds of shRNA molecules, two kinds with the complementation of DMPK coding region, the unique sequences complementation among a kind of and the 3 ' UTR, a kind of negative control that has irrelevant sequence.Preceding two kinds of shRNA can with have the multiple mutant DMPK transcript that increases be adjusted downward to about 50%, but even more effectively kytoplasm wild-type transcript is adjusted downward to about 30% or lower.It is invalid sending the synthetic siRNA that is equal to by cationic lipid.It all is invalid being proved to be two kinds of transcripts at the shRNA of 3 ' UTR sequence.Therefore, this method also the non preference target to having increased multiple mRNA type.

(iii) the third method of Furling etc. (Gene Therapy, Vol.10, p795-802,2003) has used the recombinant retrovirus of expressing the long sense-rna of 149bp to suppress DMPK mRNA level in the people DM1 sarcoplast.With retrovirus be designed to the DM1 cell provide repeat for 13 times with long (CUG) of 39bp and this repetitions after the sense-rna grown of the 149bp of 110bp regional complementarity, with the raising specificity.This method makes sudden change (tumor-necrosis factor glycoproteins has increased) DMPK transcript reduce 80%, and comparatively speaking, wild-type DMPK transcript reduces 50%, and has recovered differentiation and functional characteristic in the infected DM1 sarcoplast.Therefore, also the non preference target is to the multiple mRNA kind that increases for this method, and it depends on very long sense-rna, and only can unite use with the recombinant virus delivery technique.

Detailed Description Of The Invention

Method mentioned above and technology provide the method based on nucleic acid, and its allelotrope and unaffected (normally) allelotrope that non-selectively causes the affected tumor-necrosis factor glycoproteins of the inherited disease relevant with repeat instability and/or amplification to increase is all degraded.In addition, the prior art utilization has specific sequence to disease related gene, and the method that can be applicable to several inherited diseases relevant with the tumor-necrosis factor glycoproteins amplification is not provided.At last, prior art has only been instructed and has been related to the method for using the recombinant DNA carrier delivery system, and it need be adjusted at every kind of oligonucleotide and target cell, and still needs to be optimized.

The invention provides scheme by using short single stranded nucleic acid molecule to address these problems, described short single stranded nucleic acid molecule only comprises and the repeat region complementary sequence that increases, perhaps be made up of it, promptly it does not rely on the exon or the unique sequences in the intron of the gene that comprises tumor-necrosis factor glycoproteins and hybridizes.In addition, do not require that the nucleic acid (oligonucleotide) that is adopted reduces transcript by the degradation mechanism that RNA enzyme H mediates.

Without wishing to be bound by theory, the present invention can by change prematurity RNA transcribe post-treatment and/or montage causes that the transcript level descends.By alternative splicing and/or transcribe post-treatment and reduce the transcript level and be considered to produce and lack that excessive amplification or unsettled (three) Nucleotide repeat but still transcript with functionally active.Reduce the unusual transcript that unusual transcript can prevent that tumor-necrosis factor glycoproteins has increased in the cell and in cell, accumulate and/or translate by changing RNA processing and/or montage.

Without wishing to be bound by theory, think that also method of the present invention provides the specificity at the influenced transcript that comprises the tumor-necrosis factor glycoproteins that increases, because the kinetics of hybridizing with the tumor-necrosis factor glycoproteins that increases is more favourable.The possibility of complementary segment hybridization increases along with the increase that repeats sector sizes in the specific complementary nucleic acid oligonucleotide molecules of tumor-necrosis factor glycoproteins and RNA or the dna molecular.The inner usually pairing of RNA molecule (the RNA molecule that particularly comprises tumor-necrosis factor glycoproteins) forms the secondary structure that comprises split ring and closed hair clip part.It is come-at-able relatively to complementary nucleic acid that opening portion is only arranged.Answering section with the short weight of the incoherent wild-type transcript of disease often only is 5 to about 20-40 repetitions, because secondary structure is not easy to and the complementary nucleic acid base pairing relatively.On the contrary, the allelic repeating unit of tumor-necrosis factor glycoproteins and disease-related that increased increases 2 times usually at least, but often more, and 3,5,10 times, nearly 100 times, even concerning some unstable multiple diseases, increase above 1000 times.This amplification has improved this multiple part (temporary transient at least) and has been arranged in the possibility of open loop structure, and therefore is easier to and the complementary nucleic acid molecule base pairing with respect to wild-type allele.Therefore, although the tumor-necrosis factor glycoproteins that exists in the transcript that oligonucleotide and wild-type and tumor-necrosis factor glycoproteins have increased is all complementary and can hybridize with two kinds of transcripts in theory, but the present invention's instruction is, preferential with disease-related or cause the transcript of disease to be hybridized with repeating part complementary oligonucleotide, and normal transcription thing function is unaffected relatively.This selectivity is useful for the treatment disease relevant with repeat instability, and irrelevant with the mechanism of unusual transcript minimizing.

Therefore, the invention provides by providing only complementary and/or only can treating the method that repeats relevant inherited disease with unstable cis element DNA with the nucleic acid molecule of its hybridization with tumor-necrosis factor glycoproteins.Therefore, the preferential target of the present invention multiple transcript that increased, and normal wild-type allele is unaffected relatively.Therefore this is favourable, because normal allele provides the normal function of gene, this is ideal at least and depends on and have unstable DNA multiple specific gene and in many cases may be to being controlled cell and/or individuality is necessary.

In addition, this method is not limited only to repeat relevant inherited disease with specific unstable DNA, repeat disease but can be applicable to any known unstable DNA, such as but not limited to: have polyglutamyl amine (CAG) multiple coding region and repeat disease: Huntington chorea, Haw-River syndrome, kennedy's disease/spinal cord bulbar muscular atrophy disease, spinocerebellar ataxia; Perhaps has many L-Ala (GCG) multiple disease, for example: infantile spasm syndrome, craneocleidol dysplasia (deidocranial dysplasia), blepharophimosis/ptosis/epicanthus inversus syndrome, hand-foot-sexual organ syndrome, synpolydactyly, oculopharyngeal muscular dystrophy, holoprosencephaly.Treatment has the multiple disease at the gene non-coding region and comprises that trinucleotide repeats disease (great majority are that CTG and/or CAG and/or CCTG repeat): I type myotonia atrophica, II type myotonia atrophica, Friedreich ataxia (mainly being GAA), spinocerebellar ataxia, autism according to the present invention.In addition, the inventive method can be used for the relevant repetition disease of fragile site, comprise multiple fragile X mental retardation, Jacobson syndrome and other unstable repeat element disease, for example type ii diabetes of myoclonic epilepsy, facioscapulohumeral dystrophy and some form.

Another advantage of the present invention is the repeat region specific oligonucleotide directly can be applied to cell, and it does not rely on the delivery system based on carrier.The technology of having described in the prior art (DM1 of being used for the treatment of for example mentioned above and the technology of removing the DMPK transcript from cell) needs use the delivery system based on carrier to be applied to cell with the oligonucleotide with enough levels.The use of plasmid or virus vector is not very desirable for therapeutic purpose, this be since at present for the strict safety regulations of therapeutic recombinant DNA carrier, be used for clinical application on a large scale enough recombinant vectorss production and to use the back excessively (or non-specific) reply limited control and reversibility.However, be optimized in these fields future probably, and the virus of plasmid is sent can have favourable long-term effect.The inventor has unexpectedly found, with unique sequences in the transcript had specific oligonucleotide compare, comprise with the multiple transcript that increased in tumor-necrosis factor glycoproteins complementary sequence or owing to the amplification of its hybrid molecule target its target is had much higher avidity/avidity by the oligonucleotide that described sequence is formed.Owing to the high-affinity and the avidity of the target transcript that tumor-necrosis factor glycoproteins has been increased, more a spot of oligonucleotide just is enough to disturb degraded or change to translate the active allelotrope that tumor-necrosis factor glycoproteins has been increased of post-treatment (including but not limited to montage or exon skipping (exon skipping)) by RNA enzyme H degraded, RNA and produces inhibition fully and/or minimizing.Only can produce by synthetic, and be used for direct oligonucleotide delivery in use and when the common technology of cell and/or tissue directly is delivered to cell, have enough effective effectiveness with tumor-necrosis factor glycoproteins complementary oligonucleotide of the present invention.When needing, but method of the application of the invention and oligonucleotide molecules avoid using the recombinant vectors delivery system.

In aspect first, the present invention open and instructed only comprise with the people's gene transcript in tumor-necrosis factor glycoproteins complementary sequence or the purposes that is used for diagnosing, treating or prevent the medicine of the relevant people's inherited disease of cis element repeat instability in preparation by the oligonucleotide that described sequence is formed.Therefore, the invention provides the method that is used for the treatment of the inherited disease relevant with the cis element repeat instability.

In aspect second, the invention still further relates to can be used for first aspect present invention and/or be used for the inventive method and stop the transcript that repeats to have increased oligonucleotide in cell accumulation and/or translation.

Oligonucleotide of the present invention only can comprise and hereinafter define tumor-necrosis factor glycoproteins complementary sequence.Preferably, described tumor-necrosis factor glycoproteins is at least 50% of an oligonucleotide length of the present invention, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90% or more.In a most preferred embodiment, oligonucleotide of the present invention by only with hereinafter define tumor-necrosis factor glycoproteins complementary sequence and form.For example, oligonucleotide only can comprise and hereinafter define tumor-necrosis factor glycoproteins complementary sequence and targeting moiety, and the latter hereinafter is called as the target part.

This paper is with repetition or repeat element or tumor-necrosis factor glycoproteins or repeat at least 3,4,5,10,100,1000 times or more times repetition that section is defined as repeating unit in the open gene sequence in experimenter's (comprising people experimenter) genome or repeated nucleotide units (comprise the trinucleotide repeating unit, or the repeating unit of 4,5 or 6 Nucleotide).

Oligonucleotide can be strand or two strands.Two strands means the heterodimer that is formed by two complementary strands, for example siRNA.In a preferred embodiment, oligonucleotide is a strand.Single stranded oligonucleotide has several advantages than double-stranded siRNA oligonucleotide: estimate that (i) it is synthetic easier than two complementary siRNA chains; (ii) there is the more chemically modified of wide region might be used to optimize more effective cell absorption, better (physiology) stability and general side effect of reduction potential; (iii) siRNA more likely produce nonspecific action and over-drastic pharmacotoxicological effect (for example to the validity of treatment plan or dosage and optionally to control possibility less) and (iv) siRNA can not act in the nuclear, can not be at intron.Therefore, in a preferred embodiment of first aspect, only the present invention relates to comprise with the people's gene transcript in tumor-necrosis factor glycoproteins complementary sequence or the single stranded oligonucleotide formed by described sequence be used for diagnosing, treat or the purposes of the medicine of people's inherited disease that prevention is relevant with the element repeat instability in preparation.

Described oligonucleotide preferably comprise at least 10 to about 50 with repeat element complementary continuous nucleotides, preferred 12 to 45, more preferably 12 to 30, most preferably 12 to 25 with repeat section (preferably having trinucleotide repeating unit or tetranucleotide repeating unit) complementary Nucleotide.Described oligonucleotide can with the complementary and/or hybridization with it of repetition section in the coding region in the transcript (preferred polyglutamyl amine (CAG) or many L-Ala (GCG) coding section).Described oligonucleotide is also can be with the intron sequences that exists in non-coding region (for example 5 ' or 3 ' non-translational region) or the precursor rna molecule complementary and/or can hybridize with it.

In a preferred embodiment, the oligonucleotide that uses in the inventive method comprises and following repeat element complementary sequence, perhaps form by it, described repeat element has and is selected from (CAG) n, (GCG) n, (CUG) n, (CGG) n, (GAA) n, (GCC) n and (CCUG) the repetition nucleotide units of n is as repeating nucleotide units, and described oligonucleotide is strand or double chain oligonucleotide.Preferably, described oligonucleotide is double-stranded.

Application comprise with transcript in polyglutamyl amine (CAG) n sections complementary sequence or by of the diagnosis of its oligonucleotide of forming in following disease, particularly useful in treating and/or preventing: by people's gene HD, HDL2/JPH3, SBMA/AR, SCA1/ATX1, SCA2/ATX2, SCA3/ATX3, SCA6/CACNAIA, SCA7, SCA17, human disease's Huntington chorea that the amplification of tumor-necrosis factor glycoproteins causes among AR or the DRPLA, the spinocerebellar ataxia of some forms or Haw-River syndrome, X chain spinal cord oblongata muscular dystrophy and/or dentatorubral-pallidoluysian atrophy.

Application comprise with transcript in many L-Ala (GCG) n sections complementary sequence or by of the diagnosis of its oligonucleotide of forming in following disease, particularly useful in treating and/or preventing: by people's gene ARX, CBFA1, FOXL2, HOXA13, HOXD13, OPDM/PABP2, the human disease that the amplification of tumor-necrosis factor glycoproteins causes among TCFBR1 or the ZIC2: infantile spasm syndrome, craneocleidol dysplasia, blepharophimosis, hand-foot-sexual organ syndrome, synpolydactyly, oculopharyngeal muscular dystrophy and/or holoprosencephaly.

Application comprise with transcript in (CUG) n sections complementary sequence or particularly useful in the diagnosis of following disease, in treating and/or preventing by its oligonucleotide of forming: people's inherited disease I type myotonia atrophica that the amplification of tumor-necrosis factor glycoproteins causes in respectively by people's gene DM1/DMPK, SCA8 or JPH3,8 type spinocerebellar ataxias and/or 2 type Huntington chorea sample diseases (Huntington ' s disease-like 2).Preferably, these genes are human origin.

Application comprise with transcript in (CCUG) n sections complementary sequence or particularly useful in the diagnosis of following disease, in treating and/or preventing by its oligonucleotide of forming: people's inherited disease II type myotonia atrophica that the amplification of tumor-necrosis factor glycoproteins causes in by the DM2/ZNF9 gene.

Application comprise with transcript in (CGG) n sections complementary sequence or particularly useful in the diagnosis of following disease, in treating and/or preventing by its oligonucleotide of forming: people's fragile X mental retardation that the amplification of tumor-necrosis factor glycoproteins causes in by FRAXA/FMR1, FRAXE/FMR2 and FRAXF/FAM11A gene.

Application comprise with transcript in (CCG) n sections complementary sequence or particularly useful in the diagnosis of following disease, in treating and/or preventing by its oligonucleotide of forming: people's inherited disease Jacobson syndrome that the multiple amplification causes in by the FRA11B/CBL2 gene.

Application comprise with transcript in (GAA) n sections complementary sequence or particularly useful in the diagnosis of following disease, in treating and/or preventing by its oligonucleotide of forming: people's inherited disease Friedreich ataxia.

Application comprise with intron in (ATTCT) n sections complementary sequence or particularly useful in the diagnosis of following disease, in treating and/or preventing by its oligonucleotide of forming: people's inherited disease 10 type spinocerebellar ataxias (SCA10).

Be used for can comprising following molecule with tumor-necrosis factor glycoproteins complementary oligonucleotide or forming of the inventive method: RNA, DNA, lock nucleic acid (Locked nucleic acid by it, LNA), peptide nucleic acid(PNA) (PNA), morpholino phosphoryl diamine (morpholino phosphorodiamidate, PMO), ethidene bridged linkage nucleic acid (ethylene bridged nucleic acid, ENA) or its mixture/hybrid, it comprises the combination of n DNA and RNA molecule and synthetic modified nucleotide.In these oligonucleotide, the phosphodiester backbone chemical structure can further be replaced by other modification, for example thiophosphatephosphorothioate or methyl phosphorodithioate.Also exist other oligonucleotides-modified, new modification also might be developed and be used for practice.But all these oligonucleotide all have the oligomer character of energy sequence-specific ground in conjunction with RNA.Therefore, in a preferred embodiment, described oligonucleotide comprises following molecule or is made up of it: the RNA Nucleotide, DNA Nucleotide, lock nucleic acid (LNA) Nucleotide, peptide nucleic acid(PNA) (PNA) Nucleotide, morpholino phosphoryl diamine, ethidene bridged linkage nucleic acid (ENA) Nucleotide or its mixture that have or do not have the skeleton that comprises thiophosphatephosphorothioate.

Contain to the oligonucleotide of small part n DNA Nucleotide and can be used for inducing DNA-RNA hybrid molecule by in RNA enzyme H activity (EC.3.1.26.4) degradation of cell.

The oligonucleotide that comprises natural RNA ribonucleotide or RNA sample synthetic kernel sugar nucleotide can be used for method of the present invention, to form double-stranded RNA-RNA crossbred, it is by the effect of RNA interference or reticent (RNAi/siRNA) approach performance enzyme dependency antisense nucleic acid, by there being justice-antisense strand pairing to participate in target RNA identification, then by the reticent mixture of RNA inductive (RISC) degraded target RNA.

As an alternative or supplement, the antisense oligonucleotide (non-RNA enzyme H dependency antisense nucleic acid) of three-dimensional sealing disturbs genetic expression or other precursor RNA or the dependent cell processes of messenger RNA(mRNA), particularly (but being not limited to) RNA montage and exon skipping, this is by in conjunction with target sequence rna transcription thing and hinder such as the process of translation or sealing donor splicing site or acceptor splicing site and realize.Use modifying montage and the exon skipping technology that antisense oligonucleotide carries out and fully describe, is that ordinary skill is known, for example is found in US6,210,892, WO9426887, WO04/083446 and WO02/24906.In addition, but therefore the combination of sterically hindered arrestin matter, nf etc. helps to reduce accumulation of disease such as DM1 center or ribose nuclear speckle.

The oligonucleotide of the present invention that can comprise or modified nucleotide synthetic with (having increased) tumor-necrosis factor glycoproteins complementary can be used for the inventive method, contains the multiple transcript or makes its inactivation to reduce by siRNA/RNA interference or reticent approach.

Being used for the strand of the inventive method or double chain oligonucleotide can comprise following molecule or be made up of it: the ribonucleotide that DNA Nucleotide, RNA Nucleotide, 2 '-O replace (comprising that alkyl and methoxy ethyl replace), peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA) and morpholino (PMO) antisense oligonucleotide and ethylene bridge Nucleotide (ENA) and make up randomly are the mosaics with RNA enzyme H dependency antisense nucleic acid.The integration of lock nucleic acid in oligonucleotide changed the conformation of duplex after the base pairing, improved the stability of duplex.Therefore, the integration of LNA base in oligonucleotide sequence can be used for improving the following ability of complementary oligonucleotide of the present invention: the RNA, the degraded that promptly initiatively improve transcript in vitro and in vivo suppress the skip capability of accumulation or raising exon.Peptide nucleic acid(PNA) (PNA) is that wherein skeleton is the artificial DNA/RNA analogue of false peptide rather than sugar, owing to can form stable especially complex body with the complementary DNA oligomer in conjunction with improving with higher melting temperature(Tm).PNA also is the good reagent that is used for antisense nucleic acid of the present invention and exon skipping application.Most preferably, the oligonucleotide that is used for the inventive method comprises part or all of at least 2 '-O-methoxy ethyl thiophosphatephosphorothioate RNA Nucleotide or 2 '-O-methyl thiophosphatephosphorothioate RNA Nucleotide.Most preferably use comprises following sequence or is diagnosed by the oligonucleotide that it is formed in the present invention, treatment or prevention cis element repeat unstable inherited disease, described sequence be selected from (CAG) n, (GCG) n, (CUG) n, (CGG) n, (CCG) n, (GAA) n, (GCC) n and (CCUG) the tumor-necrosis factor glycoproteins complementation of n, length is 10 to 50, more preferably 12 to 35,12 to 25 Nucleotide most preferably, and comprise 2 '-O-methoxy ethyl thiophosphatephosphorothioate RNA Nucleotide, 2 '-O-methyl thiophosphatephosphorothioate RNA Nucleotide, LNA Nucleotide or PMO Nucleotide.

Therefore, in a preferred embodiment, of the present invention and be used for oligonucleotide of the present invention comprise be selected from (CAG) n, (GCG) n, (CUG) n, (CGG) n, (GAA) n, (GCC) n and (CCUG) n tumor-necrosis factor glycoproteins complementary sequence or form by it, length is 10 to 50 Nucleotide, also has following feature:

(a) comprise RNA thiophosphatephosphorothioate Nucleotide (for example 2 '-O-methyl or 2 '-O-methoxy ethyl RNA thiophosphatephosphorothioate Nucleotide), LNA Nucleotide or the PMO Nucleotide that 2 '-O-replaces.Described Nucleotide can anyly be used in combination and/or use with DNA thiophosphatephosphorothioate or RNA Nucleotide; And/or

(b) be single stranded oligonucleotide.

Therefore, in a further preferred embodiment, of the present invention and be used for oligonucleotide of the present invention comprise be selected from (CAG) n, (GCG) n, (CUG) n, (CGG) n, (GAA) n, (GCC) n and (CCUG) n tumor-necrosis factor glycoproteins complementary sequence or form by it, length is 10 to 50 Nucleotide, also has following feature:

(c) comprise RNA thiophosphatephosphorothioate Nucleotide (for example 2 '-O-methyl or 2 '-O-methoxy ethyl RNA thiophosphatephosphorothioate Nucleotide), LNA Nucleotide or the PMO Nucleotide that 2 '-O-replaces.Described Nucleotide can anyly be used in combination and/or use with DNA thiophosphatephosphorothioate or RNA Nucleotide; And/or

(d) be double chain oligonucleotide.

If the present invention relates to have the double chain oligonucleotide of two complementary strands, wherein antisense strand only with the people's gene transcript in the tumor-necrosis factor glycoproteins complementation, then this double chain oligonucleotide preferably is not to have sense-rna chain (CUG) ₇With adopted RNA chain (GCA) is arranged ₇SiRNA, it is applied to monkey inoblast (COS-7) or people's neuroblast (SH-SY5Y) clone of the cultivation of the human Huntington gene extron 1 that transfection or untransfected and GFP merge, as Wanzhao Liu etc. ((2003), Proc.Japan Acad, 79:293-298) described.More preferably, the present invention does not relate to double chain oligonucleotide siRNA and (has antisense strand (CUG) ₇And sense strand (GCA) ₇), do not relate to its purposes in the medicine of preparation treatment or prevention Huntington chorea (more preferably treatment or prevention contain the construct of Huntington chorea gene extron 1) yet.

Although the amount of the transcript (for example DMPK or ZNF9 transcript or its fragment of nuclear accumulation) that uses a kind of oligonucleotide may just be enough to reduce tumor-necrosis factor glycoproteins to have increased, perhaps be enough to reduce the proteic accumulation of HD that tumor-necrosis factor glycoproteins has increased, but make up 2,3,4,5 or more kinds of oligonucleotide also within the scope of the invention.Can with comprise with transcript repeating part complementary sequence or by its oligonucleotide of forming advantageously with comprise following sequence or by the combination of its oligonucleotide of forming, this sequence is with to contain unique sequences in the multiple transcript complementary and/or can hybridize with it.The inventive method and the medicine of the present invention that comprise the tumor-necrosis factor glycoproteins specific oligonucleotide also can be combined with any other treatment or medicine that is used for cis element repeat instability inherited disease.For diagnostic purpose, the oligonucleotide that uses in the inventive method can have radio-labeling or fluorescent mark, with allow in sample, in vivo in the original position cell, exsomatize or the vitro detection transcript.For myotonia atrophica, the oligonucleotide of mark can be used for diagnostic purpose, so that the nuclear aggregate of DMPK or ZNF9RNA transcript molecule and related protein develops.Fluorescent mark can comprise Cy3, Cy5, FITC, TRITC, rhodamine, GFP etc.Radio-labeling can comprise ³H, ³⁵S, ^32/33P, ¹²⁵I.Also can use enzyme and/or immunogenicity haptens, for example digoxin, vitamin H and other molecular label (HA, Myc, FLAG, VSV, lexA).Therefore, in one aspect of the method, the invention discloses the external or stripped detection and/or the diagnostic method that wherein use above-mentioned oligonucleotide.

Be used for oligonucleotide of the present invention and be suitable for being applied in the direct body and suffer from or risky generation cis element repeats cell, tissue and/or the organ of the individuality of unstable disease, but and in the body, exsomatize or externally directly use.Perhaps, described oligonucleotide can be provided by the nucleic acid carrier that this oligonucleotide is expressed in people's cell, to obtain the lasting source of described oligonucleotide.The expression vector form that oligonucleotide molecules of the present invention can make described oligonucleotide express in people's cell offers subject cell, tissue, organ and/or experimenter.Described carrier is preferably introduced in the cell by the gene delivery vehicle.The vehicle that preferably is used to send is a virus vector, for example retroviral vector, gland relevant viral vector (AAV), adenovirus carrier, Semliki forest virus carrier (SFV), EBV carrier etc.The plasmid, artificial chromosome, plasmid that are applicable to target formula homologous recombination and are integrated into people's cellular genome are also applicable to oligonucleotide delivery.The present invention preferably transcribe by the carrier of polIII promoters driven and/or wherein transcript be the form that merges mutually with U1 or U7 transcript, this obtains good result for sending little transcript.

In a preferred embodiment, use the oligonucleotide concentration of about 0.1nM to about 1 μ M.More preferably, employed concentration is about 0.3 to about 400nM, even more preferably from about 1 to about 200nM.If use several oligonucleotide, then this concentration can refer to the total concn of oligonucleotide or the concentration of every kind of oligonucleotide being added.The concentration range of the oligonucleotide that provides as mentioned is external or the preferred concentration of stripped use.Those of ordinary skills will appreciate that, according to employed oligonucleotide, target cell, gene target and expression level thereof to be treated, employed substratum and transfection and incubation conditions, the oligonucleotide concentration of using can further change, and may need to be optimized.

More preferably, be used among the present invention to prevent, treat or the oligonucleotide of diagnosing cis element to repeat unstable disease produces by synthetic, and be applied directly to cell, tissue, organ and/or patient with the form that is mixed with in the pharmaceutically acceptable composition.The delivering drugs composition is preferably undertaken by injecting at one or more parenteral at one or more positions of human body to the experimenter, for example in intravenously and/or subcutaneous and/or intramuscular and/or the canalis spinalis and/or administration in the ventricle, and preferred drug administration by injection.Administration (in the cerebrospinal fluid) preferably realizes by diffusion pump being introduced experimenter's health in the canalis spinalis or in the ventricle.Multiple filling pump is that those of ordinary skills are known.

Being used for target comprises with tumor-necrosis factor glycoproteins complementary sequence or by the pharmaceutical composition of its oligonucleotide molecules of forming and can comprise multiple vehicle, for example thinner, weighting agent, sanitas, solubilizing agent etc., for example be found in Remington:The Science and Practice of Pharmacy, the 20 edition .Baltimore, MD:Lippincott Williams; Wilkins, 2000.

For the inventive method, particularly preferred be to use help with described oligonucleotide delivery to and send the into vehicle of cell, particularly can form the vehicle of complex body, vehicle and/or the liposome that will be passed cytolemma by the compound or material of capturing and/or oligonucleotide delivery.Many such materials are known in the art.But comprising, suitable material can be self-assembled into particulate polymine (PEI), ExGen 500, synthetic amphiphilic species (SAINT-18), lipofectin TM, DOTAP and/or the viral capsid proteins of oligonucleotide delivery to cell.Lipofectin represents an example of liposome transfection agent.It is made up of two kinds of lipid composition: cation lipid N-[1-(2,3 two oily acyloxy) propyl group]-N, N, N-trimethyl ammonium chloride (DOTMA) (and being the DOTAP of its Methylsulfate) and neutral lipid dioleoyl phosphatidylethanolamine (DOPE).Discharge in the described neutral compound mediation born of the same parents.Another group delivery system is the poly nano particle.Known polycation as the DNA transfection agents (for example diethylin ethyl (DEAE)-dextran) can make up with Tisuacryl (PBCA) and the own ester of alpha-cyanoacrylate (PHCA), strides across the cationic nano-grain that cytolemma enters cell but be mixed with oligonucleotide delivery.Except these common nano-particle materials, this cationic peptide of protamine provides oligonucleotide has been mixed with the colloidal alternative method.This colloidal nano particle system can form so-called proticle, and it can prepare by simple self assembling process, discharges to pack described oligonucleotide and to mediate in its born of the same parents.Those of ordinary skills can select and adjust any above-mentioned or alternative vehicle that other is commercially available and delivery system is packed and oligonucleotide delivery, are used for the treatment of the oligonucleotide that cis element in the human body repeats unstable disease to send in the present invention.

In addition, described oligonucleotide can become to promote that taking in cell, tenuigenin and/or endonuclear target part covalently or non-covalently is connected with particular design.Such part can comprise (i) recognizing cells, tissue or organ specificity element to promote the compound (including but are not limited to peptide (sample) structure) that cell is taken in and/or (ii) can promote to take in the cell and/or the interior compound that discharges oligonucleotide of born of the same parents from vesica (for example endosome or lysosome).These target parts are also contained and are promoted oligonucleotide to pass hemato encephalic barrier and take in molecule in the brain.Therefore, in a preferred embodiment, for the nucleic acid in the medicine provides the vehicle that is used to send and/or target part and/or oligonucleotide delivery to cell and/or strengthen the device of sending in its cell at least.Therefore, this pharmaceutical composition is also contained in the present invention, and it comprises oligonucleotide of the present invention, and comprises at least a vehicle that is used to send and/or target part and/or oligonucleotide delivery to cell and/or strengthen the device of sending in its cell.

The invention still further relates to and be used for reducing the method that cell comprises multiple genetic transcription thing, described method comprises uses strand or double chain oligonucleotide molecule, described oligonucleotide molecules preferably comprises RNA thiophosphatephosphorothioate Nucleotide (for example 2 '-O-methyl or 2 '-O-methoxy ethyl RNA thiophosphatephosphorothioate Nucleotide), LNA Nucleotide or the PMO Nucleotide that 2 '-O-replaces, length is 10 to 50 Nucleotide, only with the tumor-necrosis factor glycoproteins complementation.Described Nucleotide use capable of being combined and/or use with DNA thiophosphatephosphorothioate Nucleotide.

In presents and claim thereof, verb " comprises " and version uses with non-limiting implication, means the project that comprises this speech back, but does not get rid of combination and/or the project of specifically not mentioning.In addition, the key element that does not have concrete number to modify is not got rid of the possibility of existence more than (kind) key element, has one (kind) and only has (kind) key element unless context explicitly calls for.Therefore, do not have number to modify and be often referred to " at least one (kind) ".

Legend

Fig. 1: from Northern trace with isolating RNA the myotube of different oligonucleotide or simulation contrast transfection.Myotube is derived from immortalization mouse muscle-forming cell system, and it contains adjacent (CTG) n multiple transgenic human DMPK gene that has amplification length about 500 of being and not being (CTG) multiple normal mouse DMPK gene.Trace shown people DMPK mRNA (on), mouse DMPK (mDMPK) mRNA (in) and mouse GAPDH mRNA (descending).

Fig. 2: people and the mouse DMPK signal analyzed Fig. 1 by phosphorescence imager (phosphoimager) carry out quantitatively carrying out normalization method with respect to the GAPDH signal.The result represents with respect to simulation process (being made as 100).

Fig. 3: the Northern trace of isolating total RNA from the mouse myotube that contains the chimeric DMPK gene of mouse-people, wherein 3 ' part of mDMPK gene involved (CTG) ₁₁₀The respective section of repeater DMPK gene is replaced.Detect DMPK mRNA (last figure) and mouse GAPDH mRNA (figure below) in the trace.With antisense oligonucleotide PS58 or contrast transfectional cell.

Fig. 4Show replying of DM500 myotube that the oligonucleotide PS58 with multiple concentration handled.Follow by the Northern engram analysis and the expression of hDMPK to be carried out quantitatively by the analysis of phosphorescence imager.Signal carries out normalization method with respect to the GAPDH signal, and represents with respect to replying after the simulation process.

Fig. 5Be presented at the Northern trace of different time points (2h, 4h, 8h and 48h before the results) with the total RNA of DM500 myotube of 200nM PS58 transfection.Simulation process was carried out in results in preceding 48 hours.The Northern trace shows the mRNA of people and mouse DMPK and mouse GAPDH.By the analysis of phosphorescence imager they are carried out quantitatively, normalized DMPK signal is represented with respect to simulation process.

Fig. 6Be presented at Northern trace with the total RNA of DM500 myotube of 2 days, 4 days, 6 days and 8 days results after 200nM PS58 or the simulation contrast transfection.Carry out Northern engram analysis and quantitative as mentioned above.

Fig. 7Demonstration has the Northern engram analysis of the total RNA of sarcoplast of MyoD through the conversion of oligonucleotide PS58 (20 and 200nM) or simulation control treatment.Described sarcoplast is from the inoblast that obtains from congenital I type myotonia atrophica patient, and described patient has and has the hDMPK allelotrope that length is about 1500 triplet repeat amplification protcol and 11 multiple hDMPK allelotrope with normal length.With Northern trace and people DMPK (hDMPK) probe and GAPDH mRNA probe hybridization.People DMPK signal is carried out normalization method with respect to the GAPDH signal, and represent with respect to the simulation contrast.

Fig. 8Demonstration has 200nM only (CUG) tumor-necrosis factor glycoproteins to be had specific oligonucleotide PS58, sequence in the exons 1 is had the RT-PCR that the DM500 myotube of specific oligonucleotide PS113 or simulation contrast carries out analyzes to transfection.RT-PCR analyze to use to have specific primer and carries out having following three kinds of other genetic transcription things of natural (CUG) multiple in hDMPK mRNA and the mouse: have (CUG) 6 Ptbp1mRNA, have syndecan 3 (Syndecan3) mRNA of (CUG) 6 and have (CUG) 9 slide albumen β (Taxilinbeta) mRNA.Strength of signal is carried out normalization method with respect to the Actin muscle signal, and represent with respect to the simulation contrast.

Fig. 9Show the myoblastic fish analysis of DM500 with 200nM PS58 (B) or simulation contrast (A) transfection.Handle back 48 hours of beginning, washing and fixed cell are hybridized with fluorescently-labeled oligonucleotide Cy3-(CAG) 10-Cy3 then.Use Bio-Rad MRC1024 laser confocal scanning microscope and LaserSharp2000 acquisition software to manifest indication hDMPK (CUG) ₅₀₀The ribose nuclear speckle that mRNA accumulates in nuclear.

Figure 10Show the relative cell counting that has the ribose nuclear speckle in the experiment shown in Figure 9 in the myoblastic nuclear of DM500 with PS58 or simulation contrast transfection.

Figure 11Demonstration is analyzed with the RT-PCR of hDMPKmRNA in the muscle of the DM500 mouse of PS58 or simulation control treatment.In brief, in the GPS mixture, PS58 (2nmol) is expelled in the DM500 mouse in one-year-old age, repeats this process after 24 hours.After 15 days, separate in mouse sole of the foot flesh and the gastrocnemius muscle, use total RNA that hDMPK and mouse Actin muscle are carried out RT-PCR.The intensity of hDMPK signal is carried out normalization method with respect to the Actin muscle signal, and value is represented with respect to the simulation contrast.

Figure 12Show Northern engram analysis with the DM500 myotube of different oligonucleotide (200nM) or simulation control treatment.PS58, PS146 and PS147 have 2 ' O-methyl phosphorothioate backbone completely, but the length difference, are respectively (CAG) 7, (CUG) 10 and (CUG) 5.PS142 has the skeleton of phosphorothioate dna completely that has (CAG) 7 sequences.HDMPK and mDMPK signal with respect to mouse GAPDH normalization method, and are expressed as per-cent with respect to the simulation contrast.Figure below shows quantitatively.

Embodiment

Embodiment 1

Immortalization sarcoplast system uses standard technique well known by persons skilled in the art to derive from DM500 or CTG110 mouse.The DM500 mouse derives from the mouse that derives from Paris Gourdon group.The CTG110 mouse is described hereinafter, is present in Wieringa and the Wansink group of Nijmegen.To in the DMPK gene, have different lengths (CTG) n multiple immortalization sarcoplast and be DM500 or CTG110 and grow to the Asia and converge, and at 33 ℃ of 5% CO down ₂Maintain in the atmosphere in the plate with 0.1% gelatin bag quilt.Sarcoplast is converged cultivation in the DMEM Central Asia of having added 20% FCS, 50 μ g/ml gentamicins and the 20 IFN-/ml of unit.Induce myotube to form by following steps: on plate, cultivate sarcoplast, and the sarcoplast culture that will converge to place the DMEM that is added with 5% horse serum and 50 μ g/ml gentamicins under 37 ℃ with Matrigel (BD Bioscience) bag quilt.After 5 days, on this low blood serum medium, occur the myotube of contraction in the culture, it is carried out transfection with the oligonucleotide of expecting.(ExGen 500, and Fermentas) also direct mixing is to carry out transfection to add NaCl (500mM, filtration sterilization), oligonucleotide and transfection reagent PEI by following particular order.(ExGen 500, and Fermentas), oligonucleotide transfection solution contains the ExGen 500 of every microgram oligonucleotide 5 microlitres according to specification sheets., after 15 minutes oligonucleotide transfection solution is joined in the low blood serum medium that has institute's cultivation myotube in incubated at room, and mixing gently.The oligonucleotide final concentration is 200nM.Simulate control treatment with the transfection solution that does not contain oligonucleotide.37 ℃ hatch 4 hours after, fresh culture is joined in the culture (obtain about 2.3 * diluent), continue 37 ℃ of overnight incubation.Removed the substratum that contains oligonucleotide in second day, add fresh low blood serum medium, kept again one day at 37 ℃ to myotube.Adding oligonucleotide 48 hours (converting to induce the low serum condition that myotube forms after the 7th day) behind the myotube culture, (Bio-Rad) separating and preparation RNA with " total RNA small volume of reagent box ", to be used for Northern trace and RT-PCR analysis.With Northern trace and radioactivity people DMPK (hDMPK) probe and mouse GAPDH probe hybridization.The probe that is used for DMPK is people DMPK cDNA, and it is made up of the DMPK opening code-reading frame that has complete 3 ' UTR and 11 CTG.

Come quantitative people and mouse DMPK signal by the analysis of phosphorescence imager, and carry out normalization method with respect to the GAPDH signal.The primer that is used for the RT-PCR of hDMPK mRNA is positioned at 3 ' non-translational region, its sequence is 5 '-GGGGGATCACAGACCATT-3 ' and 5 '-TCAATGCATCCAAAACGTGGA-3 ', and the primer that is used for the mouse Actin muscle is as follows: Actin muscle have justice 5 '-GCTAYGAGCTGCCTGACGG-3 ' and Actin muscle antisense 5 '-GAGGCCAGGATGGAGCC-3 '.The PCR product carries out electrophoretic analysis on sepharose, (UVP BioImaging systems, Cambridge UnitedKingdom) carry out quantitatively signal with Labworks 4.0.The intensity of each band is carried out normalization method with respect to the intensity of corresponding Actin muscle band, and represent with respect to the simulation contrast.

Comprising (CTG) n multiple transgenic human DMPK gene of having length about 500 and do not testing 13 kinds of different oligonucleotide (summary sees Table 1) on the immortalization sarcoplast DM500 clone with (CTG) multiple normal mouse DMPK gene as mentioned above.Fig. 1 shown from the Northern engram analysis with isolating RNA the myotube of described oligonucleotide transfection, wherein uses the hDMPK probe and manifests as the GAPDH probe of last sample contrast.People DMPK on the Northern trace (have CTG repeat) and mouse DMPK (not having the CTG repetition) quantitatively are shown in Fig. 2.Signal carries out normalization method with respect to mouse GAPDH, and to represent with respect to the mode of simulation contrast.

Table 2 has shown that the caused hDMPK mRNA of specific oligonucleotide level reduces.Negative (-) representative does not reduce, and band is the digitized representation hDMPK mRNA of (+) number level relatively of decomposing just.Obviously, the oligonucleotide PS58 of selectively targeted tumor-necrosis factor glycoproteins reduce or change aspect the hDMPK transcript than with strong many of other oligonucleotide of hDMPK transcript unique sequences complementary.

Fig. 3 shows PS58 from the effect in the mouse immortalization myotube of CTG110 mouse, and described mouse is for containing the mouse of knocking in of DMPK gene with the 3 ' part that comprises about 110 (CTG) repeater DMPK genes.The Northern engram analysis shows that oligonucleotide PS58 handles to have reduced and contains (CTG) 110 multiple DMPK transcripts, but it does not reduce after the simulation process.

Embodiment 2 (Fig. 4)

Cultivate, prepare the DM500 immortalization sarcoplast clone (referring to embodiment 1) that also transfection has the people DMPK gene that comprises pact (CTG) 500 repeat amplification protcols as mentioned above.In this embodiment, the PS58 with different concns carries out transfection.Handle back 84 hours of beginning, the results myotube carries out Northern engram analysis (referring to embodiment 1) to isolating RNA as mentioned above.

Fig. 4 shows that the hDMPK mRNA signal that carries out with the analysis of phosphorescence imager is quantitative, carries out normalization method with respect to the GAPDH of different concns.Under these conditions, near about 1nM, observe partly the highest effect.

Embodiment 3 (Fig. 5 and 6)

Cultivate, prepare the DM500 immortalization sarcoplast clone (referring to embodiment 1) that also transfection has the people DMPK gene that comprises pact (CTG) 500 repeat amplification protcols as mentioned above.But, in this embodiment, carry out transfection with 200nM PS58 in different time points.Usually results DM500 myotube after being transformed into low serum condition and inducing myotube to form 7 days.Standard method (in embodiment 1 and 2) is to begin to handle (transfection) in results preceding 48 hours (2 days).Now, 2 to 48 hours (Fig. 5) or 2 to 8 days (Fig. 6) handles with PS58 before results.Carry out Northern engram analysis and quantitative as described above.

Fig. 5 shows that than the simulation control treatment, the hDMPK mRNA that increases in the DM500 myotube reduced rapidly in 2 hours that handle with oligonucleotide PS58.

Fig. 6 showed the hDMPK mRNA continuous decrease that increases in the DM500 myotube at least 8 days.It should be noted that the situation for experiment in 8 days, at sarcoplast phase (about 60% degree of converging, 33 ℃, high serum) transfectional cell, they repeatedly accept fresh culture (changing into 37 ℃ of low serum when comprising after the transfection 2 days) before results.Embodiment 2 and 3 has shown the efficient inhibition ability that only realizes at the oligonucleotide of repeat amplification protcol.The intensity of this effect can be subjected to the influence that low-level relatively hDMPK expresses in these pattern cell systems, and this also is common in the human body.

Embodiment 4 (Fig. 7)

In this embodiment, used the inoblast that obtains from congenital I type myotonia atrophica (cDM1) people patient.These patient's cells have one, and to have pathogenic DMPK allelotrope that length is 1500 triplet repeat amplification protcol and multiplicity length be 11 normal DMPK allelotrope.Confirm the size of (CTG) n amplification in two allelotrope with PCR and Southern trace.

The cultivation inoblast is converged in the Asia, 37 ℃ of following 5% CO ₂Maintain in the atmosphere in the plate with 0.1% gelatin bag quilt.Converge the cultivation inoblast in the DMEM Central Asia that is added with 10%FCS and 50 μ g/ml gentamicins.Induce myotube to form by following steps: on plate, to cultivate inoblast with Matrigel (BDBiosciences) bag quilt, when 75% converges, be added with among the DMEM of 2%HS and 50 μ g/ml gentamicins with the adenovirus (Ad5Fib50MyoD that expresses MyoD, Crucell, Leiden) (MOI=100) cells infected is 2 hours.After incubation period, remove the MyoD adenovirus, add the DMEM that contains 10%FCS and 50 μ g/ml gentamicins.Described cell is maintained at 37 ℃ 5% CO in this substratum ₂Converge until 100% under the atmosphere.At this moment, cell is placed the DMEM that has added 2% FCS and 50 μ g/ml gentamicins.After 5 days, in this low blood serum medium, (ExGen 500, Fermentas) also use the PS58 transfectional cell as mentioned above for method to specifications.Final oligonucleotide concentration is 200nM and 20nM.Handle back 48 hours of beginning (be transformed into low serum condition after 7 days), (Bio-Rad) separate and preparation RNA with " total RNA small volume of reagent box ", to be used for the Northern engram analysis.With Northern trace and radioactivity people DMPK (hDMPK) probe and mouse GAPDH mRNA probe hybridization.Come quantitative DMPK signal by the analysis of phosphorescence imager, and carry out normalization method, to represent with respect to the form of simulation contrast at the GAPDH signal.

Fig. 7 shows through oligonucleotide PS58 (the 20 and 200nM) conversion of handling the myoblastic Northern engram analysis of MyoD.The result has proved morbific hDMPK (CUG) 1500RNA transcript has effectively been suppressed fully, and less normal hDMPK (CUG) 11RNA transcript only is subjected to minimal effect under two concentration.Therefore, the oligonucleotide at repeat region demonstrates at the selectivity than big repeat size (or the amplification of causing a disease).

Embodiment 5 (Fig. 8)

In this embodiment, cultivation described in the embodiment 1, transfection and analysis have and comprise the DM500 immortalization sarcoplast clone of the people DMPK gene of (CTG) 500 repeat amplification protcols approximately as mentioned.Gathered in the crops preceding 48 hours, and only (CUG) tumor-necrosis factor glycoproteins was had specific oligonucleotide PS58, sequence in the exons 1 is had specific oligonucleotide PS113 or simulation control treatment DM500 myotube with 200nM.At have in hDMPK mRNA that expresses in this mouse cell lines (referring to the primer of embodiment 1) and the mouse three kinds of other genes of natural (CUG) multiple (have (CUG) 6 Ptbp1, have the syndecan 3 of (CUG) 6 and have (CUG) 9 slide albumen β) transcript carry out RT-PCR and analyze.

The PCR the primer is as follows: Ptbp1:5 '-TCTGTCCCTAATGTCCATGG-3 ' and 5 '-GCCATCTGCACAAGTGCGT-3 '; Syndecan 3:5 '-GCTGTTGCTGCCACCGCT-3 ' and 5 '-GGCGCCTCGGGAGTGCTA-3 '; Slide albumen β: 5 '-CTCAGCCCTGCTGCCTGT-3 ' and 5 '-CAGACCCATACGTGCTTATG-3 '.The PCR product carries out agarose gel electrophoresis, and (it is quantitative UnitedKingdom) to carry out signal for UVP BioImaging systems, Cambridge to use Labworks 4.0 programs.Use corresponding Actin muscle signal that strength of signal is carried out normalization method, be expressed as the form of relative simulation contrast.

Fig. 8 shows the RT-PCR result of the highest inhibition that PS58 expresses hDMPK mRNA.Than not having the oligonucleotide PS113 of complementary sequence, have other genetic transcription thing of natural little (CUG) multiple and be not subjected to (CUG) to repeat specific oligonucleotide PS58 influence or minimal effect is only arranged with these genetic transcription things.

This embodiment confirmed, only at the oligonucleotide of repeat region to long segment repeat the amplification of disease (or cause) compared to natural than short-movie section multiple selectivity.

Embodiment 6 (Fig. 9 and 10)

In this embodiment, cultivate, have the DM500 immortalization sarcoplast system of the people DMPK gene that comprises pact (CTG) 500 repeat amplification protcols with PS58 (200nM) transfection.In the present embodiment, pair cell carries out fish analysis.Began to handle back 48 hours, with 4% formaldehyde, 5mM MgCl ₂With 1 * PBS fixed cell 30 minutes.Hybridize in the moist chambers down at 37 ℃ with fluorescently-labeled oligonucleotide Cy3-(CAG) 10-Cy3 and to spend the night.After the hybridization, detergent also is embedded among the mowiol, makes it dried overnight.Use Bio-Rad MRC1024 laser confocal scanning microscope and LaserSharp2000 acquisition software to manifest nuclear inclusion body (ribose nuclear speckle).To 50 cell countings altogether, the situation that exists of inclusion body in the nuclear of these cells is marked.

Document shows, contains (CUG) amplification multiple DMPK mRNA accumulation and gathering in nuclear, forms the ribose nuclear speckle with modulability nucleoprotein and transcription factor.Therefore, normal nuclear gene processing and cell function suffer damage.

Fig. 9 has shown the cell that contains the simulation process of ribose nuclear inclusion body at nuclear, and after handling with PS58, they no longer are present in the nucleus.Figure 10 shows, handles the nuclear per-cent that makes in the DM500 myotube visible under the collating condition contain the ribose nuclear speckle with PS58 and obviously reduces.This result proves, the triplet that the inhibition that hDMPK mRNA is expressed has also suppressed to be rich in disease-related repeats the inclusion body of (CUG).

Embodiment 7 (Figure 11)

In the present embodiment, the effect of interior evaluating PS58 in the DM500 mouse that contains the hDMPK that increases with about 500 triplets (CTG) n.The DM500 mouse derives from the DM300 mouse (for example, referring to (2007) PLoS Genet.20073 (4) such as Gomes-Pereira M: e52) by the somatocyte amplification.Confirm (CTG) triplet repeat amplification protcol of about 500 by Southern trace and pcr analysis.

In brief, according to appended specification sheets PS58 and transfection reagent ExGen 500 (Fermentas) are mixed for using in the body.PS58 (2nmol, with Exgen 500 in transfection solution) (40 μ l) is expelled in the GPS complex body of DM500 mouse in one-year-old age, repeats this process after 24 hours.In contrast, similarly with the transfection solution-treated DM500 mouse that does not contain PS58.After 15 days, put to death mouse, separating muscle separates total RNA and (uses Trizol, Invitrogen) from tissue.With mentioned above similar, carry out RT-PCR and analyze to detect the hDMPK mRNA in the muscle.(UVP Bio1maging systems, Cambridge UnitedKingdom) determine the intensity of each band, carry out normalization method with respect to the intensity of corresponding Actin muscle band to use Labworks 4.0 programs.Primer location as shown in the figure.

Figure 11 shows than simulation process, reduces the hDMPK mRNA of (CUG) n of existing containing repeat amplification protcol in sole of the foot flesh and the gastrocnemius muscle in the body strongly with PS58 processing DM500 mouse.

Embodiment 8 (Figure 12)

In this embodiment, to different oligonucleotide (different) but all only compare at the sequence of (CTG) n repeat amplification protcol in length and skeleton chemistry properties.Cultivation described in the embodiment 1 as mentioned, transfection and analysis DM500 myotube.Quantitative with the analysis of phosphorescence imager to the Northern trace, the DMPK signal pin is carried out normalization method to GAPDH.

Here, handle the DM500 myotube with following oligonucleotide (200nM), all oligonucleotide all have phosphorothioate backbone (referring to table 3).

Figure 12 shows that for all oligonucleotide of being tested, the processing of DM500 myotube all causes the hDMPK mRNA with (CUG) n amplification to reduce fully.With this understanding, the highest effect that is obtained does not rely on oligonucleotide length, backbone modification or the possible inhibition mechanism of used single stranded oligonucleotide.

Embodiment 9

From male sex's Huntington chorea patient's inoblast (GM 00305) obtain from Coriell Cell Repository (Camden, New Jersey, US), and according to appended specification sheets with well known to a person skilled in the art that standard technique cultivates.The Huntington chorea patient has allelotrope and morbific allelotrope of a health of Huntington chorea gene, and it causes expressing simultaneously two kinds of mRNA, and (CAG) with amplification quantity that have normal quantity respectively repeats.

And have with huntington mRNA in (CAG) triplet repeat 21 aggressiveness, 2 ' the O-methyl thiophosphatephosphorothioate RNA antisense oligonucleotide PS57 transfection inoblast that complementary has (CUG) 7 sequences.Indicated as manufacturers, 100 or the PEI of 200nM in the presence of carry out transfection.After the transfection 24 hours, harvested cell separated total RNA, analyzes by RT-PCR.Use the primer (5 ' GAAAG TCAGT CCGGG TAGAA CTTC 3 ' and 5 ' CAGAT ACCCG CTCCA TAGCAA 3 ') in the downstream exon 64 to determine the huntington transcript.This method all detects two types huntington mRNA (normally with the sudden change transcript with extra (CAG) amplification).Also measured GAPDH mRNA (house-keeping gene).Signal is carried out quantitatively, the total amount of huntington mRNA is carried out normalization method with respect to the amount of GAPDH mRNA in the same sample.The result is with respect to representing from the form of fibroblastic control treatment (no oligonucleotide, it is 100%) sample.

Use by oneself 100 or fibroblastic sample of 200nM PS57 transfection in, observe and be respectively remarkable lower total huntington mRNA level of about 53% and 66% than the cell of control treatment.

Therefore, only induce the reduction of huntington mRNA level at (CAG) multiple oligonucleotide PS57, these results with respect to normal Huntington chorea mRNA and selectivity mutation inhibiting body is consistent.

Table 1: the oligonucleotide of being tested is summed up

The oligonucleotide name	Modify	Sequence	The position
				PS40	2 ' OMe RNA thiophosphatephosphorothioate/FAM	GAGGGGCGUCCAGGGAUCCG	Introne 1 4-exons 15
PS41	2 ' OMe RNA thiophosphatephosphorothioate	GCGUCCAGGGAUCCGGACCG	Introne 1 4-exons 15
				PS42	2 ' OMe RNA thiophosphatephosphorothioate	CAGGGAUCCGGACCGGAUAG	Introne 1 4-exons 15
PS56	DNA	CAGCAGCAGCAGCAGCAGCAG	Repetition in the exons 15
				PS58	2 ' OMe RNA thiophosphatephosphorothioate/FAM	CAGCAGCAGCAGCAGCAGCAG	Repetition in the exons 15
PS59	2 ' OMe RNA thiophosphatephosphorothioate	UGAGUUGGCCGGCGUGGGCC	ESE exons					15
				PS60	2 ' OMe RNA thiophosphatephosphorothioate	UUCUAGGGUUCAGGGAGCGCGG	ESE exons		15
PS61	2 ' OMe RNA thiophosphatephosphorothioate	ACUGGAGCUGGGCGGAGACCC	ESE exons					15
				PS62	2 ' OMe RNA thiophosphatephosphorothioate	CUCCCCGGCCGCUAGGGGGC	ESE exons		15
PS113	The DNA thiophosphatephosphorothioate	GAGCCGCCTCAGCCGCACCTC	Exons 1
				PS114	The DNA thiophosphatephosphorothioate	GAAGTCGGCCACGTACTTGTC	Exons 1
PS115	The DNA thiophosphatephosphorothioate	GGAGTCGAAGACAGTTCTAGG	Exons					15
				PS116	The DNA thiophosphatephosphorothioate	GGTACACAGGACTGGAGCTGG	Exons		15

Table 2: oligonucleotide is transcribed the reduction of back hDMPK mRNA

Oligonucleotide	The reduction of hDMPK mRNA	SEQ?ID?No.’s
			PS40	+	1
PS41	-	2
			PS42	-	3
PS59	-	4
			PS60	-	5
PS61	+/-	6
			PS62		7
PS58	++++	8
			PS56	-	9
PS113	-	10
			PS114	-	11
PS115	+/-	12
			PS116	+	13

(-) expression does not have reduction, the reduction level of (+) expression hDMPK mRNA

The oligonucleotide that uses among table 3: the embodiment 9

#	Length	(CAG)n	Replace ribose	RNAse H decomposes possibility
					PS58	21-mer	n＝7	2 ' O-methyl	Do not have
PS146	30-mer	n＝10	2 ' O-methyl	Do not have
					PS147	15-mer	n＝5	2 ' O-methyl	Do not have
PS142	21-mer	n＝7	Ribodesose (DNA)	Have

* the equal total length of all oligonucleotide is thiophosphatephosphorothioate and replaces

Sequence table

<110>Prosensa?B.V.

＜120〉be used for the treatment of the method and the article of the relevant genetic diseases of DNA repeat instability

<130>P6010168PCT

<150>EP06118809.0

<151>2006-08-11

<150>EP06119247.2

<151>2006-08-21

<160>34

<170>PatentIn?version?3.3

<210>1

<211>20

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS40 of chemosynthesis

<400>1

<210>2

<211>20

<212<RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS41 of chemosynthesis

<400>2

<210>3

<211>20

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS42 of chemosynthesis

<400>3

<210>4

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS56 of chemosynthesis

<400>4

<210>5

<211>21

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS57 of chemosynthesis

<400>5

<210>6

<211>21

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS58 of chemosynthesis

<400>6

<210>7

<211>20

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS59 of chemosynthesis

<400>7

<210>8

<211>22

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS60 of chemosynthesis

<400>8

<210>9

<211>21

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS61 of chemosynthesis

<400>9

<210>10

<211>20

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS62 of chemosynthesis

<400>10

<210>11

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS113 of chemosynthesis

<400>11

<210>12

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS114 of chemosynthesis

<400>12

<210>13

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS115 of chemosynthesis

<400>13

<210>14

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS116 of chemosynthesis

<400>14

<210>15

<211>21

<212>DNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS142 of chemosynthesis

<400>15

<210>16

<211>30

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS146 of chemosynthesis

<400>16

<210>17

<211>15

<212>RNA

＜213〉the unknown

<220>

＜223〉the oligonucleotide PS147 of chemosynthesis

<400>17

<210>18

<211>12

<212>RNA

＜213〉the unknown

<220>

＜223〉oligonucleotide of chemosynthesis (CAG) n

<400>18

<210>19

<211>12

<212>RNA

＜213〉the unknown

<220>

＜223〉oligonucleotide of chemosynthesis (GCG) n

<400>19

<210>20

<211>12

<212>RNA

＜213〉the unknown

<220>

＜223〉oligonucleotide of chemosynthesis (CUG) n

<400>20

<210>21

<211>12

<212>RNA

＜213〉the unknown

<220>

＜223〉oligonucleotide of chemosynthesis (CGG) n

<400>21

<210>22

<211>12

<212>RNA

＜213〉the unknown

<220>

＜223〉oligonucleotide of chemosynthesis (CCUG) n

<400>22

<210>23

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉primer 1 hDMPK

<400>23

<210>24

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉primer 2 hDMPK

<400>24

<210>25

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉Actin muscle has adopted primer

<400>25

<210>26

<211>17

<212>DNA

＜213〉artificial

<220>

＜223〉Actin muscle antisense primer

<400>26

<210>27

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer 1 Ptbpl

<400>27

<210>28

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉primer 2 Ptbpl

<400>28

<210>29

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉primer 1 syndecan 3

<400>29

<210>30

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉the primer 2 syndecan 3

<400>30

<210>31

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉primer 1 β slides albumen

<400>31

<210>32

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉primer 2 β slides albumen

<400>32

<210>33

<211>24

<212>DNA

＜213〉artificial

<220>

＜223〉primer 1 huntington

<400>33

<210>34

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉primer 2 huntington

<400>34

Claims (23)

1, single stranded oligonucleotide is used for preparing the purposes of the medicine of treatment or prevention people cis element repeat instability correlated inheritance disease, described single stranded oligonucleotide only comprise with the genetic transcription thing in tumor-necrosis factor glycoproteins complementary sequence, perhaps form by described sequence.

2, according to the purposes of claim 1, wherein said repeat element is present in the encoding sequence of genetic transcription thing.

3, according to the purposes of claim 1, wherein said repeat element is present in the non-coding sequence of genetic transcription thing.

4, according to each purposes in the aforementioned claim, wherein said oligonucleotide comprises and is selected from (CAG) n, (GCG) n, (CUG) n, (CGG) n, (CCG) n, (GAA) n, (GCC) n and (CCUG) the repeat element complementary sequence of n, perhaps is made up of described sequence.

5, according to the purposes of claim 2, wherein said oligonucleotide comprises with polyglutamyl amine (CAG) n sections complementary sequence or by described sequence and forms, and wherein said repeat instability disease is Huntington chorea, spinocerebellar ataxia, Haw-River syndrome, X chain spinal cord oblongata muscular dystrophy and/or dentatorubral-pallidoluysian atrophy.

6, according to the purposes of claim 2, wherein said oligonucleotide comprises with many L-Ala (GCG) n sections complementary sequence or by described sequence and forms, and wherein said repeat instability disease is selected from infantile spasm syndrome, craneocleidol dysplasia, blepharophimosis, hand-foot-sexual organ syndrome, synpolydactyly, oculopharyngeal muscular dystrophy and/or holoprosencephaly.

7, according to the purposes of claim 3, wherein said oligonucleotide comprises and repeats the complementary sequence with (CUG) n or be made up of described sequence, and wherein said repeat instability disease is I type myotonia atrophica, 8 type spinocerebellar ataxias and/or 2 type Huntington chorea sample diseases.

8, according to the purposes of claim 3, wherein said oligonucleotide comprises and repeats the complementary sequence with (CCUG) n or be made up of described sequence, and wherein said repeat instability disease is an II type myotonia atrophica.

9, according to the purposes of claim 3, wherein said oligonucleotide comprises and repeats the complementary sequence with (CGG) n or be made up of described sequence, and wherein said repeat instability disease is a fragile X mental retardation.

10, according to the purposes of claim 3, wherein said oligonucleotide comprises (GAA) n repetition complementary sequence or is made up of described sequence, and wherein said repeat instability disease is a Friedreich ataxia.

11, according to each purposes in the aforementioned claim, the length of wherein said oligonucleotide is about 10 to about 50 Nucleotide, preferred 12 to 30 Nucleotide.

12, according to each purposes in the aforementioned claim, wherein said oligonucleotide is made of following: the RNA Nucleotide, DNA Nucleotide, lock nucleic acid (LNA) Nucleotide, peptide nucleic acid(PNA) (PNA) Nucleotide, morpholino phosphoryl diamine (PMO), ethidene bridged linkage nucleic acid (ENA) Nucleotide or its mixture that have or do not have the skeleton that comprises thiophosphatephosphorothioate.

13, according to the purposes of claim 12, wherein said oligonucleotide comprises the RNA thiophosphatephosphorothioate Nucleotide that 2 '-O-replaces.

14, according to each purposes in the aforementioned claim, the oligonucleotide in the wherein said medicine is provided by the nucleic acid carrier that can express described oligonucleotide.

15, according to each purposes in the aforementioned claim, the oligonucleotide in the wherein said medicine is at least with being used to send described oligonucleotide to cell and/or strengthen vehicle and/or the target part sent in its born of the same parents and provide.

16, single stranded oligonucleotide, its comprise be selected from (CAG) n, (GCG) n, (CUG) n, (CGG) n, (GAA) n, (GCC) n and (CCUG) n tumor-necrosis factor glycoproteins complementary sequence or form by it, length is 10 to 50 Nucleotide.

17, according to the oligonucleotide of claim 16, it also comprises RNA thiophosphatephosphorothioate Nucleotide, DNA thiophosphatephosphorothioate Nucleotide, lock nucleic acid (LNA) Nucleotide, morpholino Nucleotide and/or its combination that 2 '-O-replaces.

18, according to each defined oligonucleotide among the claim 1-17, it has radio-labeling or fluorescent mark.

19, comprise medicinal compositions according to each oligonucleotide that defines among the claim 1-18.

20, the pharmaceutically acceptable composition of claim 19, it also comprises and at least aly is used to send described oligonucleotide to cell and/or strengthen vehicle and/or the target part of sending in its born of the same parents.

21, can make the nucleic acid carrier that each defined oligonucleotide is expressed among the claim 1-18 in people's cell, the preferred virus carrier.

22, reduce the method that contains multiple genetic transcription thing in the cell, it comprises uses each defined oligonucleotide among the claim 1-18.

23, external or stripped detection and/or diagnostic method wherein use each described oligonucleotide among the claim 17-18.

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