CN1718194A - Oligonucleotide medicine for treating myophagism - Google Patents

Abstract

An oligonucleotide medicine for treating amytrophy contains 7-75 nucleotide, 7-75 adjacent ribosyls, and the 5'-terminate and 3'-terminate non-translation regions and translation start region. Said oligonucleotide includes ribooligonucleotide and deoxyribooligonucleotide. Said oligonucleotide may be antisense oligonucleotide, interfering RNA(RNAi), ribozyme and deoxyribozyme.

Description

Treat amyotrophic oligonucleotide drug

Technical field:

The oligonucleotide that the present invention relates to treat amyotrophy or strengthen muscle growth the invention still further relates to the compositions and the purposes that contain this oligonucleotide.

Background technology:

Strengthen muscle growth and be clinically with farming and animal husbandry in need the problem that solves.

Amyotrophy and relevant human body comprise muscular dystrophy, spinal cord injury, neurodegenerative disease, flesh shortage, cachexia, amyotrophy and diabetes unusually.The common symptom of these diseases is muscle minimizings and is accompanied by myasthenia that serious will cause maimed person and death.

In farming and animal husbandry was produced, for example in the raising of edible animal, people always wished to increase the ratio composition of muscle.

But the method for still not having effectively treatment amyotrophy or enhancing muscle growth at present.

Summary of the invention:

The purpose of this invention is to provide medicine or method that a class is effectively treated amyotrophy or strengthened muscle growth.

Although it is multifactorial causing amyotrophic origin, normally caused by multiple different genes abnormal expression.Obviously, by adjusting, can reach the purpose of treatment to unconventionality expression gene in this type of disease.

The present invention finds that some oligonucleotide has the effect of treatment amyotrophy or enhancing muscle growth.

The feature of described oligonucleotide is:

(1) contains 7～75 nucleotide;

(2) contain ribose groups by 7～75 adjacency of achirality 5 ' connect to key between 3 ' phosphoric acid nucleoside;

(3) be complementary to and 5 of the relevant target gene of muscular dystrophy ' end untranslated region, translation initiation district, 3 ' end untranslated region and translation termination district;

(4) has 75 ℃～115 ℃ melting temperature (Tm, 1 millimolar concentration)

When oligonucleotide of the present invention as the treatment amyotrophy or when strengthening muscle growth, can use a kind ofly, also can use two kinds or more kinds of.When using two kinds of oligonucleotide, these two kinds of oligonucleotide will be separately and one section regional complementarity of identical or different muscular dystrophy related gene.

When using two kinds of oligonucleotide, the regional complementarity in 5 ' untranslated region, translation initiation site, 3 ' untranslated region or the translation termination site of first gene in first kind of oligonucleotide and a certain group of gene, the regional complementarity in 5 ' untranslated region, translation initiation site, 3 ' untranslated region or the translation termination site of second gene in second kind of oligonucleotide and same group or another group gene.

Preferably, above-mentioned oligonucleotide is made up of 10～26 nucleotide, has 40 ℃～85 ℃ melting temperature (Tm, 1 pmol concentration).

Described oligonucleotide also can be through chemical modification, become the modification oligonucleotide that on glycosyl 2 ' position, has substituted radical, also have the effect for the treatment of amyotrophy preferably or strengthening muscle growth, these groups comprise oxygen, methoxyl group, propoxyl group, methoxy-ethyoxyl, fluorine, chlorine, bromine, iodine.

In some instances, modification property oligonucleotide is at 3 ' end or 5 ' have blocking group.

The oligonucleotide of described modification can also be 3 ' endcapped and/or 5 ' endcapped.

Oligonucleotide of the present invention comprises ribose oligonucleotide and deoxyribose oligonucleotide.The sequence of the oligonucleotide of described modification is selected from SEQ ID NO:1-SEQ ID NO:191.

Oligonucleotide of the present invention is an antisense oligonucleotide, comprises the antisense oligonucleotide of antisense oligonucleotide, the targeting animal gene of targeting people's gene, at the representative antisense oligonucleotide of people's flesh amicine.The sequence of representational antisense oligonucleotide sees Table 3, table 4 and table 5.

Oligonucleotide of the present invention can also be the RNA interfering (RNAi) of scalable flesh amicine and its expression of receptor.These RNA interfering are the double-stranded RNA that 21～25 nucleotide are formed, and at 3 of every chain ' end 2 outstanding nucleotide are arranged; Double-stranded G+C content is 30～52%; To 19 positions, be the A/U pairing at 15 on the positive-sense strand; No intramolecularly repetitive sequence.

Oligonucleotide of the present invention can also be a ribozyme.

Oligonucleotide of the present invention can also be the DNAzyme of scalable flesh amicine and its expression of receptor.This DNAzyme contains the catalysis district that sequence is GGCTAGCTAACGA, can cut connecting key between the purine that is present among flesh amicine and its acceptor gene mRNA and the pyrimidine specifically.DNAzyme is to finish by two complementary series and mRNA hybridization that are present in cleavage site 5 ' end and 3 ' end with combining of target gene mRNA.

Can prepare multiple treatment amyotrophy as active substance or strengthen the pharmaceutical composition of muscle growth with oligonucleotide of the present invention, be applicable to the animal and human.Described animal comprises mammal, as people, horse, cattle etc. and nonmammalian, as chicken etc.Can contain various pharmaceutically acceptable excipient, adjuvant in the described pharmaceutical composition.In the described pharmaceutical composition, can contain one or more described oligonucleotide.Such as, can contain two kinds, three kinds, four kinds, the modification oligonucleotide more than five kinds or five kinds.

In some example, the invention provides a kind of oligonucleotide that contains two or more regulating muscle amicine and the expression of its acceptor gene, the target gene that wherein has an oligonucleotide at least is a flesh growth inhibited plain gene, and the target gene that has an oligonucleotide at least is not a flesh growth inhibited plain gene.

In other examples, pharmaceutical composition can contain one or more from one group of material that antisense oligonucleotide, RNA interfering, ribozyme or DNAzyme are selected.

The non-flesh growth inhibited plain gene of indication of the present invention comprises: but be not limited to Activin A receptor II B, Tollo ⊥ dmetalloproteinase, NF-kappaB, TNF-alpha, MuRF1, Caspase-3, Atrogin-1, RAS, P38MAPK, MKK3, MKK6, JNK, c-myc, c-jun, ASK-1, TAK-1 Smad, Cyclin-dependent Kinase inhibitorp21, Gadd45, IL-6 and PIF (see Table 1 and table 2).In some example, the contained antisense oligonucleotide of pharmaceutical composition can be from table 3,4,5 be selected in the oligonucleotide of targeting flesh growth inhibited plain gene, and the oligonucleotide of the non-flesh growth inhibited of contained targeting plain gene can be selected from table 3 and 4.

The present invention also provides in the cell of flesh amicine gene expression, with the method for oligonucleotide regulating muscle amicine expression of the present invention.In other examples, the molecule that these regulating muscle amicines are expressed can be antisense oligonucleotide, RNA interfering, ribozyme, DNAzyme.

The present invention also provides in the cell of flesh growth inhibited plain gene and the gene expression of non-flesh amicine, with antisense oligonucleotide of the present invention, RNA interfering, ribozyme or DNAzyme, mixes to use and regulates target gene expression.

Oligonucleotide of the present invention promptly comprises above-mentioned antisense oligonucleotide, RNA interfering, ribozyme, DNAzyme, can be used for treating people's muscular dystrophy, also can be used for increasing in the consumption animal feeding ratio and the weight of muscle.

Above-mentioned antisense oligonucleotide, RNA interfering, ribozyme, DNAzyme can be used a kind of in pharmaceutical composition, also can severally mix simultaneously and use.

Oligonucleotide provided by the invention for animal or human's dosage is: per kilogram of body weight 0.01～100mg.

The detailed description of invention

The present invention relates to can be used for to regulate oligonucleotide and pharmaceutical composition thereof with the expression of amyotrophy or disease related gene, and the method that promotes animal muscle growth with this oligonucleotide.Therefore, the medicine that invention is provided both can be used for treating diseases such as amyotrophy, unusual fatty accumulation, can be used for strengthening the animal muscle growth again, for example farming animals.

Pharmaceutical composition of the present invention, method are to utilize the gene regulation oligonucleotide to regulate expression of target gene, and these gene regulation oligonucleotide (or being called regulatory molecule) comprise antisense oligonucleotide, RNA interfering (RNAi), ribozyme, DNAzyme.The mode of regulation and control can be to remove to control a kind of gene that muscle growth is grown that relates to a kind of regulatory molecule, for example, and flesh growth inhibited plain gene.Regulation and control can also be adopted the mode of multiple combination, for example, adopt the combination of dissimilar regulatory molecules; At same gene, but the multiple adjusting molecular combinations of different loci; New combination at heterogeneic multiple adjusting molecular combinations and above-mentioned various compound mode formation.

Modification oligonucleotide of the present invention has specific structure and physicochemical characteristics, for example, terminal protection, protonated processing, acid-resisting, nuclease-resistant contains achirality phosphoric acid ester bond, modification property ribose and deoxyribose substituent group.

The invention provides method of regulating gene expression in animal and human's cell and the method for using medicine composite for curing muscular dystrophy of the present invention.Described muscular dystrophy comprises that muscular dystrophy, spinal cord injury, neurodegenerative disease, apositia, flesh lack, dislike matter disease, amyotrophy, type-II diabetes, X syndrome etc.In the intact animal, use weight and the ratio that pharmaceutical composition of the present invention can increase muscle.

The effect of modification oligonucleotide provided by the invention is to regulate its target gene expression.This adjusting both can be to suppress, and also can be to promote, other variations perhaps take place, and for example, formed the gene outcome that changes.Suppress to be meant and compare that this gene expression has reduced by 1%～99% with wild type.Promote to be meant and compare that this gene expression has increased by 1%～99% with wild type.

Containing one or more in the pharmaceutical composition of the present invention can adjusting and the oligonucleotide of amyotrophy or flesh growth related gene.These oligonucleotide comprise antisense oligonucleotide, RNA interfering, ribozyme, DNAzyme.The target site of oligonucleotide can be one or more zones of a certain gene, also can be the more than one site of a plurality of genes, for example flesh amicine, flesh amicine receptor and flesh amicine transcription factor gene.

Pharmaceutical composition and methods for using them of the present invention is applicable to any gene relevant with amyotrophy, changes or regulate these expression of gene to produce therapeutic effect or useful biological effect to amyotrophy or flesh growth.Although in the growth and maintenance process of muscle, some major genes play an important role, in most of the cases, disease often relates to a plurality of genes in a plurality of signal transduction paths and each approach.Therefore, the present invention not only provides at term single gene () medicine for example, the flesh amicine, but also drug regimen at the zones of different of a plurality of genes or term single gene is provided, or the combination of above two kinds of medicines.

Listed representative target gene in table 1 and table 2, these genes can be used for designing the Gene regulation medicine.

The medicine that the A regulator gene is expressed

Any preparation based on nucleic acid all can be used for medicine of the present invention, comprising antisense oligonucleotide, ribozyme, RNA interfering and DNAzyme.The common ground of these technical methods is that mRNA combines with its target gene by Watson-Crick base pairing principle.Therefore, the present invention relates under stringent condition the hybridization between polynucleotide and the related gene sequence.Here the stringent condition of indication is that to a certain degree mispairing can cause crossbred nucleic acid to form.Polynucleotide refer to the molecule that can be used for suppressing the amyotrophy related gene expression, and it also can be used as probe and primer, are used for diagnosis and laboratory research.

Oligonucleotide refers to and contains nucleotide (ribonucleic acid, DNA (deoxyribonucleic acid) or both) molecule, these nucleotide by 5 ' with 3 ' end or 5 ' with 2 ' hold to be connected, used nucleotide can be natural, also can be the analog of chemosynthesis, these analog can form pairing with natural acid.For example, azepine (Aza) and denitrogenation (deaza) pyrimidine analogue of mixing, azepine (aza) and denitrogenation mix (deaza) purine and other heterocyclic base analog.These base analogues are included in one or more carbon nitrogen-atoms in pyrimidine or the purine by replacements such as oxygen, sulfur, selenium, phosphorus.

The included antisense oligonucleotide of oligonucleotide of the present invention, RNA interfering, ribozyme and DNAzyme below are described in detail in detail respectively.

1. antisense oligonucleotide

Antisense oligonucleotide is the short dna or the RNA of chemosynthesis, and it can combine with said target mrna by base pairing, thereby blocks the translation of mRNA or pass through activator RNA seH degraded mRNA.The effect of antisense oligonucleotide can be by splicing, translation and the nucleus of inhibition RNA and the course of conveying between the Cytoplasm.The influence that the mechanism of action is changed by the precursor structure of oligonucleotide.Antisense oligonucleotide can be complementary to whole gene order, also can be complementary to the part of target gene.

Therefore, the length of an antisense oligonucleotide of the present invention can be 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100 or more than 100 nucleotide.Be 7～75 nucleotide preferably with length.The antisense oligonucleotide preferred length is 10～26 nucleotide.

The method of typical design antisense oligonucleotide comprises: (1) selects contiguous with target region or an overlap oligonucleotide, (2) measure and the bonded Gibbs free energy of target gene level, (3) analyze hybridization melting temperature (Tm), (4) carry out the sequence library analysis, to determine its target site zone of living in, for example, 5 ' end untranslated region, translation initiation district, 3 ' end untranslated region and translation termination district.Preferred antisense oligonucleotide generally is adjacent to the translation initiation district or has a nucleotide at least and the translation starting point overlapping.This overlapping is no less than three nucleotide in some example.

Based on secondary and the higher structure feature of RNA, the design of antisense RNA or DNA generally can be selected in 5 ' end regions.In some cases, can be selected in 3 ' end regions, or around the RNA splice point.

Antisense oligonucleotide of the present invention can analyze to determine the bond strength of itself and target RNA by the Gibbs free energy.This analysis can be undertaken by computer program, and program commonly used can be at ftp: //the rna.chem.rochester.edu website finds, and can with reference to Matthews etc. (J.Mol.Biol, 1999,288,911-940 and RNA, 1999,5,1458-1469).The Gibbs free energy of oligonucleotide and target RNA hybrid molecule is general≤-20kcal (37 ℃), be preferably≤-25kcal.For the oligonucleotide of a 10-14 unit, its free energy is≤-15kcal; The hybridization free energy of the oligonucleotide of 15-17 unit is≤-20kcal; The hybridization free energy of the oligonucleotide of 18-20 unit is≤-25kcal; The hybridization free energy of the oligonucleotide of 21-23 unit is≤-30kcal; The hybridization free energy of the oligonucleotide of 24-26 unit is≤-35kcal.

Therefore, oligonucleotide length provided by the present invention is 7～75 nucleotide, and preferred length is 10～26 nucleotide, and melting temperature (Tm) is about 75 ℃～115 ℃ (1 millimolar concentrations), preferred 40 ℃～85 ℃ of melting temperatures (1 pmol concentration).Be complementary to 5 of target gene ' end untranslated region, translation initiation district or terminator; Target gene can be the optional gene in table 1 and the table 2, or other gene, and the adjusting of its expression can produce therapeutic effect to the relevant disease of amyotrophy.，

In order to use oligonucleotide effectively in vivo, it is carried out chemical modification can increase the stability that oligonucleotide is antiacid and antienzyme is degraded significantly, for example, 2 '-O-methyl, 2 '-O-pi-allyl, delivery ceremony nucleic acid (Locked nucleic acids, LNA) and peptide chain nucleic acid (peptide nucleic acids, PNA).

Antisense oligonucleotide can utilize in method well known in the art and be prepared by chemosynthesis or enzyme ways of connecting.In addition, antisense oligonucleotide can also utilize expression vector to produce in vivo.Although at table 3, the antisense oligonucleotide that is exemplified in 4,5 is DNA (deoxyribonucleic acid) to be formed, and it also can be a ribonucleic acid, for example, replaces thymus pyrimidine with uracil, replaces deoxyribosyl with ribosyl.Therefore, oligonucleotide of the present invention can be made up of DNA (deoxyribonucleic acid) fully, also can be made up by ribonucleic acid fully, or be made up of DNA (deoxyribonucleic acid) and ribonucleic acid mixing.

Antisense oligonucleotide provided by the present invention can be fully with coming from table 3, cited sequence in 4,5, but also can have percent 60,70,80,90,95 or 99 homology with listed sequence.This sequence homology analysis can adopt in method well known in the art and measure, for example, and Fasta program (Oxford Molecular Grouplnc), BLAST (WWW.ncbi.nlm.nih.gov) (Atschul etc., 1997, Nucleic Acids Res, 25,3389-3402).All employing programs of analytical parameters of program recommend to make.

The G of antisense oligonucleotide of the present invention: the C composition is generally greater than 35%, and preferred ratio is greater than 40%, and optimal proportion is greater than 45%.

2. ribozyme

Ribozyme of the present invention refer to a kind of can specificity the nucleic acid of cutting RNA molecule, it can be RNA, DNA, the combination of nucleic acid analog or RNA, DNA etc.Ribozyme comprises several different types, for example, and hammerhead ribozyme (Rossi, Pharmac.Ther.50:245-254 such as J, 1991) (Foster and Symons, Cell 48:11-220; 1987) (Haseloff and Gerlach, Nature 328:596-600,1988) (Wallbot and Bruenning, Nature334:196,1988) (US Pat No.5 such as Haseloff, 254,678), hair fastener type ribozyme (Hampel etc., NucleicAcid Res 18:299-304; US Pat No.5,254,678), Delta hepatitis virus ribozyme (Perotta and Been, Biochem.31:16,1992), the I group includes subtype ribozyme (Cech etc., US Pat NO.4,987,071) and RNase P ribozyme (Takada etc., Cell 35:849,1983).Other list of references comprises: WO 95/29241 and WO 95/31551.In these several different ribozyme types, hammerhead ribozyme is because its simplicity and practicality, is widely used in Gene regulation and the possible disease treatment.

The targeted rna of hammerhead ribozyme contains NUH, and wherein N is any among G, U, C, the A, and H is C, U or A.This ribozyme is a binding sequence by 5～15 nucleotide that are positioned at catalysis conserved sequence both sides to the identification of targeted rna.The DNA of ribozyme and encoding ribozyme can synthesize by method well known in the art (Heidenreich etc., J.FASEB 70 (1): 90-96,1993; Sproat, Current Opin, Biotechnol 4 (1) 20-28,1993).In the building-up process of ribozyme, can introduce different chemical modifications, strengthening the stability of ribozyme, as introduce Deoxydization nucleotide, D2EHDTPA key or 2 '-the O-methyl.

Therefore, the invention provides and can suppress the ribozyme of expressing with amyotrophy relevant disease related gene, target gene can be a table 1, listed arbitrary gene or multiple combination in 2.The design of ribozyme can be based on tup type or hair fastener pattern type, and it can be synthetics (has or be not with chemical modification), also can be to produce in vivo by expression vector.The ribozyme of these regulating muscle atrophys or flesh growth all can be used as one of composition of pharmaceutical composition of the present invention.

3. RNA interfering (RNAi)

It is transfered cell by double-stranded RNA that RNA disturbs, thus mediation the degraded of corresponding mRNA.This technology has been proved to be a kind of instrument (Elbashir etc., Nature 411:494-498 2001 of effective inhibition target gene rna expression; Tuschl etc., Genes and Development, 13:3191-3197,1999; Zamore, Cell 101:25-33,2000; US pat No.6,506,539).

The interferential mechanism of action of RNA is by will be corresponding to the double-stranded RNA transfered cell of target gene RNA, and in cell, double-stranded RNA is digested short RNA interfering (siRNA), and it is incorporated into " the inductive gene inhibition complex of RNA-" (RISC).RISC guides siRNA and homologous target RNA complementation to combine then, finally causes the degraded (Sharp etc., Genes Dev 15-485-490,2001) of target RNA.

Drug regimen provided by the invention contains the RNA interfering at the amyotrophy related gene, with regulating muscle amicine or its receptor, or other Expression of Related Genes.This RNA interfering is the double-stranded RNA of 21-25 nucleotide, has two outstanding nucleotide at 3 of every chain ' end, and double-stranded G+C content is 30-52%, on the 15th～19 position on the positive-sense strand, is the A/U pairing, and does not have the intramolecularly repetitive sequence.In this way, can prepare disturbance RNA molecule at people's flesh growth inhibited plain gene.

4. DNAzyme

Using DNAzyme is a kind of novel gene inhibition technology, and it has the chemical stability of antisense oligonucleotide, have the function of the catalyze cleavage RNA of ribozyme again simultaneously, and its synthetic expense is very low.Based on these unique advantages, this technology has been widely used in the body and external gene regulation (Pharmacol.Rev.52 (3): 325-347 such as Sun; US patNo 6,617,438; US pat No 6,326,174).

Therefore, the invention provides can inhibition and the DNAzyme expressed of amyotrophy or flesh growth related gene.The structure of this DNAzyme comprises: (1) catalysis group, its nucleotides sequence is classified as: GGCTAGCTACAACGA.(2) be positioned at the binding sequence of catalysis group 5 ' end; (3) be positioned at the binding sequence of catalytic group 3 ' end.DNAzyme combines with the RNA that flesh amicine or its acceptor gene are expressed specifically by two binding sequences, induces cutting to target RNA by catalytic group at purine and pyrimidine binding site.

B. the chemical modification of nucleic acid

Oligonucleotide provided by the present invention can be modified with the mode of chemistry.Described modification is on molecular level the natural acid molecular structure to be carried out a place or many places chemical modification.

Comprise following chemical modification:

1) to the modification of the phosphoric acid ester bond between base, glycosyl, the nucleotide, reaching increases some substituent groups, for example, and diamidogen (diamine), cholesterol or other lipophilic group.

2) terminal protection: the end at molecule adds blocking group, to prevent the degraded of molecule.This protection can be 5 ' end, also can be 3 ' end, or two ends is all protected.

3) protonated: as when this molecule is dissolved in neutral water, can to cause the degraded of pH value.Protonated can after molecule is handled in acid solution, finishing.By this process, the proton acceptor that oligonucleotide had is by the proton combination, described proton acceptor be present in phosphate group replacement or unsubstituted or be present in central group and terminal blocking group on.Under low pH (for example pH1-3) is all unstable for the trunk structure of many nucleic acid.The present invention modifies by introducing some, can make nucleic acid under the condition of pH1～pH2, still has satisfied stability, and these modifications comprise: 2 '-halogenide; 2 '-the O-alkyl, 3 '-the O-alkyl; 2 '-O-alkyl-n (O-alkyl) etc.Because antacid increase makes the easier pharmaceutical composition that is made into of these oligonucleotide.

The present invention also provides the nucleic acid molecules of separating enzymatic degradation in the anti-nucleic acid.This antienzyme characteristic can be modified by number of chemical and be obtained, comprise 2 '-O-methyl acid phosphate diester linkage, 2 '-O-methane, 2 '-O-methane-n (O-methane), 2 '-fluorine, heterozygosis connecting key, the modification of other trunk structure.In addition, these modify the modification that also comprises base, for example, and methylated base, methylolation base etc.

The present invention also provides anti-nucleic acid to separate the nucleic acid molecules of enzymatic degradation outward.Oligonucleotide can be by obtaining this antienzyme characteristic to its 5 ' end and 3 ' terminal modified.These end modified comprising: oppositely connect base, dideoxy nucleotide, METHAPHOSPHORIC ACID base, alkyl, aryl, cordycepin, cytosine arabanoside, 2 '-methoxyl group, ethyoxyl nucleotide, the phosphorothioate connecting key, 3 '-O-methyl base, fluorescein, peptide bond connects, two pyridyls, cholesterol, biotin, acridine, rhodamine psoralen, glyceryl, the butanols base, butyl, hexanol base and 3 '-the O-alkyl.Preferred butanols based structures is OH-CH ₂CH ₂CH ₂CH ₂-.

Modification nucleotide also comprises the modification to glycosyl, for example in 2 ' position 3 ' and the ribonucleic acid or the DNA (deoxyribonucleic acid) that replace.Connecting key between ribosyl and the mononucleotide is called the trunk structure of nucleic acid.The modification of trunk structure is comprised that connecting key and other can enhanced stability and the modification of affinity, for example, to the modification of sugared structure.Concrete example has, and when being reverse with respect to natural dextroisomer sugar, can use left-handed (L-) isomer deoxyribose in base; Or with 2 of glycosyl '-hydroxyl groups, 2 '-halogens, 2 '-O-alkyl, 2 '-O-alkyl-n (O-alkyl) replaces; Perhaps use following connecting key: 2 '-O-methyl acid phosphate diester linkage, 2 '-the O-ethyl, 2 '-O-propyl group, butyl, 2 '-O-alkyl-n (alkyl), 2 '-methoxyethoxy, 2 '-fluorine, 2 '-deoxidation erythropentofuranosyl, 3 '-O-methyl, isobutyl group oligonucleotide, 2 '-O-(CH ₂CH ₂) _xCH ₃And Butyne connecting key.Oligonucleotide of the present invention can have part to be modified, or all modifies, or the combination of different modifying.

The present invention preferably connecting key of modification property oligonucleotide is achirality (achiral), can contain at least 3～8 successive achirality connecting keys, is preferably 7～12 successive connecting keys of achirality; Most preferably whole connecting keys is achiral.In some cases, achirality connecting key and chirality connectivity can alternately exist, and for example, connect a chirality connecting key behind two continuous achirality connecting keys, again with two continuous achirality connecting keys; Or chirality connecting key of three successive achirality keyed jointings; Or four successive achirality connecting keys are followed two chirality connecting keys etc.The achirality connecting key includes, but not limited to phosphodiester bond, D2EHDTPA diester linkage.

Phosphodiester bond can be 5 ' to 3 ', 5 ' to 2 ' or 5 ' to 5 ' or above combinations of directions.The modification of this connecting key can be intramolecular (single or repetition), also can be the end at RNA or DNA.

The functional effect of pharmaceutical composition provided by the present invention can be measured with method well known in the art.These methods comprise that body is interior and external, for example, analyze pharmaceutical composition to the influence of expression of target gene and the influence of pair cell phenotype in cell culture system; In model and the clinical trial, measure the biological effect of pharmaceutical composition in vivo, comprise drug effect, pharmacology, medicament, toxicity etc.The body inner model comprises the disease animal model and the intact animal of foundation.

The formation of C pharmaceutical composition

The oligonucleotide of more than one that pharmaceutical composition provided by the invention contains that promising adjusting and amyotrophy or flesh growth related gene express.According to the quantity of oligonucleotide, type, target gene etc., oligonucleotide can have multiple different compound mode in the pharmaceutical composition.The target gene of oligonucleotide can be selected from table 1 and 2.

Can only contain single oligonucleotide in the pharmaceutical composition, its target gene can be flesh amicine or its receptor, also can be non-flesh growth inhibited plain gene.

The oligonucleotide that can contain more than one in the pharmaceutical composition, the kind of oligonucleotide can reach below 100 kinds, preferably 2～20 kinds.

Oligonucleotide in the pharmaceutical composition can be one type, for example is selected from antisense oligonucleotide or ribozyme respectively; Or RNA interfering, or DNAzyme.

Also can comprise dissimilar oligonucleotide in the pharmaceutical composition, for example, the combination of antisense oligonucleotide and RNA interfering, the combination of antisense oligonucleotide and DNAzyme etc.

Oligonucleotide in the pharmaceutical composition can be the targeting term single gene, it can be one type a oligonucleotide, antisense oligonucleotide for example, the more than one target site of targeting, for example 5 ' end untranslated region, translation initiation district, 3 ' end untranslated region, translation termination district etc.

Oligonucleotide in the pharmaceutical composition also can the targeting several genes, for example, and the oligonucleotide more than two kinds, targeting flesh amicine and its acceptor gene; Oligonucleotide more than two kinds, the non-flesh growth inhibited of targeting plain gene; Oligonucleotide more than two kinds, hybrid target is to flesh amicine and non-flesh growth inhibited plain gene.

The gene that oligonucleotide can the targeting people in the pharmaceutical composition also can targeting different animals gene, for example cattle, pig, chicken, Mus, horse, Canis familiaris L. and fish.

D is medicinal, nourishing additive agent and hahnemannian oligonucleotide composition

It is medicinal that the present invention also further provides, nourishing additive agent and hahnemannian oligonucleotide composition.

1. pharmaceutical composition

The present invention includes pharmaceutical composition and contain at least a oligonucleotide provided by the present invention.In some pharmaceutical composition, can contain oligonucleotide different below 100 kinds, for example 2,3,4,5,6,7,8,9,10,15,20,30,40,50,75 kinds.In some pharmaceutical composition, oligonucleotide is antisense oligonucleotide, ribozyme, RNA interfering, DNAzyme, or above combination.In some pharmaceutical composition, one or more oligonucleotide are modification property oligonucleotide.In some chemical compound, contain more than one antisense oligonucleotide, it can be the pharmaceutical composition of partly or entirely modifying.Also contain in the pharmaceutical composition and be suitable for medicinal excipient and other adjuvant.

Target gene of the present invention can be single, also can be the combination of several genes.The pharmaceutical composition of targeting several genes can be regulated and control simultaneously to different signal transduction paths, will be more more effective than the method for targeting term single gene.In addition, because the amount of each oligonucleotide in a plurality of oligonucleotide that use is relatively low, therefore can reduce toxigenous probability greatly.The oligonucleotide that the present invention uses is for modification property, and is therefore more stable than natural oligonucleotide, and compares with method of modifying commonly used at present, for example, the D2EHDTPA diester linkage, the toxicity of oligonucleotide of the present invention is lower.

Pharmaceutical composition of the present invention can adopt parenteral, oral or partial administering mode.

Pharmaceutical composition of the present invention can be prepared by technology well known in the art.The selection of pharmaceutical carrier can be according to preparation type and route of administration, for example, intravenous injection, oral, the part, spray, suppository, non-intestinal, or bone marrow injection etc.Excipient can contain several pharmaceutical carriers.Can adopt some can increase the preparation of take advantage of a situation curative effect or mitigation symptoms for its adjuvant of homeopathic drug compositions.In addition, contained stabilizing agent, antiseptic and other composition is generally 0.5%～2% of pharmaceutical composition weight in the pharmaceutical composition.Those skilled in the art can determine suitable administering mode and delivery system.

Pharmaceutical composition of the present invention can be made different dosage forms on request, comprises liquid, tablet, pill, granular, powder, ointment, Emulsion, injection or suppository.Any medicine acceptable medium and adjuvant all can use, for example, water, glycerol, oils, ethanol, flavoring agent, antiseptic, food dye etc. are used for liquid preparation, and starch, sugar, diluent, granule, lubricant, binding agent, dispersant etc. are used to prepare solid preparation.

Injection preparation is the oligonucleoside aqueous acid, and solvent is water or the normal saline that meets medicinal standard.Injection liquid can also contain suitable liquid medium, suspending agent and can regulate the preparation of osmotic pressure, antiseptic etc.Actual fabrication method and program are known for a those skilled in the art.

Local application's dosage form can be made Emulsion, dressing, gel, washing liquid, ointment, liquid etc.Can add surfactant, penetrate with the deep layer that increases medicine.These surfactants comprise natural, also can be chemosynthesis, for example different third myristate.In some cases, pharmaceutical composition can adopt the preparation composition of used for cosmetic.

The preparation of spray-type can be adopted and oligonucleotide is dissolved in or is suspended in hair spray or hair spray and the solvent, and for example ethanol is as solvent.In local application's dosage form and spray-type, drug ratios (oligonucleotide) is generally 0.001%～40% of gross weight.

The preparation of suppository can mix oligonucleotide and make with lipid medium, for example theobroma oil, cocoa butter, glycerol, gelatin or polyoxyethylene glycol.

Oligonucleotide can also be made liposome, to increase the oligonucleotide half-life in vivo.Used lipid includes, but not limited to Cardiolipin, dimyristoyl, dipalmitoyl, dioleoyl phosphatidyl choline, phosphatidyl glycerol, palmitoyloleoyl phosphatidyl choline or phosphatidylglycerol, phosphatidic acid, lysophosphatidic acid, phosphatidyl serine, phosphatidyl inositol, cholesterol.

The dosage of pharmaceutical composition of the present invention is the described oligonucleotide of per kilogram of body weight 0.01～100mg.Preferred dosage is per kilogram 0.01mg～10mg.

When oral administration, but dosage unit of administration in per 1～10 day, or administration every day, every day 1～10 dosage unit, up to healing, or symptom is alleviated.In some cases, dosage is every day 1～4 time.In some cases, patient is by 2～3 dosage units of doctor's advice medication every day.

2. nourishing additive agent

Nourishing additive agent of the present invention refer to can supplements pharmaceutical composition, except that containing one or two or more kinds oligonucleotide of the present invention, also can comprise any edible material that can be used for humans and animals, these materials can strengthen food absorption, concentrate, metabolism etc.Nourishing additive agent.Dosage form comprises types such as tablet, capsule, powder, jelly, liquid.

Nourishing additive agent provided by the invention is very effective to suffering from amyotrophic patient.Also can be used as general population's nourishing additive agent.

Nourishing additive agent of the present invention can also add other nourishing additive agent, for example, and antioxidant, vitamin, edible cellulose, mineral etc.

Nourishing additive agent can also contain some medicinal mediums, for example, and lactose, glucose, fructose, corn starch, potato starch, cellulose etc.According to different preparation requirements, also can add diluent, binding agent, charges in the nourishing additive agent, change the flavor agent, eat pigment, antiseptic, stabilizing agent, regulator, emulsifying agent etc.

Because human body is very high to the tolerance degree of oligonucleotide, oligonucleotide amount contained in the nourishing additive agent can be very big.Every day, the intake of per kilogram of body weight was 0.1mg～100mg.

3. homeopathic drug compositions

The homeopathic therapeutic method refers to and gives the therapy that patient can make the medicine of similar this disease symptoms of healthy person generation on a small quantity.Pharmaceutical composition of the present invention can be used for this therapy, and its preparation method is well known in the art.For example, 1 part of solid oligonucleotide mixed to produce the 1X solid preparation with 9 parts of lactose, repeat this process and can obtain 2X, 3X, 4X ... .. wait the preparation of variable concentrations.Use similar method, can prepare liquid preparation, for example,, produce 1X liquid the liquid mixing of oligonucleotide and 10 times of volumes of 1 part of weight; Repeat this process and can obtain the low concentration preparation, for example, get 1 milliliter of 1X liquid,, can obtain 2X liquid with 9 milliliters of mixing diluents.

Homeopathic drug compositions of the present invention contains the combination of one or more oligonucleotide, and contained oligonucleotide can be through chemical modification.In some cases, the homeopathic drug compositions can be passed through the placebo administration, is about to oligonucleotide agent and drops in or be sprayed on the placebo tablet by the lactose preparation, and is oral then.

Homeopathic therapeutic method's preparation can also comprise the medium that some help relief of symptoms, for example at US pat No.5, the medium described in 603,915.It also can add a kind of electromagnetic medium, and as US pat No 5,830,140 is described.

Listed hahnemannian dosage commonly used in the table 7.

II method of the present invention

The present invention further provides the method for using medicine composite for curing disease provided by the invention.These methods comprise the method that this pharmaceutical composition is regulated gene expression in the cell of using, method with this medicine composite for curing muscular dystrophy, be used for allopathy, nutrition interpolation, hahnemannian method, be used to increase the method for edible animal and farming animals muscle growth, and the method for using auxiliary other treatment of this pharmaceutical composition.

Muscular dystrophy refers to, and is the disease of main idiopathy shape with amyotrophy, for example, muscular dystrophy, spinal cord injury, neurodegenerative disease, flesh lack, dislike matter disease, amyotrophy etc., can also be the disease that some fat and muscle ratios increase unusually, for example, type-II diabetes, X syndrome.

In adult and animal, the atrophy of skeletal muscle can be owing to lack to use muscle, the symptom that wear out, hunger and disease causes, for example, septicemia, muscular dystrophy, acquired immune deficiency syndrome (AIDS), aging and cancer etc.The main feature that muscle reduces is, the protein in the myocyte reduces, and is unable, fatigue, and diameter of muscle fiber diminishes.Proteinic minimizing in the myocyte is because the quickening of synthetic reduction and decomposition.

Muscular atrophy diseases generally all is accompanied by change that series of genes expresses or unusual.This unconventionality expression often causes the pathologic of body to change, and can be determined by the clinician who knows this area.For example, to a diabetics, the clinician can diagnose its body weight to be higher than 20% of crowd's average weight; Whether to a wasting patient, the clinician can measure muscle strength to it, and compares with people's cluster mean, be amyotrophy thereby make a definite diagnosis.

Target gene of the present invention can be single, also can be the combination of several genes.The pharmaceutical composition of targeting several genes can be regulated and control simultaneously to different signal transduction paths, will be more more effective than the method for targeting term single gene.In addition, because the amount of each oligonucleotide in a plurality of oligonucleotide that use is relatively low, therefore can reduce toxigenous probability greatly.The oligonucleotide that the present invention uses is for modification property, and is therefore more stable than natural oligonucleotide, and compares with method of modifying commonly used at present, for example, the D2EHDTPA diester linkage, the possible toxicity of oligonucleotide of the present invention is very low.

A amyotrophy symptom

Utilize pharmaceutical composition of the present invention to can be used for following (but being not limited to) symptom of treatment.

Muscular dystrophy.Muscular dystrophy is a class neuromuscular disease, comprise Erb's atrophy (DMD), pleiomorphism limb-girdle muscular dystrophy (LGMD) and congenital muscular dystrophy (CMD), the symptom of these diseases is carrying out property muscle injury and minimizing, tissue inflammation, fiber and fatty tissue replace muscular tissue, cause a series of complication, even dead.

Flesh lacks.It is a kind of disease of muscle weight, strength and the loss function relevant with aging that flesh lacks, generally at 75 years old with sequela.Paathogenic factor comprises and can't take action that motor changes, and hormonal readiness reduces, and the pancake of albumen synthetic water is low etc.Except that can't taking action, above-mentioned factor is all relevant with genetic control gene expression.Therefore, use the relevant gene of oligonucleotide adjusting and can delay or reverse muscle loss.

Evil matter disease.Disliking the matter disease is a kind of disease relevant with numerous disease, for example, cancer, acquired immune deficiency syndrome (AIDS), septicemia and congestive heart failure, these sick cardinal symptom characteristics are a large amount of simultaneously minimizings of skeletal muscle and fatty tissue, but are not because malnutrition causes.Disliking the matter disease is the reason of 1/3rd cancer patient's death.In pathogenic course, the gene outcome that the cytokine and the tumor factor suppress muscle growth causes amyotrophy.Some dislikes prime factor can optionally act on MHC in myotubule and Mus muscle, tumor necrosis factor (TNF α) and IFN-can be by a kind of mechanism of RNA dependent form, reduce synthetic J.Clin lnvest.114:370-378 such as (, 2004) Acharyya of myosin greatly.The carrying out property of tumour patient is weak to cause them more responsive to the toxicity of chemotherapy and radiation, and many patients fatal position is in the sick relevant symptom of evil matter, rather than tumor itself.Therefore, pharmaceutical composition of the present invention can reverse by disliking the amyotrophy that the matter disease causes, and promises to be a kind of effective Therapeutic Method.

Diabetes.Diabetes are a kind of modal metabolic diseases.Type i diabetes is many in the teenager outbreak, shows as the destruction of beta Cell of islet, generally needs insulinize.Type ii diabetes is many in the manhood outbreak, accounts for more than 80% of whole diabetes.Although type ii diabetes can improve by changing living habit, for example, lose weight and increase muscle growth etc., studies show that regulating some Expression of Related Genes can improve some symptom (US pat NO 6,656,475).

Above-mentioned only is to illustrate by the regulator gene expression to improve the disease symptoms relevant with amyotrophy.Other disease also includes, but is not limited to spinal cord injury, neurodegenerative disease, and wound, ischemic pneumonopathy (COPD), apositia, acquired immune deficiency syndrome (AIDS) is because amyotrophy and any disease that is caused by muscle injury, adenomyosis that bed etc. cause.

B. treat the target gene of usefulness

The target gene that is used for the treatment of has been listed table 1 and table 2 in, but is not limited only in the table listed.Being described in detail as follows of related gene.

Flesh amicine (myostatin).This gene is to regulate muscle growth, directly or indirectly relates to the major gene of nearly all amyotrophy disease.As a kind of secreting type somatomedin, it can regulate the growth of muscle in the intramuscular cell negativity of humans and animals.It belongs to the class in the transforming growth factor, is expressed in developmental pattern and sophisticated muscle.In the mice that lacks this gene expression, the muscle increase reaches 25～30% (Mcpherron etc., Nature, 387:83-90,1997; Whittemore etc., Biochem.Biophys.Res.Commun.300:965-971,2003; Grobet etc., Genesis, 35:227-238,2003).

The flesh amicine circulates in blood, and increasing all appears in the flesh amicine of suffering among the amyotrophic patients serum.Therefore, this gene has been considered to a kind of special medicine target, regulates the effective ways that this gene expression can be used as treatment and amyotrophy relevant disease.

Report shows recently, can improve the amyotrophy symptom significantly with flesh amicine antibody in the mdx mice.In subject mice, not only normal myocyte's number increases to some extent, and muscle power also strengthens greatly.Although the mechanism of this curative effect is not clear fully, obviously, it is relevant with adjusting/reduction flesh amicine level.Therefore, the blocker of flesh amicine is the amyotrophic means of a kind of treatment that gets a good chance of.

In the flesh deficiency disease, high-level flesh amicine is considered to the biomarker that a kind of and old and feeble relevant flesh lacks in the serum.Suppressing this expression of gene can be the means of improving and treating prevention flesh deficiency disease.

Flesh amicine receptor

The signal conduction of flesh amicine is similar to Activin and TGF-β.It can cause the phosphorylation to the Smad product, thereby controlling gene be expressed and the effect of mediation flesh amicine effectively in conjunction with II receptor ActRllB and I receptor ALK or ALK5 formation heterozygosis complex.Smads is a class signal transduction molecule, relevant signal can be imported in the nucleus from cell surface, by transcribing in order to regulator gene with the work of DNA and transcription factor.

Transcription factor

Myogenicity transcription factor MyoD is a kind of regulatory factor of regulating muscle amicine function.A series of transcription factor of expressing in muscle cell comprise MyoD, myogenin, myf-5 and MRF-4/herculin/myf-6.When above arbitrary factor is introduced in the non-myogenous cell, this cell can be divided into the myocyte.MyoD particularly, the cell of its bootable muscle generates, and suppresses propagation, activates differentiation, induces the contraction phenotype.

The Dystrophin gene

The Dystrophin gene has been proved to be in different duchenne muscular dystrophy and has played mastery reaction, for example, Erb's atrophy (Duchenne muscular dystrophy, DMD), Becker muscular dystrophy etc.The dystrophin gene all has different sudden changes in these duchenne muscular dystrophy.Utilize oligonucleotide, for example antisense oligonucleotide is regulated the expression of these mutant genes, or sudden change is proofreaied and correct, and can reach the treatment to muscular dystrophy.

Dislike the sick related gene of matter

Dislike the matter disease and related to many different approach and regulatory factor, for example, tumor necrosis factor (TNF).Injection TNF produces to mice and dislikes the matter disease model.Activins and flesh amicine are expressed in the cancer of several types with in infecting and are all increased.More than these genes all can be used as the medicine target that the matter disease is disliked in treatment.Recently, a kind of new albumen is called cancer and dislikes prime factor, is proved to be with to dislike matter sick relevant, also can be used as a new medicine target.

The diabetes related gene

Normal muscle function can keep the balance of sugar in the body.The expression of regulating muscle amicine and the growth that strengthens muscle can be played the function of control of diabetes.The Activin-follistatin system relates to the growth of pancreas, thus the metabolism of influence sugar.Some the secretion peptides of recent findings and the generation and the metabolism of the equal scalable sugar of BMP-9.Therefore, except the fat cellulation factor (for example leptin and adiponectin), the member of a plurality of TGF-β gene clusters (comprising the flesh amicine, activin BMP-9) all participates in intravital sugared balance and diabetes.

In sum, method provided by the invention is to utilize medicine of the present invention to regulate a kind of or a plurality of expression of gene in cell, and these genes are all relevant with muscular atrophy, and representative gene is listed in table 1 and table 2.

In some cases, can be with cell, tissue or organ come out in in-vitro separation, then it use drug treating of the present invention, then with the cell of handling, organize or the conveyed ex vivo in.

The curative effect of indication is to cure among the present invention, alleviates and prevents pairing symptom.For example, with medicine composite for curing amyotrophy of the present invention (muscular dystrophy, flesh shortage, spinal cord injury, neurodegenerative disease, apositia, evil matter disease etc.), its effective in cure referring to increases muscle volume and weight, strengthens muscle strength etc.For prophylactic effects, pharmaceutical composition of the present invention can be used for having the possible people of potential trouble amyotrophy, as the long-term bed patient, or suffers from the patient who easily causes the amyotrophy disease, or is not diagnosed as the tumor patient of disliking the matter disease as yet.Therefore, Therapeutic Method of the present invention comprises any treatment to the animal and human, comprises following content:

(1) prevent that N from going out, but the symptom that might occur;

(2) suppress the symptom development;

(3) alleviate or reverse symptom;

(4) keep homoiostasis.

The dosage that is used for the oligonucleotide drug compositions of humans and animals depend on the physiological situation of the symptom of being treated, route of administration, treated individual and treatment be trophism or disease resistance, or hahnemannian.The dosage that produces the trophism curative effect refers to a kind of avirulence, and greater than the oligonucleotide dosage that can keep desirable physiological situation minimum.The dosage that produces the disease resistance curative effect refers to a kind of avirulence, and greater than the oligonucleotide dosage of the minimum that can produce desirable physiological effect.The take advantage of a situation dosage of curative effect of generation refers to a kind of nontoxic and can produce the lowest dose level of the oligonucleotide of desirable physiological effect.

In table 6, enumerated single or combination target gene for example, in order to prepare different oligonucleotide, to cure various disease.In the heterogeneic combination, with "/" separately, for example A/B/C represents oligonucleotide target gene A, B, the combination of C between each gene in the table.

Pharmaceutical composition of the present invention also can be used for treating some animals, for example, birds, Fish and mammal (particularly chicken, pig, sheep, Canis familiaris L., horse, cat etc.), the target-gene sequence of oligonucleotide correspondence is that kind is special in pharmaceutical composition.

In further using, method provided by the invention can be used for regulating edible animal or the sports expression of the intravital flesh amicine of animal, thereby strengthens the growth of muscle.Using method and dosage are similar to treatment of diseases.In some cases, used dosage, the route of administration and the course of treatment are according to the animal species that is used for and wish that the effect that reaches adjusts to some extent.

The representative people's target gene of table 1

Gene title (Genes)	Gene data bank code (Accession Number)
		Myostatin	AF104922
MyoD	NM002478
		ARIIb	NM001106
TLL2	AF059516
		ID-1	NM002165
TACE	NM003183
		TNF-R1	NM001065
IL6R	NM000565&NM181359
		MMP2	NM004530
FOXO1	NM002015
		Bcl-3	NM005178
FOXO-3	NM001455&NM201559
		E3alphaII	AY061884
Nfkb-1	NM003998
		IL1b	NM000576
NF-IL6	M83667
		NF-B	M62399&L19067
TNF-	NM000594
		Caspase-3	NM004346
Caspase-1	NM033292
		Atrogin-1	NM058229&NM148177
Smad-2	AF027964
		PIF	AY90150
IL-6	NM000600

The representative animal target gene of table 2

Gene title (Genes)	Gene data bank code (Accession Number)
		Myostatin (cattle) Cow	NM001001525
Myostatin (horse), Horse	AB033541
		Sheep, Sheep Myostatin	AF019622
Goat Myostatin	AY436347
		Rabbit Myostatin, Rabbit	AY169410
Myostatin (pig), Pig	NM214435&AF033855
		Myostatin (chicken), Chicken	AF019621&AF346599
Myostatin (Canis familiaris L.) Dog	AY367768
		Myostatin (fish), Fish	AY825248
Mus Myostatin, Mouse	NM01834
		Mus TLII	AF073526
Mus AcRIIB	NM007397
		Mus MyoD1	NM010866
Mus Atrogin-1	AF441120
		Mus E3alpha-II	XM358323
Mus Foxo3	NM019740
		Mus Foxo1	NM019739
Mus MuRF1	AF294790
		Mus Caspase3	NM009810
Mus NFkB	NM003998
		Mus TNF-alpha	NM013693
Mus TNF-R1	NM011609
		Mus Caspase-1	BC008152
Mus TACE	AB021709
		Mus IL-1b	NM008361
Mus IL-6	NM031168

Mus MMP2	NM008610
		Mus p65	M61909
Mus IL-6R	X53802
		Mus Smad2	NM010754
Mus Cathepsin-B	BC006656

The Antisensedigonucleotsequence sequence of table 3 targeting people's gene

Target gene title (Genes)	Gene data bank code (Accession Number)	Representative Antisensedigonucleotsequence sequence (Antisense Oligonucleotide Sequences)	Nucleic acid sequence number Seq ID#
				Myostatin	AF104922	5′-TgCCACACCAgTgAATCT-3′	1
5′-gCAgTTTTTgCATgATTTTAA-3′	2
		5′-TAAATCTCATgAgCACCC-3′	3
MyoD	NM002478					5′-CAgAgAgTggCCggAACT-3′	4
		5′-TggCgCggCACggTCCTg-3′	5
				5′-AgTAgCTCCATATCCTgg-3′	6
		5′-AgAgCACCTggTATATCg-3′	7
				ARIIb	NM001106	5′-CAgggCgCCgTCATgTTC-3′	8
5′-gggTggCTCgTACgTgAC-3′	9
		5′-TgTCCTgggCTTAgATgC-3′	10
TLL2	AF059516					5′-gCggCgCCCCCTgTCTTC-3′	11
		5′-AgggAgTTgCgTgCACgA-3′	12
				5′-ggCATggTggCgCggggC-3′	13
		5′-TCCCAgggCTgTgCAggA-3′	14
				ID-1	NM002165	5′-AgCCCgAAgCAgATACgg-3′	15
5′-TCATgATTCTTggCgACT-3′	16
		ID-1	NM002165			5′-CATgATTCTTggCgACTg-3′	17
5′-CCACTggCgACTTTCATg-3′	18
				5′-TCAgCgACACAAgATgCg-3′	19

		5′-CTTCAgCgACACAAgATg-3′	20
				TACE	NM003183	5′-TTCTACCgCCAggCTCgA-3′	21
5′-ACTgCCTCATgTTCCCgg-3′	22
		5′-CCTCATgTTCCCggCCCC-3′	23
5′-ACTAAATTAgCACTCTgT-3′	24
		TNF-R1	NM001065			5′-CAgTTgAgggTTgAgACT-3′	25
5′-gCCCATgCCAgACAgCTA-3′	26
				5-gCgCAgCCTCATCTgA-3′	27
IL6R	NM000565&NM181359					5′-TCCCTCTggCCCggCTCA-3′	28
		5′-CCgACggCCAgCATgCTT-3′	29
				5′-CATTCTggggAAgAAgTA-3′	30
		MMP2	NM004530			5′-CAgCCTCCAgCCACCgCC-3′	31
5′-gCCTCCATCgTAgCgCTC-3′	32
				5′-gCAgCCTAgCCAgTCggA-3′	33
FOXO1	NM002015					5′-ggCCgCTTgCTCTCCCCA-3′	34
		5′-AgAgggggAgAACgCAgC-3′	35
				5′-CTCggCCATggTgACCCC-3′	36
		5′-ACCCTCAgCCTgACACCC-3′	37
				Bcl-3	NM005178	5′-gCggggCATCggggCATg-3′	38
5′-gCCCCCCCATCCCCCTCA-3′	39
		FOXO-3	NM001455&NM201559			5′-ggggAgggACgTggACgC-3′	40
5′-CATCTTCgCCgCCCgC-3′	41
				5′-CCTTCAgCCTggCACCCA-3′	42
E3alphaII	AY061884					5′-CAATCAgTCCCggAgCCg-3′	43
		5′-TCCTCCCCAgCgCTACCg-3′	44
				5′-CTCTagCTCCgACgCCAT-3′	45
		E3alphaII	AY061884			5′-TTggTggTgCAATAATTA-3′	46
Nfkb-1	NM003998			5′-TCTCgCTCACTCTCTCAC-3′	47
		5′-gggAAgggCAggggAAgC-3′	48

		5′-gCgCgggCggAgggAAgC-3′	49
				5′-CTgCCATTCTgAAgCCgg-3′	50
		5′-gAAATTgTCAgCAggCTA-3′	51
				IL1b	NM000576	5′-gAgAATCCCAgAgCAgCC-3′	52
5′-CCATggCTgCTTCAgACA-3′	53
		5′-ggTACAgCTCTCTTTAgg-3′	54
NF-IL6	M83667					5′-AgAgCgCggCCgTCATgg-3′	55
		5′-gggAAgggCAggggAAgC-3′	56
				5′-CCTTTTCTAgCCCCGg-3′	57
		NF-B	M62399&L19067			5′-gAACAgTTCgTCCATg-3′	58
5′-AgCCATTCGCCggAATTC-3′	59
				5′-gTgCACTACAgACgAgCC-3′	60
5′-CTgCCATTCTgAAgCCgg-3′	61
				5′-ggCCCAgCTgCgACCCgg-3′	62
TNF-	NM000594					5′-gggggTCTgTagTTgCTT-3′	63
		5′-CCAggggAgAgAgggTgg-3′	64
				5′-gCTCATggTgTCCTTTCC-3′	65
		5′-CATgCTTTCAgTgCTCAT-3′	66
				5′-gATgTTCgTCCTCCTCAC-3′	67
		Caspase-3	NM004346			5′-AggAgCCgCgTCTgCACT-3′	68
5′-TTCTACAACCgCCTCACA-3′	69
				5′-TCTCCATggATACCTTTA-3′	70
5′-ACCAACCATTTCTTTAgT-3′	71
				Caspase-1	NM033292	5′-ACCAACCATTTCTTTAgT-3′	72
5′-TATTTTAATgTCCTgggA-3′	73
		Atrogin-1	NM058229&NM148177			5′-ggATggggAgACggggCC-3′	74
Atrogin-1	NM058229&NM148177			5′-ggAATggCATggCACCgC-3′	75
		5′-TTCTACAACCgCCTCACA-3′	76
				Smad-2	AF027964	5′-TgTATCgAACCTCCCggC-3′	77

		5′-gACgACATgTTCTTACCA-3′	78
				5′-gCAAgATggACgACATgT-3′	79
		5′-TTTATgACATgCTTgTTgAgC-3′	80
				5′-TggTgAAgCTTTATgACA-3′	81
		PIF	AY90150			5′-CTgTgTgCTggAgTgggT-3′	82
5′-ATgAACCTCATgCTTCTg-3′	83
				5′-TCTCCTTACAgCTATAgT-3′	84
IL-6	NM000600					5′-CTCTTTCgTTCCCggTgg-3′	85
		5′-AAGgAgTTCATAgCTggg-3′	86
				5′-CCATgCTACATTTgCCgA-3′	87

The Antisensedigonucleotsequence sequence of table 4 targeting animal gene

Target gene title (Gene)	Gene data bank code (Accession Number)	Representative Antisensedigonucleotsequence sequence (5 ' → 3 ') (Antisense Oligonucleotide Sequences)	Nucleic acid sequence number Seq ID#
				Chicken Myostatin	AF019621&AF346599	CAgATCCgggACAgCAAATgC	88
CCCCCTCTCCCTTTCCCCTTT	89
		gCATTTTTTCTTACACCTCAC	90
CATAGACTGCTAgCTTTTgCA	91
		CTgCCATCCAgAgCCACCggA	92
TCTgCTTCCACgTACAAgCAT	93
		CTTgTTCCAggCgCAgTTTgC	94
TgCAgTggAggAgCTTTgggT	95
		TCgTCTTCCAAAgAgCCATCg	96
CagACTCCgTaggCATTgTgA	97
		Chicken Myostatin	AF019621&AF346599			TagAgCTAAACTTAAAgAAgC	98
CCgTTgTaggTTTTTggACTT	99
				TCTgCCAgATACCAgTgCCTg	100
TgTTTgAgCCAATTTTgCAgC	101

		gCAAgATCTCgTCCAgTCTCA	102
				gTgTCTgTAACTCTgACCTCT	103
		CgggTAgCgACAACATCgggA	104
				gTgCTATAATCCAgTCCCATC	105
		AATTCgCATTCTCCggAgCAg	106
				gCCTgCTgAgCCTCTgggATT	107
		TACAgCATgTTTATAggggAC	108
				ACgATCTACAACCATggCTgg	109
		TCATgAgCACCCgCAACgATC	110
				TCTCACgTCAgCCAAAATTCA	111
		Pig Myostatin	NM214435&AF033855			TgCCTgCACTgTCTgAgAgAC	112
TgCTTTTgAgTAACgCCAAgC	113
				CATgATTTTAAAATCAATACA	114
TgCAgTTTTTgCATgATTTTA	115
				CATAgATTTgCAgTTTTTgCA	116
TCAgATCCACgggACCAgCAA	117
				ACATgCATTACACAgCCCCTC	118
TCCAggCgAAgTTTACTgAgg	119
				gATCAATCAgTTCCCggAgTG	120
TTCcAAggAgCCATCACTgCT	121
				AgAAgATCAgACTCTgTAggC	122
TAAAgAAgCAgCATTTgggTT	123
				TCTTGACgggTCTCAgATATA	124
TgggTTTgATgAgTCTCAggA	125
				CagTgCCTgggTTCATgTCAA	126
Pig Myostatin	NM214435&AF033855					TgAgCCAATTTTgCAACACTg	127
		TgACCATTCTCATCTAAAgCT	128
				ggATTCAgCCCATCTTCTCCT	129
		gAgTgCTCATCACAgTCgAgT	130

		TgCAATAATCCAgTCCCATCC	131
				ACAAgATgAgTgTgAgggTAT	132
		TgTgggAgTACAgCAggggCC	133
				ACCATggCTggAATTTTCCCA	134
		AATCTCATgAgCACCCACAgC	135
				Cattle Myostatin	NM001525	gAgTAAcGcCAAgCCAAACgT	136
TCCCTTgTTCTTACTTCTTCC	137
		gCAgTTTTTgCATggTTTTAA	138
CCTTCTgCTCgCTgTTCTCAT	139
		gTTTTCCCTCCACAAACATgC	140
TTTgCTgATgTTAggAgCTgT	141
		gCTggCATCTCTCTggACATC	142
AtgACCgTTTCCgTCCTggCg	143
		gTTTTCCTTCCACTTgCgTTA	144
CACAgTTgggCCTTTACTAgT	145
		ACACTgTCgCAggAgTCTTgA	146
TCCAgTATACCTTgTACCgTC	147
		TCTgCCAAATACCAgTgCCTg	148
AgATCATggCCATTCTCATCT	149
		TCCATCTTCTCCTggTTCTgg	150
CCTAgATcTTTTTggTgTgTC	151
		TagAgggTAACgACAgCATCg	152
TCTCCAGAGCAgTAATTggCC	153
		AggAgTACAgCAggggCCggC	154
TCATgAACACCCACAgCgATC	155
		Horse Myostatin	AB033541			TgAgTAACgCCAAgCCAA	156
CAgTTTTTgCATggTTTT	157
				TCATgAACACCCACAgCg	158
Sheep	AF019622					TgAgTAACgCCAAgCCAA	159

Myostatin		CAgTTTTTgCATggTTTT	160
				TCATgAACACCCACAgCg	161
		Goat Myostatin	AY436347			TgAgTAACgCCAAgCCAA	162
CAgTTTTTgCATggTTTT	163
				TCATgAACACCCACAgCg	164

Table 5 is at the representative antisense oligonucleotide of people's flesh amicine

Antisensedigonucleotsequence sequence (5 ' → 3 ') * (Oligonucleotide Sequences)	Serial number Seq ID#
		ACTTgCCACACCAgTgAATCT	165
TCTTgTTCTTgTTTCTTCCTT	166
		gTTgCAgTTTTTgCATgATTT	167
ACACAgAgTTgCAgTTTTTgC	168
		AATCAgCATAAACAggTAAAT	169
AgATCCACTggACCAgCAACA	170
		ACACAgCCCCTCTTTTTCCAC	171
gAgCTgTTTCCAgACgAAgTT	172
		AgTggAggAgCTTTgggTAAA	173
TCgCTgCTgTCATCCCTCTgg	174
		CCgTTgTagCgTgATAATCgT	175
AGAAAATCAgACTCTgTAggC	176
		CATAgTTgggCCTTTACTACT	177
TTgTAggAgTCTCgACgggTC	178
		CATAggTTTgATgAgTCTCAg	179
AgTgCCTgggTTCATgTCAAg	180
		TCTTCACATCAATgCTCTgCC	181
ATgCCTAAgTTggATTCAggT	182
		gCCCATCTTCTCCTggTCCTg	183
gTgTgTCTgTTACCTTgACCT	184

TgTTgAgTgCTCATCACAgTC	185
		ATCCAATCCCATCCAAAAgCT	186
TCCAgAgCAgTAATTggCCTT	187
		gTgTACCAgATgAgTATgAgg	188
TgggAgTACAgCAAgggCCTg	189
		TCgCTggAATTTTCCCATATA	190
ATATAAATCTCATgAgCACCC	191

* the design of antisense oligonucleotide is based on people's flesh growth inhibited plain gene (myostatin) (AF104922).

Table 6 illustrates and is used for the treatment of amyotrophic oligonucleotide drug compositions

Oligonucleotide combination (Combinations of oligonucleotides targeting the following genes)
	Myo
Myo/myoD
	Myo/ActRIIB
Myo/myoD/ActRIIB
	Myo/ActRIIB/Smad2
Myo/NF-kB
	Myo/Atrogin-1
Myo/MuRF1
	Myo/Atrogin-1/MuRF1
Atrogin-1/FOXO1
	Atrogin-1/FOXO3
Atrogin-1/FOXO1/FOXO3
	Myo/MyoD/ActIIB/Samd2/NF-kB
Atrogin-1/FOXO1/FOXO3/MuRF1

Table 7: the RNA/DNA dosage that the homeopathic therapeutic method uses

Dose dilution multiple/effectiveness Dilution/Potency	Microgram/kg body weight μ g/kg
		2x	50
3x	5
		4x	0.5
5x	0.05
		6x	0.005

The specific embodiment:

Listing of following examples is in order to describe the present invention in detail, the content to invention is not limited, as table 3,4, listed Antisensedigonucleotsequence sequence is representational molecule in 5, any other can produce the antisense oligonucleotide that the amyotrophy associated conditions is produced curative effect, all can be used in the pharmaceutical composition of the present invention.

Embodiment 1 cellular level is to the inhibition of flesh amicine (Myostatin)

1. experiment purpose

This experiment is for the activated RNA oligonucleotide of screening in mouse muscle cell line.The index that detects is to observe the influence that the RNA oligonucleotide is expressed the flesh amicine by Western blotting.

2. experiment material

1) cell: C2C12 cell line derives from the mouse muscle cell, is incubated in the DMEM culture fluid, adds 15ml hyclone, 100 unit penicillins, 100 microgram streptomycins in per 100 milliliters of DMEM complete culture solutions.Condition of culture is 37 ℃, 5%CO ₂The differentiation culture of cell adopts 2% horse serum culture medium, and condition of culture is 37 ℃, 5%CO ₂

2) RNA oligonucleotide: seven kinds of targeting flesh growth inhibited plain gene zones of different RNA oligonucleotide, each molecule all have 3 ' end butane protecting group.The RNA oligonucleotide that is used for the experiment of cell transfecting efficient has the FITC of fluorescence group labelling at 5 ' end.

3. experimental procedure

1) transfectional cell: this process is that the RNA oligonucleotide is gone into cell by the liposome road.

2) analysis of cells transfection efficiency: the efficient of utilizing the flow cytometry analysis transfection.

3) detection of gene expression dose: utilize Western blotting to analyze of the influence of RNA oligonucleotide to expression of target gene.

4. experimental result

1) FACS (fluorescence activating cell sorting) quantitative analysis cell transfecting efficient

Transfection passage the previous day 3 * 10 ⁵In 6 orifice plates, transfection is undertaken by following process:

A) in the A-pipe, mix 250 μ l opti-MEM and 1 μ l oligonucleotide (final concentration 2 μ M), room temperature 5 minutes;

B) in the B-pipe, mix 250 μ l opti-MEM and 4 μ l Lipofectamine-2000, room temperature 5 minutes;

C) solution in A, the B pipe is mixed room temperature 20-30 minute.

D) cell that goes down to posterity is washed twice with PBS, mixed solution is added to cell surface, gently mixing.

E) place after 4-6 hour for 37 ℃, change complete medium, cultivation is carried out related detection after a few hours.

With the RNA-oligo transfection C2C12 cell (2M) of FITC labelling after 24 hours, outwell culture fluid, PBS washes cell twice, and the trypsinization collecting cell is in centrifuge tube, suction pipe is blown and beaten cell dispersion repeatedly, centrifugation cell, PBS is resuspended, again centrifugation, wash repeatedly twice, remove free F ITC-RNA oligonucleotide, 400 order steel meshes filter and make cell be single cell suspension, the efficient of flow cytometry analysis transfection.

Cell with untransfected is a blank, and fluorescigenic cell accounts for the proportional representation transfection efficiency of total cell.The result shows that under this experiment condition, the transfection efficiency of RNA oligonucleotide in the C2C12 cell is 45-50%.

2) Western blotting (Western blotting) detects the RNA oligonucleotide to the inhibition of acid to the gene expression of flesh amicine

Cell transfecting as mentioned above.The C2C12 cell of distinguishing transfection with seven kinds of RNA oligonucleotide is through using cell pyrolysis liquid (50mM Tris-HCl, 1mM EDTA, 2%SDS, 5mM DTT, 10mM PMSF) cracking.The 30s Ultrasonic Pulverization, boiling water bath degeneration 10min, 13, the centrifugal removal cell debris of 000g, (Pierce Chemical Co, Rockford IL) measure protein concentration with BCA assay reagent in the supernatant.100 μ g albumen are concentrating glue through 12%, separation gel SDS-PAGE electrophoretic separation, and electrotransfer is to nitrocellulose filter, 5% defatted milk powder sealing nonspecific binding site, film and a corresponding antibody are hatched 12h for 4 ℃ then, wash; Two anti-incubated at room 2h, washing, and the adding chemical luminous substrate (Supersignal WestDura Extended Duration Substrate, PIERCE), darkroom X-ray sheet exposure, development, photographic fixing show band, and special protein B eta-actin band is as internal reference.The concentration of flesh amicine one antibody is 2 μ g/ml, the antibody concentration of Beta-actin 1: 500,0.2 μ g/ml.

The result shows, test seven kinds of used RNA antisense oligonucleotides and all can suppress flesh amicine myostatin expression of gene specifically, and the pair cell reference protein (expression of β-actin) does not have influence.This result shows that antisense rna oligonucleotide provided by the present invention can enter cell effectively, combines with the mRNA of target gene (flesh amicine myostatin), and makes its degraded, finally causes the reduction of its protein expression.In seven kinds of used RNA antisense oligonucleotides of experiment, molecule A, B, the specificity of C suppress effect maximum (A:ATg Tag CgT CCg AgA gAC; B:TgC ATC ATT TTA AAA ATC AgC; C:ACC TAA TgC AAA gCT CAT).Test used oligonucleotide be 2 '-the O-methyl modifies and has a protection of 3 ' end butoxy.

Embodiment 2 single and combined RNA oligonucleotide to the rejection ratio of mouse muscle growth

1. experiment purpose: research RNA oligonucleotide is single or be used in combination in the condition lower body influence to muscle growth.

2. experiment material: three kinds of RNA oligonucleotide are 5 ' end untranslated region (A) of targeted muscles growth inhibited plain genes respectively; Translation initiation district (B); Translation termination district (C).Every kind of RNA oligonucleotide is dissolved in normal saline by 1mg/ml concentration.Every kind of RNA oligonucleotide is mixed and made into combined RNA oligonucleotide by equal-volume.

3. experimental procedure: experiment divides five groups to be carried out, and every group contains six 6-8 week female mices.Per three days one time tail vein injection is adopted in experiment, and injection volume is 100 μ l (5mg/kg body weight).Peeling off leg muscle after experiment is finished weighs.

4. experimental result: experiment is grouped into:

A, RNA oligonucleotide A (ATg Tag CgT CCg AgA gAC);

B, RNA oligonucleotide B (TgC ATC ATT TTA AAA ATC AgC);

C, RNA oligonucleotide C (ACC TAA TgC AAA gCT CAT);

D, RNA oligonucleotide A+B+C;

E, the normal saline contrast.

The flesh amicine can suppress the growth of muscle, suppresses this expression of gene and will cause promoting muscle growth.This experiment is injected in the mice body with different RNA oligonucleotide combination vein, and the influence that mouse muscle is grown sees the following form.Muscle growth promotion rate (%) is (processed group-normal saline group)/normal saline group.

The comparison that table 8 is single and combination flesh amicine RNA oligonucleotide promotion mouse muscle is grown.

	A	B	C	A+B+C
					Muscle growth promotion rate (%)	0.8	2.4	7.2	8.8

5. experiment conclusion

From last table as seen, merge and use the RNA oligonucleotide,, can more effectively suppress flesh amicine expression of gene, thereby promote the growth of mouse muscle with respect to independent use.

Embodiment 3 RNA oligonucleotide compositions are to the influence of mouse muscle growth

1. experiment purpose

Utilize three kinds of RNA oligonucleotide A that filter out, B, C further inquire into merge to use three kinds of oligonucleotide to the mouse muscle growth and in muscular tissue to and the influence of flesh auxin gene expression.

2. experiment material

1) laboratory animal: totally 24 mices, 6-8 age in week female Mus.

2) oligonucleotide: with each oligonucleotide dissolving.Three kinds of RNA oligonucleotide (5 ' end untranslated region, translation initiation district, the translation termination district of targeting targeting myostatin respectively) equal-volume mixes, and injection volume is 5mg/kg, every Mus 100 μ l; Four kinds of DNA oligonucleotide (with comparing) equal-volume mixes, and injection volume is 5mg/kg, every Mus 100 μ l;

3. experimental technique and step

Experiment is divided into three groups of A, B, C, every group of 8 Mus.Wherein:

A group: experimental group, the compositions of injecting above-mentioned three kinds of RNA oligonucleotide;

The B group: the oligonucleotide matched group, inject four kinds of DNA oligonucleotide reference composition;

C group: handle matched group, injecting normal saline.

Concrete experimental procedure is as follows:

1) 24 of mices (6-8 week) are stablized a week in Animal House

2) the 8th day, after weighing, every mice of tail vein injection 100 μ l oligonucleotide or normal saline

3) the 9th day, the lumbar injection isodose

4) the 10th day, lumbar injection was with the 9th day

5) repeat: vein-abdominal cavity-lumbar injection circulates eight times

6) the 34th day, put to death rapidly behind every the mice of weighing, get gastrocnemius and musculus quadriceps, weigh.Get the fritter fixation of tissue to treat histology, all the other cooled with liquid nitrogen are stored, and Western blot Western Blotting is stand-by.

2. experimental result

1) flesh auxin gene antisense RNA oligonucleotide composition is to the influence of mouse muscle growth

Put to death when experiment finishes that mice is got gastrocnemius and musculus quadriceps is weighed, data are handled, muscle growth promotion rate (%) is (processed group-normal saline group)/normal saline group.The results are shown in following table:

The combination of table 9 RNA oligonucleotide is to the promotion of mouse muscle growth

	The combination of RNA oligonucleotide	Control oligonucleotide
			Muscle growth promotion rate (%)	11.3	-0.4

From last table as seen, the average weight of its leg muscle of experimental group of injection targeting flesh auxin gene antisense RNA oligonucleotide composition is significantly higher than control experiment group and DNA oligonucleotide matched group, thereby proved that antisense rna oligonucleotide can suppress flesh amicine expression of gene, and the growth of its muscle is accelerated.

2) flesh auxin gene antisense RNA oligonucleotide composition is to the influence of mice average weight

24 mices (female) grouping is that its average body weight average presents slight ascendant trend at random, but does not have significant difference between group.This result shows that the effect of RNA oligonucleotide is a muscle specific.

3) flesh auxin gene antisense RNA oligonucleotide composition influence that flesh auxin gene is expressed in muscular tissue

Experiment has been extracted further and has been organized total protein, to detect the expression of flesh growth inhibited plain gene in muscular tissue.Muscular tissue is in cell pyrolysis liquid (50mM Tris-HCl, 1mM EDTA, 2%SDS, 5mM DTT, 10mMPMSF) middle homogenate, cracking.The 30s Ultrasonic Pulverization, boiling water bath degeneration 10min, 13, the centrifugal removal cell debris of 000g, (Pierce Chemical Co, Rockford IL) measure protein concentration with BCA assay reagent in the supernatant.100 μ g albumen are concentrating glue through 12%, separation gel SDS-PAGE electrophoretic separation, and electrotransfer is to nitrocellulose filter, 5% defatted milk powder sealing nonspecific binding site, film and a corresponding antibody are hatched 12h for 4 ℃ then, wash; Two anti-incubated at room 2h, washing, and the adding chemical luminous substrate (Supersignal West Dura Extended Duration Substrate, PIERCE), darkroom X-ray sheet exposure, development, photographic fixing show band, and special protein B eta-actin band is as internal reference.The concentration of flesh amicine one antibody is 2 μ g/ml, the antibody concentration of Beta-actin 1: 1000, and 0.2 μ g/ml, two resist 1: 10000,0.1 μ g/ml.

The testing result of Western blot Western Blotting shows that the RNA-oligonucleotide can suppress flesh amicine expression of gene significantly in the mouse muscle tissue, and histiocyte internal reference albumen is not had influence, thereby proof, the RNA oligonucleotide produces by suppressing the gene expression of flesh auxin the promotion of mouse muscle growth.

The combination of embodiment 4 flesh amicine RNA oligonucleotide is by the inhibition of different way of administration to the mouse muscle growth

1. experiment purpose

Research RNA oligonucleotide composition effect in the body under the different way of administration is promptly to the promotion of muscle growth.

2. experiment material

1) animal, totally 24 mices, 6-8 age in week, female normal mouse was divided into A, B, C, D group, every group of 6 Mus.

2) oligonucleotide: each oligonucleotide is dissolved in the normal saline, and three kinds of RNA oligonucleotide equal-volumes mix, and it is 5 ' end untranslated region of targeting targeting flesh growth inhibited plain gene myostatin respectively; The translation initiation district; The translation termination district, injected dose is 5mg/kg, every Mus 100 μ g/100 μ l.

3. experimental procedure and method

Experiment divides four groups and carries out.Experiment is around whole process is.

The A group is intravenous injection, per three days injection 100 μ l mixing oligonucleotide (A+B+C);

The B group is injected 100 μ l mixing oligonucleotide (A+B+C) every day for vein and alternately injection of abdominal cavity;

C is the physiology saline control, and injecting method such as B organize;

D is oral, and every day is by pouring into 100 μ l oligonucleotide (A+B+C) in the mice mouth

Put to death when experiment finishes that mice is got gastrocnemius and musculus quadriceps is weighed, data are handled, muscle growth promotion rate (%) is (processed group-normal saline group)/normal saline group.

4. experimental result

Table 10 RNA oligonucleotide makes up the promotion of different injecting pathways to the mouse muscle growth

	Intravenous injection	Vein+lumbar injection	Oral
				Muscle growth promotion rate (%)	25.91	22.38	18.55

From last table as seen, the RNA oligonucleotide is compared with matched group (normal saline) under the condition of different way of administration, all can suppress flesh amicine expression of gene effectively, thereby promotes the growth of mouse muscle.

Claims (16)

1. oligonucleotide for the treatment of amyotrophy or strengthening muscle growth, feature is:

(1) contains 7～75 nucleotide;

(2) contain ribose groups by 7～75 adjacency of achirality 5 ' connect to key between 3 ' phosphoric acid nucleoside;

(3) be complementary to and 5 of the relevant target gene of muscular dystrophy ' end untranslated region, translation initiation district, 3 ' end untranslated region and translation termination district;

(4) has 75 ℃～115 ℃ melting temperature (Tm, 1 millimolar concentration)

2. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide is made up of 10～26 nucleotide, has 40 ℃～85 ℃ melting temperature (Tm, 1 pmol concentration).

3. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide comprises ribose oligonucleotide and deoxyribose oligonucleotide.

4. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide is the modification oligonucleotide through chemical modification.

5. oligonucleotide as claimed in claim 4 is characterized in that: described modification oligonucleotide has substituted radical on glycosyl 2 ' position, described substituted radical is selected from oxygen, methoxyl group, propoxyl group, methoxy-ethyoxyl, fluorine, chlorine, bromine, iodine.

6. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide 3 ' end and/or 5 ' have blocking group.

7. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide is 3 ' endcapped and/or 5 ' endcapped.

8. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide is an antisense oligonucleotide.

9. oligonucleotide as claimed in claim 8 is characterized in that: described antisense oligonucleotide comprise the targeting people's gene antisense oligonucleotide, targeting animal gene antisense oligonucleotide and at the representative antisense oligonucleotide of people's flesh amicine.

10. oligonucleotide as claimed in claim 8 is characterized in that: the sequence of this oligonucleotide is selected from SEQ ID NO:1～SEQ ID NO:191.

11. oligonucleotide as claimed in claim 1 is characterized in that: this oligonucleotide is RNA interfering (RNAi), ribozyme or DNAzyme.

12. the oligonucleotide composition for the treatment of amyotrophy or strengthening muscle growth contains one or more in the described oligonucleotide of arbitrary claim among the claim 1-11.

13. compositions as claimed in claim 12 is characterized in that: when using two kinds of oligonucleotide, these two kinds of oligonucleotide will be separately and one section regional complementarity of identical or different muscular dystrophy related gene; Wherein, the regional complementarity in 5 ' untranslated region, translation initiation site, 3 ' untranslated region or the translation termination site of first gene in first kind of oligonucleotide and a certain group of gene, the regional complementarity in 5 ' untranslated region, translation initiation site, 3 ' untranslated region or the translation termination site of second gene in second kind of oligonucleotide and same group or another group gene.

14. purposes as described oligonucleotide preparation treatment animal or human's amyotrophy medicine of arbitrary claim among the claim 1-11 or nutritional supplement.

15. described oligonucleotide preparation strengthens the medicine of animal or human's muscle growth or nutritional supplement's purposes as arbitrary claim among the claim 1-11.

16. purposes as claimed in claim 15, wherein said animal comprises mammal and nonmammalian.