Rnai inhibition of CTGF for treatment of ocular diseasesTechnical Field
The present invention relates to the field of interfering RNA compositions that inhibit the expression of Connective Tissue Growth Factor (CTGF) in ophthalmic disorders.
Background
Most ophthalmic diseases are associated with cellular processes including cell proliferation, survival, migration, differentiation and angiogenesis. CTGF is a secreted cytokine and a major mediator of these cellular processes. Specifically, CTGF is known to increase extracellular matrix production primarily by increasing collagen I and fibronectin deposition. Overexpression of CTGF is considered to be a major causative factor of diseases such as scleroderma, fibroproliferative disease, scar formation, etc., and excessive accumulation of extracellular matrix components is present in these diseases.
Excessive accumulation of extracellular matrix material in the Trabecular Meshwork (TM) region is a hallmark of many types of glaucoma, and this increase is thought to increase resistance to aqueous outflow, resulting in elevated intraocular pressure. International patent application No. PCT/US2003/012521, published as WO 03/092584 at 11/13 of 2003 and assigned to Fieenor et al, Alcon corporation, describes an increase in CTGF mRNA in glaucoma TM cells as compared to normal TM cells. Therefore, CTGF is thought to play a role in extracellular matrix production by trabecular meshwork cells.
Macular degeneration is the loss of photoreceptor cells in the central retinal portion (known as the macular region) responsible for high-precision vision. Macular degeneration is associated with abnormal deposition of extracellular matrix components in the membrane between the retinal pigment epithelium and the vascular choroid. This clastic material is called drusen. Drusen can be observed by funduscopic examination. The macular area of the normal eye is free of drusen, and drusen may be abundant around the retina. If there is no loss of macular vision and soft drusen appear in the macular area, AMD can be considered early.
Choroidal neovascularization often occurs in macular degeneration and is associated with proliferation of choroidal endothelial cells, overproduction of extracellular matrix, and formation of the fibrovascular subretinal membrane, among other ophthalmic diseases. Proliferation of retinal pigment epithelial cells and production of angiogenic factors appear to effect choroidal neovascularization.
Diabetic retinopathy is an ophthalmic disease occurring in diabetes mellitus due to thickening of capillary basement membrane and lack of contact between pericapillary cells and endothelial cells. The loss of pericytes increases capillary leakage, resulting in a disruption of the blood-retinal barrier.
Proliferative vitreoretinopathy is associated with cell proliferation of cell membranes and fibrotic membranes within the vitreous membrane and at the retinal surface. Proliferation and migration of retinal pigment epithelial cells are common in this ophthalmic disease. Membranes associated with proliferative vitreoretinopathy include extracellular matrix components such as collagen types I, II and IV, fibronectin, and these membranes become progressively fibrotic.
Wound healing disorders can lead to severe ocular tissue damage by activating inflammatory cells, releasing growth factors and cytokines, proliferation and differentiation of ocular cells, increased capillary permeability, alteration of basement membrane matrix components, increased deposition of extracellular matrix, fibrosis, neovascularization, and tissue remodeling.
Thus, overexpression of CTGF is considered to be a major causative factor in these ocular diseases. Current therapeutic approaches do not directly address the pathogenesis of these diseases.
Summary of The Invention
The present invention relates to interfering RNAs capable of targeting and interfering with expression of CTGF mRNA. Such interfering RNAs of the invention are useful for treating CTGF-associated ocular diseases such as glaucoma, macular degeneration, diabetic retinopathy, choroidal neovascularization, proliferative vitreoretinopathy and abnormal wound healing.
One embodiment of the present invention provides a method for attenuating the expression of connective tissue growth factor in the eye of a subject. The method comprises administering to the eye of the subject a composition comprising an effective amount of an interfering RNA, such as a double-stranded (ds) siRNA or a single-stranded (ss) siRNA of 19-49 nucleotides in length, and a pharmaceutically acceptable carrier.
The double stranded siRNA comprises a sense nucleotide sequence, an antisense nucleotide sequence, and a region of at least nearly perfect sequential complementarity of at least 19 nucleotides. Furthermore, under physiological conditions, the antisense sequence hybridizes to a nucleic acid sequence corresponding to SEQ ID NO: 1 (sense strand sequence of human connective tissue growth factor DNA, Genebank reference NM — 001901) and having at least 19 nucleotides that hybridize to a sequence corresponding to SEQ ID NO: 1 at least close to the region of complete continuous complementarity. Administration of such a composition can attenuate the expression of connective tissue growth factor mRNA in the eye of the subject.
The single stranded siRNA has a length of 19-49 nucleotides and is capable of hybridizing to a polynucleotide corresponding to SEQ ID NO: 1, starting at nucleotide 379, 691, 801, 901, 932, 937, 969, 986, 1119, 1170, 1201, 1346, 1473, 1478, 1481, 1488, 1626, 1660, or 1666, and has a sequence capable of hybridizing to the corresponding nucleotide at position 379, 691, 801, 901, 932, 937, 969, 1473, 1478, 1481, 1488, 1626, 1660, or 1666 of the mRNA of SEQ ID NO: 1 at least close to the region of complete complementarity.
In an embodiment of the invention, the antisense strand of the double-stranded interfering RNA is designed to target a nucleic acid sequence corresponding to SEQ ID NO: 1 from or comprises a nucleotide sequence of nucleotides 379, 691, 801, 901, 932, 937, 969, 986, 1119, 1170, 1201, 1346, 1473, 1478, 1481, 1488, 1626, 1660, or 1666.
Another embodiment of the invention provides a method of treating a connective tissue growth factor-related ophthalmic disease in a subject in need thereof. The method comprises administering to the eye of the subject a composition comprising an effective amount of a 19-49 nucleotide interfering RNA comprising a sense strand, an antisense strand, and an at least nearly perfectly contiguous complementary region of at least 19 nucleotides, and a pharmaceutically acceptable carrier. The antisense sequence hybridizes under physiological conditions with a sequence corresponding to SEQ ID NO: 1 and having at least 19 nucleotides capable of hybridizing to a portion of an mRNA corresponding to SEQ ID NO: 1 at least close to the region of complete continuous complementarity. Thereby treating connective tissue growth factor-related ophthalmic diseases.
Brief Description of Drawings
FIG. 1A shows SITOXTMData, together with the data of figure 1B, demonstrate that trabecular meshwork cell transfection efficiency is not rate limiting. By SITOXTM(Dharmacon) transfection control GTM3 cells were transfected. Determination of SITOX after 24 hours by Trypan blue exclusion assayTMThe number of viable cells remaining in the culture, in turn, reflects the relative transfection efficiency. Hollow bar shape: no transfection; solid bar shape: by SITOXTM。
FIG. 1B shows SIGLO of siRNA taken up by GTM3 cellsTMImages, together with the data of figure 1A demonstrate that transfection efficiency is not rate limiting. Using LIPOFECTAMINE 2000TMMixing SIGLOTMsiRNA was transfected into GTM3 cells. Detection of SIGLO 24 hours later by fluorescence microscopyTMUptake of siRNA (red irregular shape). Individual nuclei (blue circular areas) were identified using DAPI (4', 6-diamidino-2-phenylindole), a dye for double-stranded DNA. As shown by the data in FIG. 1A and the image in FIG. 1B, almost all cells or dead (SITOX)TM) Or emit fluorescence (SIGLO)TM)。
Figure 2A is a schematic showing the exon (square box) and intron (line) structures of the CTGF gene, as well as the positions of siRNA S1, S2, S3 and QPCR primer/probe sets Q1 and Q2 relative to Genebank CTGF sequence NM — 001901, as shown in SEQ ID NO: 1 provides the sequence NM _ 001901. The sequences of the siRNA and primer/probe sets are provided in example 1.
FIG. 2B shows QPCR amplification of CTGF mRNA using primer/probe set Q2 for exon 5. With the sirnas of S1 and S4, no significant down-regulation of CTGF mRNA levels was detected.
Figure 2C shows QPCR for CTGF mRNA with primer/probe set Q1 spanning exon 4/5. Downregulation of CTGF mRNA was observed for each siRNA, whereas downregulation of-90% was observed with S2 siRNA.
FIG. 3 shows titration studies to detect CTGF mRNA level downregulation using different concentrations of S2 siRNA. Downregulation of CTGF mRNA was assessed by QPCR amplification with primer/probe set Q1. As described in example 1, IC was observed after 24 hours in GTM3 cells treated with 0, 1, 3, 10, 30 and 100nM S2siRNA50At-2.5 nM.
Detailed Description
RNA interference, known as "RNAi", is a method of achieving reduced expression of a gene of interest as a small single-or double-stranded RNA molecule. Interfering RNAs include double-or single-stranded small interfering RNAs (dssiRNAs or ss siRNAs), microRNAs (miRNAs), small hairpin RNAs (shRNAs), and the like. Without wishing to be bound by theory, it appears that RNA interference occurs in vivo, with double-stranded RNA precursors being cleaved into small RNAs of about 20-25 nucleotides in length. Cleavage is accomplished by RNAeIII-RNA helicase dicer. The sense strand of the siRNA, i.e., the strand having a sequence identical to the sequence of the mRNA of interest, is removed, leaving the "antisense strand" complementary to the sequence of the mRNA of interest to reduce mRNA expression. The antisense strand of the siRNA appears to direct a protein complex called RISC (RNA-induced silencing complex) to the mRNA, which is then cleaved by the Argonaute protein of RISC, thereby reducing protein production from the mRNA. Interfering RNA is catalytically active, and a sub-stoichiometric amount of mRNA-associated interfering RNA reduces mRNA expression. The reduction of mRNA expression can also occur through the mechanisms of transcription and translation.
The present invention relates to the use of interfering RNA in inhibiting connective tissue growth factor expression in ophthalmic diseases. In accordance with the present invention, the tissues of the eye, and in particular the trabecular meshwork cells of the eye, are siRNA silenced by exogenously supplied siRNA. In addition, aspects of the invention have determined that in using a PCR-based method to determine siRNA downregulation efficacy, the amplification primers of the PCR should be designed to include siRNA targeting sequences to accurately determine silencing.
Unless otherwise indicated, all nucleic acid sequences referred to herein are written in the 5 'to 3' direction. The term "nucleic acid" as used herein refers to DNA or RNA, or modified forms thereof, comprising purine or pyrimidine bases present in DNA (adenine "A", cytosine "C", guanine "G" and thymine "T"), or purine or pyrimidine bases present in RNA (adenine "A", cytosine "C", guanine "G" and uracil "U"). Interfering RNAs provided herein may include "T" bases, particularly at the 3' end, although "T" bases do not naturally occur in RNA. "nucleic acid" includes the terms "oligonucleotide" and "polynucleotide", and may refer to a single-stranded molecule or to a double-stranded molecule. The double-stranded molecules are formed between the A and T bases, the C and G bases, and the A and U bases according to the Watson-Crick base pairing principle. The two strands of a double-stranded molecule may be partially, substantially or completely complementary to each other and will form a duplex hybrid molecule, the strength of the bond depending on the nature of the base sequence and the degree of complementarity. Knowing the sequence of the sense or antisense strand of the DNA encoding the mRNA, the sequence of the mRNA can be readily determined. For example, seq id NO: 1 provides the DNA sense strand sequence corresponding to connective tissue growth factor mRNA. The "T" base is replaced by a "U" residue and the mRNA sequence is identical to the sense strand of DNA. Thus, the sequence can be represented by SEQ ID NO: 1 the mRNA sequence of connective tissue growth factor.
Connective tissue growth factor mRNA: GenBank database of National Center for Biotechnology (National Center for Biotechnology) of ncbi.nlm.nih.gov provides DNA sequences corresponding to messenger RNA of human connective tissue growth factor, reference NM — 001901, as follows in SEQ ID NO: 1 is provided. The coding sequence of connective tissue growth factor is nucleotides 146 to 1195.
SEQ ID NO：1：
1 tccagtgacg gagccgcccg gccgacagcc ccgagacgac agcccggcgc gtcccggtcc
61 ccacctccga ccaccgccag cgctccaggc cccgcgctcc ccgctcgccg ccaccgcgcc
121 ctccgctccg cccgcagtgc caaccatgac cgccgccagt atgggccccg tccgcgtcgc
181 cttcgtggtc ctcctcgccc tctgcagccg gccggccgtc ggccagaact gcagcgggcc
241 gtgccggtgc ccggacgagc cggcgccgcg ctgcccggcg ggcgtgagcc tcgtgctgga
301 cggctgcggc tgctgccgcg tctgcgccaa gcagctgggc gagctgtgca ccgagcgcga
361 cccctgcgac ccgcacaagg gcctcttctg tgacttcggc tccccggcca accgcaagat
421 cggcgtgtgc accgccaaag atggtgctcc ctgcatcttc ggtggtacgg tgtaccgcag
481 cggagagtcc ttccagagca gctgcaagta ccagtgcacg tgcctggacg gggcggtggg
541 ctgcatgccc ctgtgcagca tggacgttcg tctgcccagc cctgactgcc ccttcccgag
601 gagggtcaag ctgcccggga aatgctgcga ggagtgggtg tgtgacgagc ccaaggacca
661 aaccgtggtt gggcctgccc tcgcggctta ccgactggaa gacacgtttg gcccagaccc
721 aactatgatt agagccaact gcctggtcca gaccacagag tggagcgcct gttccaagac
781 ctgtgggatg ggcatctcca cccgggttac caatgacaac gcctcctgca ggctagagaa
841 gcagagccgc ctgtgcatgg tcaggccttg cgaagctgac ctggaagaga acattaagaa
901 gggcaaaaag tgcatccgta ctcccaaaat ctccaagcct atcaagtttg agctttctgg
961 ctgcaccagc atgaagacat accgagctaa attctgtgga gtatgtaccg acggccgatg
1021 ctgcaccccc cacagaacca ccaccctgcc ggtggagttc aagtgccctg acggcgaggt
1081 catgaagaag aacatgatgt tcatcaagac ctgtgcctgc cattacaact gtcccggaga
1141 caatgacatc tttgaatcgc tgtactacag gaagatgtac ggagacatgg catgaagcca
1201 gagagtgaga gacattaact cattagactg gaacttgaac tgattcacat ctcatttttc
1261 cgtaaaaatg atttcagtag cacaagttat ttaaatctgt ttttctaact gggggaaaag
1321 attcccaccc aattcaaaac attgtgccat gtcaaacaaa tagtctatct tccccagaca
1381 ctggtttgaa gaatgttaag acttgacagt ggaactacat tagtacacag caccagaatg
1441 tatattaagg tgtggcttta ggagcagtgg gagggtacca gcagaaaggt tagtatcatc
1501 agatagctct tatacgagta atatgcctgc tatttgaagt gtaattgaga aggaaaattt
1561 tagcgtgctc actgacctgc ctgtagcccc agtgacagct aggatgtgca ttctccagcc
1621 atcaagagac tgagtcaagt tgttccttaa gtcagaacag cagactcagc tctgacattc
1681 tgattcgaat gacactgttc aggaatcgga atcctgtcga ttagactgga cagcttgtgg
1741 caagtgaatt tcctgtaaca agccagattt tttaaaattt atattgtaaa tattgtgtgt
1801 gtgtgtgtgt gtgtatatat atatatatat gtacagttat ctaagttaat ttaaagttgt
1861 ttgtgccttt ttatttttgt ttttaatgct ttgatatttc aatgttagcc tcaatttctg
1921 aacaccatag gtagaatgta aagcttgtct gatcgttcaa agcatgaaat ggatacttat
1981 atggaaattc tctcagatag aatgacagtc cgtcaaaaca gattgtttgc aaaggggagg
2041 catcagtgtc cttggcaggc tgatttctag gtaggaaatg tggtagctca cgctcacttt
2101 taatgaacaa atggccttta ttaaaaactg agtgactcta tatagctgat cagttttttc
2161 acctggaagc atttgtttct actttgatat gactgttttt cggacagttt atttgttgag
2221 agtgtgacca aaagttacat gtttgcacct ttctagttga aaataaagta tattttttct
2281 aaaaaaaaaa aaaaacgaca gcaacggaat tc.
The equivalent of the CTGF mRNA sequence described above is an alternative splice form, allelic form or homologue thereof. Homologues are homologous to SEQ ID NO: 1 homologous connective tissue growth factor mRNA from another mammalian species. And SEQ ID NO: 1 is a sequence of GeneBank accession nos. AK092280, AK125220, AY395801, AY550024, BT019794, BT019795, CR541759, M92934, U14750 and X78947, and SEQ ID NO: 1, incorporated herein by reference.
Attenuation of mRNA expression: as used herein, the phrase "attenuation of mRNA expression" refers to the administration of an amount of interfering RNA to a cell that results in a decrease in the level of full-length mRNA transcripts of a target gene in the cell relative to a control RNA having misordering, such that translation of the mRNA produces less protein. The reduction of mRNA expression is commonly referred to as mRNA "down-regulation" (knock-down). Embodiments herein contemplate that down-regulation of expression levels between 50% and 100%, including 50% and 100%. However, it is not necessary for the purposes of the present invention to achieve such a level of downregulation. Moreover, the effect of two groups of interfering RNAs when administered alone on down-regulation is minor, but simultaneous administration can be significantly more effective. In one embodiment, a single ds siRNA is effective down-regulated by at least up to 70%. In another embodiment, two or more ds sirnas together are effective to downregulate by at least up to 70%.
The detection of down-regulation is usually by measuring mRNA levels using Quantitative Polymerase Chain Reaction (QPCR) amplification, or by measuring protein levels using western blot or enzyme-linked immunosorbent assay (ELISA). Analysis of protein levels provides an estimate of mRNA degradation and translational inhibition of RNA-induced silencing complex (RISC). Other techniques for detecting down-regulation include RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with microarrays, antibody binding, radioimmunoassays, and fluorescence activated cell assays. Yet another method of detection is to overexpress TGF-beta 2, which induces CTGF, add CTGF siRNA back, and then detect down-regulation of CTGF mRNA/protein by any of the methods described above.
By observing the improvement of ocular diseases in humans or mammals, it can also be concluded that CTGF is inhibited. For example, in age-related macular degeneration, slowing or reversing vision loss indicates that CTGF is inhibited, silencing of CTGF mRNA in glaucoma patients results in decreased intraocular pressure, delaying or preventing the onset of symptoms in subjects with progressive glaucoma.
The interfering RNA of the embodiments of the invention proceeds catalytically, i.e., the interfering RNA is able to achieve inhibition of the mRNA of interest at sub-stoichiometric doses. Significantly less interfering RNA is required to exert a therapeutic effect compared to antisense therapy.
Double-stranded interfering RNA: as used herein, a double-stranded interfering RNA (also referred to as ds siRNA) has a sense nucleotide sequence and an antisense nucleotide sequence, the sense and antisense nucleotide sequences comprising a region of at least nearly complete sequential complementarity of at least 19 nucleotides. Interfering RNAs comprise 19 to 49 nucleotides in length and may comprise a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides. The antisense strand of ds siRNA hybridizes under physiological conditions to a nucleic acid sequence corresponding to SEQ ID NO: 1, having at least 19 nucleotides capable of hybridizing to a portion of an mRNA corresponding to SEQ ID NO: 1 at least close to the region of complete continuous complementarity.
The antisense strand of the siRNA is an active guide of the siRNA because it binds to the RISC complex in the cell, guiding the binding complex to specifically bind to the sequence of the mRNA that is complementary to the sequence of the antisense RNA, thereby allowing cleavage of the mRNA by the binding complex to occur.
Techniques for selecting target sequences for sirnas are provided by Tuschl, t.et al, "The siRNA user, revised 5/6 2004, available from The rockfiller University website, or by Technical bulletin #506," siRNA DesignGuidelines, "at Ambion's website, obtained from Invitrogen's website using The search parameters" minimum 35%, maximum 55% G/C content, "and obtained by Dharmacon's website. The sequence of interest may be located in the coding region or in the 5 'or 3' untranslated region of the mRNA.
In one embodiment, the DNA target sequence of CTGF is set forth in SEQ ID NO: nucleotide 1488 to 1506 of 1:
5’-ggttagtatcatcagatag-3’SEQ ID NO：18.nt 1488。
targeting SEQ ID NO: 18 and a 3' UU overhang on each strand is:
5’-gguuaguaucaucagauagUU-3’ SEQ ID NO：25
3’-UUccaaucauaguagucuauc-5’ SEQ ID NO：26.。
the 3' overhang may have a number of "U" residues, for example, a number of "U" residues between and including 2, 3, 4, 5 and 6. 5 'end also has a nucleotide 5' overhang. The targeting SEQ ID NO: 18 and a 3' TT overhang on each strand is:
5’-gguuaguaucaucagauagTT-3′ SEQ ID NO：27
3’-TTccaaucauaguagucuauc-5’ SEQ ID NO：28.
。
the two strands of the double-stranded siRNA can be connected by a hairpin loop to form a single-stranded siRNA as follows:
5’-gguuaguaucaucagauagUUNNN\
N
3’-UUccaaucauaguagucuaucNNNNN/ SEQ ID NO：29.
。
n is nucleotide A, T, C, G, U, or a modified form known to those skilled in the art. The number of nucleotides N is between 3-23, or 5-15, or 7-13, or 4-9, or 9-11, and includes 3, 23, 5, 15, 7, 13, 4, 9, or 9, 11, or the number of nucleotides N is 9.
Table 1 lists SEQ ID NOs: 1, the sirnas of the present invention are derived from the CTGF DNA of SEQ ID NO: 1 designed.
TABLE 1 CTGF target sequences of siRNAs
| Target sequence | Relative to SEQ ID NO: 1 starting nucleotide number | SEQ ID NO： |
| GGGCCTCTTCTGTGACTTC | 379 | 2 |
| CCGACTGGAAGACACGTTT | 691 | 3 |
| CCCGGGTTACCAATGACAA | 801 | 4 |
| GGGCAAAAAGTGCATCCGT | 901 | 5 |
| TCCAAGCCTATCAAGTTTGAGCTTT | 932 | 6 |
| GCCTATCAAGTTTGAGCTT | 937 | 7 |
| GCATGAAGACATACCGAGCTAAATT | 969 | 8 |
| GCTAAATTCTGTGGAGTAT | 986 | 9 |
| GCCATTACAACTGTCCCGGAGACAA | 1119 | 10 |
| GGAAGATGTACGGAGACAT | 1170 | 11 |
| GAGAGTGAGAGACATTAACTCATTA | 1201 | 12 |
| GCCATGTCAAACAAATAGTCTATCT | 1346 | 13 |
| GGGTACCAGCAGAAAGGTT | 1473 | 14 |
| CCAGCAGAAAGGTTAGTAT | 1478 | 15 |
| GCAGAAAGGTTAGTATCAT | 1481 | 16 |
| GCAGAAAGGTTAGTATCATCAGATA | 1481 | 17 |
| GGTTAGTATCATCAGATAG | 1488 | 18 |
| GGTTAGTATCATCAGATAGCTCTTA | 1488 | 19 |
| GAGACTGAGTCAAGTTGTTCCTTAA | 1626 | 20 |
| GCAGACTCAGCTCTGACAT | 1660 | 21 |
| TCAGCTCTGACATTCTGATTCGAAT | 1666 | 22 |
| TCCTGTCGATTAGACTGGACAGCTT | 1712 | 23 |
| GCTTGTGGCAAGTGAATTT | 1733 | 24 |
As cited in the examples above, one skilled in the art can use the target sequence information provided in table 1, with reference to SEQ ID NO: 1, and adding or deleting a sequence identical to SEQ ID NO: 1 complementary or near complementary nucleotides, and designs interfering RNAs that are longer or shorter than the sequences provided in table 1.
The cleavage reaction to the target RNA guided by ds or ss siRNA is highly specific. Generally, an siRNA containing a sense nucleotide sequence identical to a portion of the target mRNA and an antisense portion fully complementary to the sense strand is an embodiment that inhibits CTGF mRNA. However, in the specific practice of the invention, it is not required that the antisense strand of the siRNA have 100% sequence complementarity with the target mRNA. Thus, the present invention contemplates sequence variations that may result from genetic mutations, strain polymorphisms, or evolutionary divergence. For example, siRNA sequences having insertions, deletions, or single point mutations relative to the target sequence are all inhibitory.
The antisense sequence of the siRNA has at least 19 nucleotides that are nearly completely complementary to the mRNA target sequence. As used herein, "near-complete" means that the antisense sequence of the siRNA is "substantially complementary" to at least a portion of the target mRNA and the sense sequence of the siRNA is "substantially identical" to at least a portion of the target mRNA. "identity" as known to those skilled in the art refers to the degree of sequence relatedness between nucleotide sequences as determined by the order of nucleotides between the matched sequences. In one embodiment, antisense RNAs that have 80% and 80% to 100% complementarity to a target mRNA sequence are considered to have near complete complementarity and can be used in the present invention. "complete" continuous complementarity refers to adjacent base pairing standard Watson-Crick base pairing. "at least near perfect" consecutive complementarity includes "perfect" complementarity as used herein. Computer methods designed to determine identity or complementarity provide the greatest degree of matching of nucleic acid sequences, such as BLASTP and BLASTN (Altschul, S.F., et al (1990) J.mol.biol.215: 403-410), and FASTA.
SEQ ID NO: 1 can be in the 5 'or 3' untranslated region of an mRNA and the coding region of an mRNA.
One or both strands of a double-stranded interfering RNA can have a 3' overhang of 1 to 6 nucleotides, which can be ribonucleotides, or deoxyribonucleotides, or a mixture thereof. The nucleotides of the overhang are not base-paired. In one embodiment of the invention, the interfering dsRNA comprises a 3' overhang of TT or UU.
The sense and antisense strands of a double-stranded siRNA can be in the form of a duplex consisting of two single strands as described above, or can be a single molecule in which complementary regions base pair and are covalently linked by a hairpin or loop to form a single strand. The hairpin is believed to be cleaved within the cell by a protein called dicer to form an interfering RNA consisting of two single base-paired RNA molecules.
Interfering RNAs may differ from naturally occurring RNAs by the addition, deletion, substitution, or modification of one or more nucleotides. The non-nucleotide species can be bound to the interfering RNA at the 5 'end, 3' end, or internally. Such modifications are typically designed to increase resistance of interfering RNAs to nucleases, enhance cellular uptake, enhance cellular targeting, aid in tracking of interfering RNAs, or further increase stability. For example, interfering RNA can comprise purine nucleotides at the overhang. For example, the siRNA can also be stabilized by linking cholesterol to the 3' end of the sense strand of the siRNA molecule via pyrrolidine. Other modifications include, for example, 3' biotin molecules, peptides known to have cell penetrating properties, nanoparticles, peptidomimetics, fluorescent dyes, or dendrimers.
Nucleotides may be modified on the base portion, sugar portion, or phosphate portion of the molecule and function in embodiments of the invention. Modifications include, for example, substitution with alkyl, alkoxy, amino, deaza, halo, hydroxy, thiol, or combinations thereof. Nucleotides may be substituted with analogs having greater stability, such as replacement of U with 2 ' deoxy-T, or with sugar modifications, such as substitution of the 2 ' hydroxyl group with a 2 ' amino or 2 ' methyl, 2 ' methoxyethyl, or 2 ' -O, 4 ' -C methylene bridge. Examples of purine or pyrimidine analogues of nucleotides include xanthine, hypoxanthine, azapurine, methylthioadenine, 7-deaza-adenosine, O-and N-modified nucleotides. The phosphate group of a nucleotide may be modified by replacing one or more oxygens on the phosphate group with nitrogen or sulfur (phosphorothioates).
There may be a sequence on the antisense siRNA that corresponds to the sequence of SEQ ID NO: 1, a region in which a part of the mRNA of the above-mentioned gene is not complementary. The non-complementary region can be at the 3 'end, the 5' end, or both ends of the complementary region.
The interfering RNA can be produced synthetically, such as in vitro transcription, siRNA expression vectors, or PCR expression cassettes. Interfering RNA that functions well as transfected siRNA also functions well as siRNA expressed in vivo.
Interfering RNA can be chemically synthesized using protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer, and can also be obtained from commercial suppliers such as Ambion Inc (Austin, Texas), Invitrogen (Carlsbad, CA), or Dharmacon (Lafayette, colo., USA). The interfering RNA can be purified, for example, by solvent extraction or resin, precipitation, electrophoresis, chromatography, or a combination of these methods. Alternatively, interfering RNA that is hardly purified can be used to avoid losses during sample processing.
The interfering RNA can be provided to the subject by expression from recombinant plasmids using constitutive or inducible promoters such as the U6 or H1 RNA pol III promoter, the cytomegalovirus promoter, SP6, T3, or T7 promoters, all of which are known to those skilled in the art. For example, psiRNA from Invivogen (san Diego, Calif.)TMAllowing production of siRNA from the RNA pol III promoter within the cell. Interfering RNA expressed using recombinant plasmids can be isolated using standard techniques.
The viral vector expressing the interfering RNA may be derived from adenovirus, adeno-associated virus, vaccinia virus, retrovirus (e.g., lentivirus, rhabdovirus, murine leukemia virus), herpes virus, and the like, using the above promoters, such as those used for plasmids. Selection of viral vectors, methods of expressing interfering RNA by the vectors, and methods of delivering viral vectors are within the ability of those skilled in the art.
Expression of interfering RNA can also be achieved by using SILENCER EXPRESSTM(Ambion, Austin, Texas) was provided by expression cassettes (SECs) with human H1, human U generated by PCR6 or mouse U6 promoter. The silencing expression cassette is a PCR product comprising a promoter and a hairpin siRNA template flanking a termination sequence. Hairpin siRNA are expressed from PCR products and induce specific silencing after transfection into cells.
Hybridization under physiological conditions: "hybridization" is a technique in which single-stranded nucleic acids (DNA or RNA) are allowed to interact such that those nucleic acids having complementary or near complementary base sequences form hydrogen-bonded complexes called hybrids. The hybridization reaction is sensitive and selective, identifying specific sequences of interest when present at low concentrations in the sample. The specificity (e.g., stringency) of hybridization is controlled by the concentration of salt or formamide in the in vitro prehybridization and hybridization solutions, and the hybridization temperature, and is known in the art. Specifically, stringency can be increased by decreasing the salt concentration, increasing the concentration of formamide, or increasing the hybridization temperature.
For example, high stringency conditions can occur at 37 ℃ to 42 ℃ with about 50% formamide. Low stringency conditions can occur at about 30 ℃ to 35 ℃, about 35% to 25% formamide. In Sambrook, j., 1989, molecular cloning: examples of stringent hybridization conditions are provided in the laboratory manual, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.. Additional examples of stringent hybridization conditions include: 400mM NaCl, 40mM PIPES pH6.4, 1mM EDTA, at 50 ℃ or 70 ℃ for 12-16 hours, followed by washing, or hybridization at 70 ℃ in 1XSSC or at 50 ℃ in 1XSSC, 50% formamide, followed by washing at 70 ℃ in 0.3XSSC, or hybridization at 70 ℃ in 4XSSC or at 50 ℃ in 4XSSC, 50% formamide, followed by washing at 67 ℃ in 1 XSSC. The temperature of hybridization is about 5 to 10 ℃ below the melting temperature (Tm) of the hybrid, and when the length of the hybrid is between 19 and 49 base pairs, the melting temperature is calculated using the following equation: tm ℃81.5+16.6 (log)10[Na+]) +0.41 (% G + C) - (600/N), where N is the number of bases in the hybrid, [ Na +]Is the concentration of sodium ions in the hybridization buffer.
In embodiments of the invention, the antisense strand of interfering RNA that hybridizes to CTGF mRNA under highly stringent conditions in vitro also specifically binds under physiological conditions in vivo. The identification or isolation of related nucleic acids that do not hybridize to nucleic acids under highly stringent conditions is performed under conditions of reduced stringency.
Single-stranded interfering RNA: as mentioned above, interfering RNA ultimately functions in a single stranded form. Ss siRNAs were found to achieve mRNA silencing, albeit less efficiently than double-stranded RNA. Accordingly, embodiments of the present invention also provide administration of ss siRNAs wherein the single stranded siRNA hybridizes under physiological conditions to a nucleic acid sequence corresponding to SEQ ID NO: 1, having a sequence that hybridizes to a portion of an mRNA corresponding to seq id NO: 1 of at least 19 nucleotides of the hybridizing portion of the mRNA of 1. The length of the single stranded siRNA is 19-49 nucleotides with respect to the double stranded siRNA introduced above. The single stranded siRNA has a 5 'phosphate or is phosphorylated at the 5' position in situ or in vivo. The term "5 ' phosphorylated" is used to describe a polynucleotide or oligonucleotide (e.g., 5 ' ribose or deoxyribose, or analogs thereof) having a phosphate group such as the C5 hydroxyl group linked to a 5 ' sugar by an ester linkage. The single stranded siRNA may have a single, double, or triple phosphate group.
Single stranded siRNA can be produced by chemical synthesis, or by a vector as double stranded siRNA. The 5 'phosphate group may be added via a kinase, or the 5' phosphate may be the result of cleavage of the RNA by a nuclease. Delivery was performed as double stranded siRNA. In one embodiment, single stranded siRNA with protected ends and nuclease resistance modifications are administered for silencing. The single stranded siRNA may be stored dry or dissolved in an aqueous solution. To remain stable, the solution may contain a buffer or salt to inhibit annealing or for stabilization.
Hairpin interfering RNA: hairpin interfering RNA is single-stranded, in a single strand of both sense and antisense strand. For expression by the DNA vector, a corresponding DNA oligonucleotide of at least 19 nucleotides corresponding to the sense siRNA sequence is linked to its reverse complementary antisense sequence by a short spacer. The 3 'terminal T', and the nucleotides forming the restriction site, can be increased if required by the chosen expression vector. The resulting RNA transcript folds upon itself to form a stem-loop structure.
The application mode is as follows: the interfering RNA can be delivered directly to the eye by injection into ocular tissue, such as periocular, conjunctival, sub-tenon, intracameral, intravitreal, subretinal, retrobulbar, or intratubular injection; or directly to the eye using a catheter or other placement device, such as a retinal pellet, intraocular insert, suppository, or implant comprising a porous, non-porous, or gelatinous substance; by topical eye drops or ointments; or by a slow release device in the cul-de-sac, or by a slow release device implanted adjacent the sclera (transscleral) or within the eye. Intraocular injection may be through the cornea to the anterior chamber, allowing the agent to reach the trabecular meshwork. The intravessel injection may enter the venous collection channel, empty the schlemm's canal or enter the schlemm's canal.
Subject: a subject in need of treatment for, or at risk of developing, an ocular disorder refers to a human or other mammal having, or at risk of developing, a condition associated with, e.g., a CTGF expression or activity, such as a CTGF-associated ocular disorder. These ophthalmic diseases may include: such as glaucoma, macular degeneration, diabetic retinopathy, choroidal neovascularization, proliferative vitreoretinopathy and wound healing, and conditions of scar overproduction, endothelial cell proliferation, fibroproliferation. The ocular structures associated with these diseases may include: such as the retina, choroid, lens, cornea, trabecular meshwork, rod, cone, ganglion, macula, iris, sclera, chamber of the eye, vitreous cavity, ciliary body, optic disc, or fovea.
Formulation and dosage: the medicament contains up to 99% by weight of the interfering RNA or salt thereof of the present invention, which is mixed with a physiologically acceptable ophthalmic carrier vehicle such as water, buffer, saline, glycine, hyaluronic acid, mannitol, and the like.
In the present invention, the interfering RNA is administered as a solution, suspension or emulsion. The following are examples of possible formulations embodied in the present invention.
| In% by weight |
| Interfering RNA | Up to 99; 0.1 to 99; 0.1 to 50; 0.5-10.0 |
| Hydroxypropyl methylcellulose | 0.5 |
| Sodium chloride | .8 |
| Benzalkonium chloride | 0.01 |
| EDTA | 0.01 |
| NaOH/HCl | To pH7.4 |
| In% by weight |
| Interfering RNA | Up to 99; 0.1 to 99; 0.1 to 50; 0.5-10.0 |
| Phosphate buffer | 1.0 |
| Benzalkonium chloride | 0.01 |
| Polysorbate 80 | 0.5 |
| Purified water | To 100 percent |
| Interfering RNA | Up to 99; 0.1 to 99; 0.1 to 50; 0.5-10.0 |
| Sodium dihydrogen phosphate | 0.05 |
| In% by weight |
| Disodium hydrogen phosphate (anhydrous) | 0.15 |
| Sodium chloride | 0.75 |
| EDTA disodium salt | 0.05 |
| Cremophor EL | 0.1 |
| Benzalkonium chloride | 0.01 |
| HCl and/or NaOH | PH7.3-7.4 |
| Purified water | To 100 percent |
| In% by weight |
| Interfering RNA | Up to 99; 0.1 to 99; 0.1 to 50; 0.5-10.0 |
| Phosphate buffer | 1.0 |
| Hydroxypropyl-beta-cyclodextrin | 4.0 |
| Purified water | To 100 percent |
In general, an effective amount of interfering RNA in embodiments of the invention comprises an intercellular concentration of from 200pM to 100nM, or from 1nM to 50nM, or from 5nM to about 25nM, at or near the eye. The topical composition is delivered to the ocular surface one to four times per day according to the routine judgment of a skilled clinician. The pH of the formulation is about 4-9, or pH4.5 to pH 7.4.
Although the precise protocol is left to the clinician's judgment, interfering RNA may be dropped in one to four drops per eye per day or under the direction of the clinician. The effective amount of the preparation depends on factors such as the age, race, sex, etc. of the subject, or the severity of the ophthalmic disease. In one embodiment, the interfering RNA is delivered locally to the eye at a therapeutic dose to the trabecular meshwork, retina or optic nerve head, thereby ameliorating the CTGF-associated disease process.
Acceptable carriers: ophthalmically acceptable carriers are those which cause at most, little or no ocular irritation, provide suitable preservation, if desired, and deliver one or more interfering RNAs of the invention in a uniform dose. Acceptable carriers for administering interfering RNAs of embodiments of the invention include: mirus TransITTKO siRNA transfection reagent (Mirus Corporation, Madison, Wisconsin), LIPOFECTIN、lipofectamine、OLIGOFECTAMINETM(Invitrogen，Carisbad，CA)、CELLFECTINDHARMAFECTTM(Dharmacon, Chicago, IL) or a polycation such as polylysine, liposomes or a fat-soluble substance such as cholesterol. Liposomes are formed from standard vesicle-forming lipids and steroids such as cholesterol, and may include a target molecule such as a monoclonal antibody having binding affinity for an endothelial cell surface antigen. Alternatively, the liposomes may be pegylated liposomes.
For ophthalmic delivery, the interfering RNA can be formed into an aqueous sterile ophthalmic suspension or solution with an ophthalmically acceptable preservative, co-solvent, surfactant, thickener, penetration enhancer, buffer, sodium chloride, or water. Ophthalmic solution formulations can be prepared by dissolving the inhibitor in a physiologically acceptable isotonic aqueous buffer. Additionally, the ophthalmic solution may include an ophthalmically acceptable surfactant to facilitate dissolution of the inhibitor. Viscosity aids (building agents) such as hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, polyvinylpyrrolidone, and the like may be added to the compositions of the present invention to enhance retention of the compounds.
To prepare sterile ophthalmic ointment formulations, the interfering RNA is combined with a preservative in a suitable carrier such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations interfering RNA can be suspended from, e.g., CARBOPOL, according to methods known in the art for other ophthalmic formulations-940(BF Goodrich, Charlotte, NC) and the like. Such as VISCOAT(Alcon LabRobots, inc., Fort Worth, TX) may be used for intraocular injection. Other compositions of the invention may contain penetration enhancers such as cremephor and TWEEN when the penetration of interfering RNA into the eye is insufficient80 (polyoxyethylene sorbitan monolaurate, Sigma Aldrich, St. Louis, Mo.).
The kit comprises: embodiments of the invention provide kits comprising agents for attenuating CTGF mRNA expression in a cell. The kit contains a DNA template having two different promoters such as the T7 promoter, the T3 promoter or the SP6 promoter, each capable of operably linking nucleotide sequences encoding two complementary single stranded RNAs corresponding to interfering RNAs. The RNA is transcribed from the DNA template and annealed to form double-stranded RNA, which effectively attenuates the expression of the target mRNA. The kit optionally contains amplification primers for amplifying DNA sequences from a DNA template, and nucleoside triphosphates (e.g., ATP, GTP, CTP, and UTP) for RNA synthesis. Optionally, the kit contains two RNA polymerases, each capable of binding to a promoter on the DNA template and effecting transcription of a nucleotide sequence to which the promoter is operably linked, a purification column for purifying single-stranded RNA, such as a size exclusion chromatography column, one or more buffers, such as a buffer that anneals single-stranded RNA to produce double-stranded RNA, and RNase A or RNase T for purifying double-stranded RNA.
Example 1
Standards for silencing interfering RNA for CTGF in trabecular meshwork cells and detecting silencing
The invention detects the down-regulation capacity of CTGF interfering RNA to endogenous CTGF expression level in human Trabecular Meshwork (TM) cells. The invention also provides a standard for determining the efficacy of interfering RNA on mRNA levels when measured using QPCR primers.
Standard in vitro concentrations (100nM) of CTGF interfering RNA and LIPOFECTAMINE at a 1: 1(w/v) ratioTM2000(Invitrogen, Carlsbad, California), transfectionTransformed human TM cell lines designated GTM3 or HTM-3 (see Pang, I.H.et al, 1994, Curr.Eye Res.13: 51-63). Use of a commercially designed pool of unknown sequence interfering RNAs (siGENOME SMARTPOOL)CTGF interfering RNA (referred to herein as siRNA S4), Dharmacon, Lafayette, Colorado) targeted CTGF. Disordered and lamin A/CsiRNA (Dharmacon) were used as controls.
The control experiment results in a lamin a/C down-regulation efficiency of approximately 90% with lamin a/C interfering RNA compared to the scrambled interfering RNA control. Initial studies showed the use of siGENOME smartCTGF siRNA M-012633-00-0020(siRNA S4) the downregulation efficiency of CTGF was about 20% to 30% with primer/probe set Q2 directed to the 3' UTR of CTGF mRNA in exon 5. Q2 is QRCR TAQMAN from ABI (Applied Biosystems Foster City, Calif.)Primer/probe sets.
To determine the cause of CTGF siRNA inefficiency, several variables were measured. Testing dose response of CTGF interfering RNA to determine whether suboptimal interfering RNA concentration or suboptimal interfering RNA was used: lipid ratio. The resulting data indicate that low CTGF mRNA downregulation is correlated with the concentration of interfering RNA or interfering RNA used: the lipid ratio is irrelevant. The transfection efficiency of TM cells was measured under the above conditions, taking into account the importance of cellular uptake for siRNA activity and the inherent difficulties of transfecting TM cells. By SITOXTM(Dharmacon) delivery to cytoplasmic induced cell death or SIGLOTM(Dharmacon) cellular fluorescence measured by cytoplasmic fluorescence was measured to reflect the transfection efficiency of the cells. In both cases, almost all cells did not die (FIG. 1A; SITOX)TM) Is fluorescent (fig. 1B; SIGLOTM) Indicating that the transfection efficiency was almost constantQuantitative and in this process is not the rate limiting step.
Further, two different QPCR TAQMAN were combined, designated Q2 and Q1(ABI, Applied biosystems Foster City, Calif.)Three additional separate CTGFsiRNA sequences, designated siRNA S1, S2 and S3, were tested for primer/probe sets, ambion inc. The target sequences for Ambion siRNA are as follows, using GenBank reference No. NM — 001901 for nucleotides (nts) of CTGF:
target of S1: (nts 379-397): gggcctcttctgtgacttc SEQ ID NO: 2
Target of S2: (nts 901-919): gggcaaaaagtgcatccgt SEQ ID NO: 5
Target of S3: (nts 1488-1506): ggttagtatcatcagatag SEQ ID NO: 18
The double-stranded siRNA with 3' TT overhangs on each strand of each target sequence above is:
siRNA S1：
5′-gggccucuucugugacuucTT-3′ SEQ ID NO：30
3’-Ttcccggagaagacacugaag-5′ SEQ ID NO：31
siRNA S2：
5′-gggcaaaaagugcauccguTT-3′ SEQ ID NO：32
3′-TTcccguuuuucacguaggca-5′ SEQ ID NO：33
SiRNA S3：
5′-gguuaguaucaucagauagTT-3′ SEQ ID NO：27
3′-TTccaaucauaguagucuauc-5′ SEQ ID NO：28
QPCR Q1 primerIs ABI ASSAY ON DEMANDTMThe proprietary sequence hs00170014_ ml (applied biosystems).
The QPCR Q2 forward primer has the sequence: 5'-CAGCTCTGACATTCTGATTCGAA-3' SEQ ID NO: 34
The Q2 reverse primer has the sequence: 5'-TGCCACAAGCTGTCCAGTCT-3' SEQ ID NO: 35
The Q2 probe has the sequence:
5’-AATCGACAGGATTCCGATTCCTGAACAGTG-3’SEQ ID NO：36
and has a FAM group (6-carboxyfluorescein) at the 5 'end and a TAMRA group (Applied bioSystems) at the 3' end.
The position of the primer/probe set relative to the siRNA target site of each siRNA is shown in fig. 2A. FIG. 2A is a schematic diagram also showing the structure of the exons (boxes) and introns (lines) of the CTGF gene, and the positions of the siRNA S1, S2, S3 and QPCR primer/probe sets Q1 and Q2 corresponding to the GenBank CTGF sequence NM-001901 (provided as SEQ ID NO: 1).
FIG. 2B shows QPCR amplification of CTGFmRNA using exon 5 primer/probe set Q2 and siRNA S1-S4. When S1 and S4 sirnas were used, the Q2 primer/probe set did not detect significant downregulation of CTGF mRNA levels. Demonstrating downregulation by siRNA S2 and S3. The primer/probe set Q2 was closer to the targets of the S2 and S3 sirnas than to the target of the S1 siRNA.
Figure 2C shows QPCR amplification of CTGF mRNA with primer/probe set Q1 spanning exon 4/5. Each siRNA demonstrated CTGF mRNA down-regulation, about 90% of which was observed with S2siRNA when detected with Q1 primer/probe set. Primer/probe set Q1 appeared to be more effective than the Q2 primer/probe set in demonstrating siRNA down-regulation.
The data in fig. 2B and fig. 2C indicate that the specific region amplified with primer/probe set Q2 directed to the 3' -UTR can be relatively stable, and therefore there is less choice to estimate the cleavage and degradation of CTGF mRNA by targeted siRNA. Therefore, in the specific case where the amplification region of QPCR is located outside the siRNA targeting region, the efficacy of siRNA may be underestimated.
To reduce non-specific off-target effects, the lowest possible siRNA concentration that inhibited CTGF mRNA expression was measured. CTGF mRNA down-regulation was estimated by QPCR using primer/probe set Q1. FIG. 3 shows dose response of CTGF S2siRNA in GTM3 cells. IC was observed after 24 hours of treatment of GTM3 cells with S2siRNA at doses ranging from 0, 1, 3, 10, 30 and 100nM50At-2.5 nM. Data were fitted using GraphPad Prism 4 Software (GraphPad Software, inc., san Diego, CA) using a variable slope, sigmoidal dose response algorithm, maximum constraint 100%.
The results of this example demonstrate that: i) trabecular meshwork cells for siRNA silencing, ii) all sirnas cited herein achieve a degree of silencing, and iii) PCR amplification primers are designed to include siRNA target sequences to optimize silencing detection when using PCR-based methods to determine siRNA down-regulation efficacy.
Cleavage of target mRNA by RISC endonucleases has been shown to occur near the center of the siRNA target sequence (Elbashir, S.M., et al, 2001.Genes Dev 15: 188-144200) and is accomplished by Argonaute RNase H activity (Liu, J., et al Science 305: 1437-1441). However, complete degradation of the remaining mRNA does not appear to be guaranteed. The stable mRNA fragments remaining after Argonaute cleavage, amplification of either of these fragments by QPCR, may underestimate the siRNA efficacy shown here. The present invention provides an embodiment wherein the QPCR primer set comprises siRNA target sequences to ensure optimal siRNA efficiency read-out.
The references cited herein are specifically incorporated by reference to the extent that they provide exemplary steps or other details supplementary to those set forth herein.
As will be understood by those skilled in the art in light of this disclosure, obvious modifications can be made to the embodiments disclosed herein without departing from the spirit and scope of the invention. In light of the present disclosure, all embodiments in accordance with the present disclosure can be made and practiced without undue experimentation. The full scope of the invention is given in this disclosure and its equivalents. This description is not to be construed as unduly narrowing the full scope of the invention to which it is entitled.
The terms "a" and "an," as used herein, mean "one," "at least one," or "one or more," unless otherwise specified.
Sequence listing
<110> ai Er kang Gong department
<120> RNAi inhibition of CTGF for treatment of ocular diseases
<130>34576.40
<150>60/638,705
<151>2004-12-23
<160>36
<170> PatentIn version 3.3
<210>1
<211>2312
<212>DNA
<213> human (Homo sapiens)
<400>1
tccagtgacg gagccgcccg gccgacagcc ccgagacgac agcccggcgc gtcccggtcc 60
ccacctccga ccaccgccag cgctccaggc cccgcgctcc ccgctcgccg ccaccgcgcc 120
ctccgctccg cccgcagtgc caaccatgac cgccgccagt atgggccccg tccgcgtcgc 180
cttcgtggtc ctcctcgccc tctgcagccg gccggccgtc ggccagaact gcagcgggcc 240
gtgccggtgc ccggacgagc cggcgccgcg ctgcccggcg ggcgtgagcc tcgtgctgga 300
cggctgcggc tgctgccgcg tctgcgccaa gcagctgggc gagctgtgca ccgagcgcga 360
cccctgcgac ccgcacaagg gcctcttctg tgacttcggc tccccggcca accgcaagat 420
cggcgtgtgc accgccaaag atggtgctcc ctgcatcttc ggtggtacgg tgtaccgcag 480
cggagagtcc ttccagagca gctgcaagta ccagtgcacg tgcctggacg gggcggtggg 540
ctgcatgccc ctgtgcagca tggacgttcg tctgcccagc cctgactgcc ccttcccgag 600
gagggtcaag ctgcccggga aatgctgcga ggagtgggtg tgtgacgagc ccaaggacca 660
aaccgtggtt gggcctgccc tcgcggctta ccgactggaa gacacgtttg gcccagaccc 720
aactatgatt agagccaact gcctggtcca gaccacagag tggagcgcct gttccaagac 780
ctgtgggatg ggcatctcca cccgggttac caatgacaac gcctcctgca ggctagagaa 840
gcagagccgc ctgtgcatgg tcaggccttg cgaagctgac ctggaagaga acattaagaa 900
gggcaaaaag tgcatccgta ctcccaaaat ctccaagcct atcaagtttg agctttctgg 960
ctgcaccagc atgaagacat accgagctaa attctgtgga gtatgtaccg acggccgatg 1020
ctgcaccccc cacagaacca ccaccctgcc ggtggagttc aagtgccctg acggcgaggt 1080
catgaagaag aacatgatgt tcatcaagac ctgtgcctgc cattacaact gtcccggaga 1140
caatgacatc tttgaatcgc tgtactacag gaagatgtac ggagacatgg catgaagcca 1200
gagagtgaga gacattaact cattagactg gaacttgaac tgattcacat ctcatttttc 1260
cgtaaaaatg atttcagtag cacaagttat ttaaatctgt ttttctaact gggggaaaag 1320
attcccaccc aattcaaaac attgtgccat gtcaaacaaa tagtctatct tccccagaca 1380
ctggtttgaa gaatgttaag acttgacagt ggaactacat tagtacacag caccagaatg 1440
tatattaagg tgtggcttta ggagcagtgg gagggtacca gcagaaaggt tagtatcatc 1500
agatagctct tatacgagta atatgcctgc tatttgaagt gtaattgaga aggaaaattt 1560
tagcgtgctc actgacctgc ctgtagcccc agtgacagct aggatgtgca ttctccagcc 1620
atcaagagac tgagtcaagt tgttccttaa gtcagaacag cagactcagc tctgacattc 1680
tgattcgaat gacactgttc aggaatcgga atcctgtcga ttagactgga cagcttgtgg 1740
caagtgaatt tcctgtaaca agccagattt tttaaaattt atattgtaaa tattgtgtgt 1800
gtgtgtgtgt gtgtatatat atatatatat gtacagttat ctaagttaat ttaaagttgt 1860
ttgtgccttt ttatttttgt ttttaatgct ttgatatttc aatgttagcc tcaatttctg 1920
aacaccatag gtagaatgta aagcttgtct gatcgttcaa agcatgaaat ggatacttat 1980
atggaaattc tctcagatag aatgacagtc cgtcaaaaca gattgtttgc aaaggggagg 2040
catcagtgtc cttggcaggc tgatttctag gtaggaaatg tggtagctca cgctcacttt 2100
taatgaacaa atggccttta ttaaaaactg agtgactcta tatagctgat cagttttttc 2160
acctggaagc atttgtttct actttgatat gactgttttt cggacagttt atttgttgag 2220
agtgtgacca aaagttacat gtttgcacct ttctagttga aaataaagta tattttttct 2280
aaaaaaaaaa aaaaacgaca gcaacggaat tc 2312
<210>2
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>2
gggcctcttc tgtgacttc 19
<210>3
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>3
ccgactggaa gacacgttt 19
<210>4
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>4
cccgggttac caatgacaa 19
<210>5
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
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tccaagccta tcaagtttga gcttt 25
<210>6
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>6
tccaagccta tcaagtttga gcttt 25
<210>7
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>7
gcctatcaag tttgagctt 19
<210>8
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>8
gcatgaagac ataccgagct aaatt 25
<210>9
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>9
gctaaattct gtggagtat 19
<210>10
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>10
gccattacaa ctgtcccgga gacaa 25
<210>11
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>11
ggaagatgta cggagacat 19
<210>12
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>12
gagagtgaga gacattaact catta 25
<210>13
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>13
gccatgtcaa acaaatagtc tatct 25
<210>14
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>14
gggtaccagc agaaaggtt 19
<210>15
<211>19
<212>DNA
<213> Artificial
<220>
<223> targeting sequence
<400>15
ccagcagaaa ggttagtat 19
<210>16
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>16
gcagaaaggt tagtatcat 19
<210>17
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>17
gcagaaaggt tagtatcatc agata 25
<210>18
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>18
ggttagtatc atcagatag 19
<210>19
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>19
ggttagtatc atcagatagc tctta 25
<210>20
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>20
gagactgagt caagttgttc cttaa 25
<210>21
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>21
gcagactcag ctctgacat 19
<210>22
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>22
tcagctctga cattctgatt cgaat 25
<210>23
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>23
tcctgtcgat tagactggac agctt 25
<210>24
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> targeting sequence
<400>24
gcttgtggca agtgaattt 19
<210>25
<211>21
<212>RNA
<213> Artificial sequence
<220>
<223> sense strand
<400>25
gguuaguauc aucagauagu u 21
<210>26
<211>21
<212>RNA
<213> Artificial sequence
<220>
<223> antisense strand
<400>26
cuaucugaug auacuaaccu u 21
<210>27
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> sense strand with 3' TT
<220>
<221>misc_RNA
<222>(1)..(19)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(20)..(21)
<223> deoxyribonucleotides
<400>27
gguuaguauc aucagauagt t 21
<210>28
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> antisense strand having 3' TT
<220>
<221>misc_RNA
<222>(1)..(19)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(20)..(21)
<223> deoxyribonucleotides
<400>28
cuaucugaug auacuaacct t 21
<210>29
<211>51
<212>DNA
<213> Artificial sequence
<220>
<223> hairpin duplex having loop
<220>
<221>misc_RNA
<222>(1)..(21)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(22)..(30)
<223>any，A，T/U，C，G
<220>
<221>misc_feature
<222>(31)..(51)
<223> ribonucleotides
<400>29
gguuaguauc aucagauagu unnnnnnnnn cuaucugaug auacuaaccu u 51
<210>30
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> sense strand with 3' TT
<220>
<221>misc_RNA
<222>(1)..(19)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(20)..(21)
<223> deoxyribonucleotides
<400>30
gggccucuuc ugugacuuct t 21
<210>31
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> antisense strand having 3' TT
<220>
<221>misc_RNA
<222>(1)..(19)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(20)..(21)
<223> deoxyribonucleotides
<400>31
gaagucacag aagaggccct t 21
<210>32
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> sense strand with 3' TT
<220>
<221>misc_RNA
<222>(1)..(19)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(20)..(21)
<223> deoxyribonucleotides
<400>32
gggcaaaaag ugcauccgut t 21
<210>33
<211>21
<212>DNA
<213> Artificial
<220>
<223> antisense strand having 3' TT
<220>
<221>misc_RNA
<222>(1)..(19)
<223> ribonucleotides
<220>
<221>misc_feature
<222>(20)..(21)
<223> deoxyribonucleotides
<400>33
acggaugcac uuuuugccct t 21
<210>34
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> Probe/primer sequences
<400>34
cagctctgac attctgattc gaa 23
<210>35
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> Probe/primer sequences
<400>35
tgccacaagc tgtccagtct 20
<210>36
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> Probe/primer sequences
<400>36
aatcgacagg attccgattc ctgaacagtg 30