AGENT FOR SUPPRESSING EXPRESSION OF DOMINANT MUTANT GENE

• US 20130197061A1
• Filed: 09/28/2011
• Published: 08/01/2013
• Est. Priority Date: 09/30/2010
• Status: Active Grant

• Current Assignee: Lsip, LLC
• Original Assignee: National Center of Neurology and Psychiatry
• Inventors: Hohjoh, Hirohiko, Takahashi, Masaki
• Granted Patent: US 9,238,812 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/34 Allele or polymorphism specific uses

Abstract

An RNAi molecule that can selectively and effectively suppress only the expression of a particular dominant mutant gene, while permitting the expression of the wild-type gene or a desired mutant gene, and a design method thereof is presented.

Claims

1. An agent for suppressing the expression of a dominant mutant gene, comprising an RNAi molecule with an ASP score of 0.4 or higher as an active ingredient, wherein

the ASP score is calculated according to the following equation:

ASP score=[(relative ratio of a normalized expression level of a normal gene treated with the RNAi molecule to a normalized expression level of the normal gene treated with a control RNAi molecule)−

(relative ratio of a normalized expression level of a mutant gene treated with the RNAi molecule to a normalized expression level of the mutant gene treated with the control RNAi molecule)]×

(1−

the relative ratio of the normalized expression level of the mutant gene treated with the RNAi molecule to the normalized expression level of the mutant gene treated with the control RNAi molecule)

wherein the control RNAi molecule represents an RNAi molecule that does not influence the expression of the normal gene and the mutant gene, and

the RNAi molecule comprises;

an RNAi sense strand region comprising at least one point of discontinuity to be generated on a transcript of the targeted dominant mutant gene and a nucleotide sequence identical to a sequence of 16 to 30 consecutive bases of the transcript; and

an RNAi antisense strand region comprising a nucleotide sequence complementary thereto, wherein

any one of the 4th to 15th bases downstream from a base 3′

flanking any one point of discontinuity on the RNAi sense strand region constitutes the 3′

terminal base of the RNAi sense strand region.

3. The suppressing agent according to claim 1 or 2, wherein TT or UU is further added to each of the 3′

ends of the RNAi sense strand region and the RNAi antisense strand region.

4. The suppressing agent according to any one of claims 1 to 3, wherein the RNAi molecule is an siRNA.

5. The suppressing agent according to any one of claims 1 to 3, wherein the RNAi molecule is an shRNA.

6. The suppressing agent according to any one of claims 1 to 5, wherein the mutation in the dominant mutant gene is selected from the group consisting of a base deletion, a base insertion, a base substitution capable of destroying a splice site, a gene duplication, a gene translocation, and a chromosomal inversion.

7. The suppressing agent according to any one of claims 1 to 6, wherein the dominant mutant gene is a gain of function type.

8. The suppressing agent according to any one of claims 1 to 7, wherein the dominant mutant gene is involved in the onset of a disease.

9. The suppressing agent according to claim 8, wherein the disease is malignant neoplasm.

10. The suppressing agent according to claim 9, wherein the malignant neoplasm is non-small cell lung cancer, and the targeted dominant mutant gene thereof is a mutant EGFR gene;

the malignant neoplasm is colon cancer, and the targeted dominant mutant gene thereof is a mutant CTNNB1 gene;

the malignant neoplasm is stomach cancer, and the targeted dominant mutant gene thereof is a mutant CDH1 gene;

the malignant neoplasm is breast cancer, and the targeted dominant mutant gene thereof is a mutant BRCA1 gene or a mutant BRCA2 gene;

the malignant neoplasm is autoimmune polyendocrine syndrome type I, and the targeted dominant mutant gene thereof is a mutant AIRE gene;

the malignant neoplasm is autoimmune lymphoproliferative syndrome, and the targeted dominant mutant gene thereof is a mutant TNFRSF6/APT1/FAS gene;

the malignant neoplasm is chronic myeloid leukemia or acute lymphocytic leukemia, and the targeted dominant mutant gene thereof is a BCR-ABL chimeric gene;

the malignant neoplasm is Burkitt'"'"'s lymphoma, and the targeted dominant mutant gene thereof is a c-myc-IgH chimeric gene;

the malignant neoplasm is anaplastic large cell lymphoma, and the targeted dominant mutant gene thereof is a NPM-ALK chimeric gene;

the malignant neoplasm is lung cancer, and the targeted dominant mutant gene thereof is an EML4-ALK chimeric gene;

the malignant neoplasm is dermatofibrosarcoma protuberans, and the targeted dominant mutant gene thereof is a PDGFB-COL1A1 chimeric gene;

the malignant neoplasm is congenital fibrosarcoma, and the targeted dominant mutant gene thereof is an ETV6-NTRK3 chimeric gene;

the malignant neoplasm is low-grade fibromyxoid sarcoma, and the targeted dominant mutant gene thereof is a FUS-CREB3L2 chimeric gene;

the malignant neoplasm is extraskeletal myxoid chondrosarcoma, and the targeted dominant mutant gene thereof is an EWS-CHN chimeric gene;

the malignant neoplasm is Ewing'"'"'s sarcoma or desmoplastic small cell tumor, and the targeted dominant mutant gene thereof is a chimeric gene whose translocation partner is EWSR1 gene;

the malignant neoplasm is alveolar rhabdomyosarcoma, and the targeted dominant mutant gene thereof is a chimeric gene whose translocation partner is SYT gene or SSX gene;

the malignant neoplasm is inflammatory myofibroblastic tumor, and the targeted dominant mutant gene thereof is a chimeric gene whose translocation partner is ALK gene;

the malignant neoplasm is liposarcoma, and the targeted dominant mutant gene thereof is a chimeric gene whose translocation partner is CHOP gene;

or the malignant neoplasm is clear cell sarcoma of soft tissue or malignant fibrous histiocytoma, and the targeted dominant mutant gene thereof is a chimeric gene whose translocation partner is ATF1 gene.

11. The suppressing agent according to claim 10, wherein the malignant neoplasm is non-small cell lung cancer, and the targeted dominant mutant gene thereof is a mutant EGFR gene.

12. The suppressing agent according to claim 11, wherein the sense strand region of the RNAi molecule consists of a nucleotide represented by SEQ ID NO:

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 59, 61, 63, 65, 67, 129, 131, 133, 135, 137, 139, 141, 143, or 145.

13. The suppressing agent according to claim 10, wherein the malignant neoplasm is chronic myeloid leukemia or acute lymphocytic leukemia, and the targeted dominant mutant gene thereof is a BCR-ABL chimeric gene.

14. The suppressing agent according to claim 13, wherein the sense strand region of the RNAi molecule consists of a nucleotide represented by SEQ ID NO:

97, 99, 101, 103, 105, 107, 109, 111, or 113.

15. The suppressing agent according to claim 8, wherein the disease is a disease caused by a human autosomal dominant mutation.

16. The suppressing agent according to claim 15, wherein the disease caused by a human autosomal dominant mutation is congenital night blindness, and the targeted dominant mutant gene thereof is RHO gene;

the disease caused by a human autosomal dominant mutation is deafness nonsyndromic autosomal dominant 2 (DFNA2), and the targeted dominant mutant gene thereof is KCNQ4 gene or GJB gene;

the disease caused by a human autosomal dominant mutation is Waardenburg'"'"'s syndrome, and the targeted dominant mutant gene thereof is MITF gene;

the disease caused by a human autosomal dominant mutation is nonsyndromic deafness, and the targeted dominant mutant gene thereof is DIAPH1/DFNA1 gene or POU4F3 gene;

the disease caused by a human autosomal dominant mutation is hypertrophic cardiomyopathy, and the targeted dominant mutant gene thereof is TNNT2 gene;

the disease caused by a human autosomal dominant mutation is familial hypertrophic cardiomyopathy, and the targeted dominant mutant gene thereof is MYBPC3 gene;

the disease caused by a human autosomal dominant mutation is apical hypertrophic cardiomyopathy, and the targeted dominant mutant gene thereof is TNNI3 gene;

the disease caused by a human autosomal dominant mutation is Charcot-Marie-Tooth disease type 1A, and the targeted dominant mutant gene thereof is PMP22 gene;

the disease caused by a human autosomal dominant mutation is Charcot-Marie-Tooth disease type 1B, and the targeted dominant mutant gene thereof is MPZ gene;

the disease caused by a human autosomal dominant mutation is long QT syndrome, and the targeted dominant mutant gene thereof is KCNQ1 gene, KCNH2 gene, SCN5A gene, ANK2 gene, KCNE1 gene, KCNE2 gene, KCNJ2 gene, CAV3 gene, SCN48 gene, AKAP9 gene, or ANTA1 gene;

the disease caused by a human autosomal dominant mutation is short QT syndrome, and the targeted dominant mutant gene thereof is KCNH2 gene or KCNJ2 gene;

the disease caused by a human autosomal dominant mutation is Brugada syndrome, and the targeted dominant mutant gene thereof is SCN5A gene, GPD1L gene, CACNA1C gene, CACNB2B gene, or SCN1B gene;

the disease caused by a human autosomal dominant mutation is catecholaminergic polymorphic ventricular tachycardia, and the targeted dominant mutant gene thereof is RYR2 gene;

the disease caused by a human autosomal dominant mutation is cardiac conduction disorder, and the targeted dominant mutant gene thereof is SCN5A gene or SCN1B gene;

the disease caused by a human autosomal dominant mutation is amyotrophic lateral sclerosis, and the targeted dominant mutant gene thereof is TDP43 gene;

the disease caused by a human autosomal dominant mutation is Noonan syndrome, and the targeted dominant mutant gene thereof is PTPN11 gene;

or the disease caused by a human autosomal dominant mutation is hypocalcemia, and the targeted dominant mutant gene thereof is CaR gene.

17. The suppressing agent according to claim 8, wherein the disease is myotonic dystrophy, and the targeted dominant mutant gene thereof is DMPK gene;

the disease is spinal muscular atrophy, and the targeted dominant mutant gene thereof is SMN1 gene;

the disease is congenital myasthenic syndrome, and the targeted dominant mutant gene thereof is CHRNE gene;

the disease is frontotemporal dementia, and the targeted dominant mutant gene thereof is MAPT gene;

or the disease is isolated growth hormone deficiency type II, and the targeted dominant mutant gene thereof is GH1 gene.

18. A pharmaceutical composition comprising at least one suppressing agent according to any one of claims 1 to 17 as an active ingredient.

19. The pharmaceutical composition according to claim 18 dependent on claim 11 or 12, further comprising, as an active ingredient:

an RNAi molecule whose sense strand region consists of a nucleotide represented by SEQ ID NO;

83 or 85, and/or an expression vector comprising an operably linked DNA encoding the nucleotide; and

/or an RNAi molecule whose sense strand region consists of a nucleotide represented by SEQ ID NO;

89, and/or an expression vector comprising an operably linked DNA encoding the RNAi molecule.

2. An agent for suppressing the expression of a dominant mutant gene, comprising an expression vector comprising an operably linked DNA encoding an RNAi molecule with an ASP score of 0.4 or higher as an active ingredient, wherein

the ASP score is calculated according to the following equation:

ASP score=[(relative ratio of a normalized expression level of a normal gene treated with the RNAi molecule to a normalized expression level of the normal gene treated with a control RNAi molecule)−

(relative ratio of a normalized expression level of a mutant gene treated with the RNAi molecule to a normalized expression level of the mutant gene treated with the control RNAi molecule)]×

(1−

the relative ratio of the normalized expression level of the mutant gene treated with the RNAi molecule to the normalized expression level of the mutant gene treated with the control RNAi molecule)

wherein the control RNAi molecule represents an RNAi molecule that does not influence the expression of the normal gene and the mutant gene, and

the RNAi molecule comprises;

an RNAi sense strand region comprising at least one point of discontinuity to be generated on a transcript of the targeted dominant mutant gene and a nucleotide sequence identical to a sequence of 16 to 30 consecutive bases of the transcript; and

an RNAi antisense strand region comprising a nucleotide sequence complementary thereto, wherein

any one of the 4th to 15th bases downstream from a base 3′

flanking any one point of discontinuity on the RNAi sense strand region constitutes the 3′

terminal base of the RNAi sense strand region.

20. An agent for suppressing the expression of a point mutant EGFR gene, comprising an RNAi molecule whose sense strand region consists of a nucleotide represented by SEQ ID NO:

89, and/or an expression vector comprising an operably linked DNA encoding the RNAi molecule.

21. A method for designing an RNAi molecule selectively suppressing the expression of a dominant mutant gene generating a point of discontinuity on a transcript, the method comprising the steps of:

(a) selecting bases 5′ and

3′

flanking the point of discontinuity on the transcript as first and second reference bases, respectively;

(b) selecting the 3′

terminal base of an RNAi sense strand such that the 3′

-terminal base corresponds to any one of the 4th to 15th bases downstream from the base corresponding to the second reference base on the transcript;

(c) selecting a nucleotide sequence as an RNAi sense strand region, the nucleotide sequence comprising 16 to 30 consecutive bases comprising the first and second reference bases in the transcript from the dominant mutant gene; and

(d) selecting a nucleotide sequence as an RNAi antisense strand region, the nucleotide sequence comprising a nucleotide sequence complementary to the selected nucleotide sequence of the RNAi sense strand region.

22. The design method according to claim 21, further comprising the step of (e) screening for an RNAi molecule with an ASP score of 0.4 or higher, wherein

the ASP score is calculated according to the following equation:

ASP score=[(relative ratio of a normalized expression level of a normal gene treated with the RNAi molecule to a normalized expression level of the normal gene treated with a control RNAi molecule)−

(relative ratio of a normalized expression level of a mutant gene treated with the RNAi molecule to a normalized expression level of the mutant gene treated with the control RNAi molecule)]×

(1−

the relative ratio of the normalized expression level of the mutant gene treated with the RNAi molecule to the normalized expression level of the mutant gene treated with the control RNAi molecule)

wherein the control RNAi molecule represents an RNAi molecule that does not influence the expression of the normal gene and the mutant gene.

23. The design method according to claim 21 or 22, wherein TT or UU is further added to each of the 3′

ends of the RNAi sense strand region and the RNAi antisense strand region.