TYROSINASE ANTISENSE OLIGONUCLEOTIDES

Abstract

Provided are peptide nucleic acid derivatives targeting a 3′ splice site of the human tyrosinase pre-mRNA. The peptide nucleic acid derivatives potently induce a splice variant of the human tyrosinase mRNA in cells, and are useful to safely treat dermatological indications or conditions involving the human tyrosinase protein upon topical administration.

Claims

1. A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof: ##STR00014## wherein, n is an integer between 10 and 21; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA, or partially complementary to the human tyrosinase pre-mRNA with one or two mismatches from the entire compound of Formula I; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n independently represent deuterido, hydrido, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical; X and Y independently represent deuterido, hydrido [H], formyl [H—C(═O)—], aminocarbonyl [NH.sub.2—C(═O)—], aminothiocarbonyl [NH.sub.2—C(═S)—], substituted or non-substituted alkyl, substituted or non-substituted aryl, substituted or non-substituted alkylacyl, substituted or non-substituted arylacyl, substituted or non-substituted alkyloxycarbonyl, substituted or non-substituted aryloxycarbonyl, substituted or non-substituted alkylaminocarbonyl, substituted or non-substituted arylaminocarbonyl, substituted or non-substituted alkylaminothiocarbonyl, substituted or non-substituted arylaminothiocarbonyl, substituted or non-substituted alkyloxythiocarbonyl, substituted or non-substituted aryloxythiocarbonyl, substituted or non-substituted alkylsulfonyl, substituted or non-substituted arylsulfonyl, substituted or non-substituted alkylphosphonyl radical, or substituted or non-substituted arylphosphonyl radical; Z represents hydrido, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted amino, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and, at least four of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases with a substituted or non-substituted amino radical covalently linked to the nucleobase moiety.

2. The peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof: wherein, n is an integer between 10 and 21; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA, or partially complementary to the human tyrosinase pre-mRNA with one or two mismatches from the entire compound of Formula I; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n independently represent deuterido or hydrido radical; X and Y independently represent deuterido, hydrido, formyl, aminocarbonyl, aminothiocarbonyl, substituted or non-substituted alkyl, substituted or non-substituted aryl, substituted or non-substituted alkylacyl, substituted or non-substituted arylacyl, substituted or non-substituted alkyloxycarbonyl, substituted or non-substituted aryloxycarbonyl, substituted or non-substituted alkylaminocarbonyl, substituted or non-substituted aryl aminocarbonyl, substituted or non-substituted alkylaminothiocarbonyl, substituted or non-substituted arylaminothiocarbonyl, substituted or non-substituted alkyloxythiocarbonyl, substituted or non-substituted aryloxythiocarbonyl, substituted or non-substituted alkylsulfonyl, substituted or non-substituted arylsulfonyl, substituted or non-substituted alkylphosphonyl radical, or substituted or non-substituted arylphosphonyl radical; Z represents hydrido, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, or substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least four of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV: ##STR00015## wherein, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently selected from hydrido, and substituted or non-substituted alkyl radical; L.sub.1, L.sub.2 and L.sub.3 are a covalent linker represented by Formula V covalently linking the basic amino group to the nucleobase moiety: ##STR00016## wherein, Q.sub.1 and Q.sub.m are substituted or non-substituted methylene (—CH.sub.2—) radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m−1 are independently selected from substituted or non-substituted methylene, oxygen (—O—), sulfur (—S—), and substituted or non-substituted amino radical [—N(H)—, or —N(substituent)-]; and, m is an integer between 1 and 15.

3. The peptide nucleic acid derivative according to claim 2, or a pharmaceutically acceptable salt thereof: wherein, n is an integer between 11 and 18; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n are hydrido radical; X and Y independently represent hydrido, substituted or non-substituted alkyl, substituted or non-substituted aryl, substituted or non-substituted alkylacyl, substituted or non-substituted arylacyl, substituted or non-substituted alkyloxycarbonyl, or substituted or non-substituted aryloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least four of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently selected from hydrido, and substituted or non-substituted alkyl radical; Q.sub.1 and Q.sub.m are substituted or non-substituted methylene radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m−1 are independently selected from substituted or non-substituted methylene, oxygen, and amino radical; and, m is an integer between 1 and 11.

4. The peptide nucleic acid derivative according to claim 3, or a pharmaceutically acceptable salt thereof: wherein, n is an integer between 11 and 16; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n are hydrido radical; X and Y independently selected from hydrido, substituted or non-substituted alkylacyl, or substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least four of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently selected from hydrido, and substituted or non-substituted alkyl radical; Q.sub.1 and Q.sub.m are methylene radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m−1 are independently selected from methylene, oxygen, and amino radical; and, m is an integer between 1 and 9.

5. The peptide nucleic acid derivative according to claim 4, or a pharmaceutically acceptable salt thereof: wherein, n is an integer between 11 and 16; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n are hydrido radical; X and Y independently selected from hydrido, substituted or non-substituted alkylacyl, or substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; at least four of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.3, and R.sub.5 are hydrido radical, and R.sub.2, R.sub.4, and R.sub.6 independently represent hydrido, or substituted or non-substituted alkyl radical; Q.sub.1 and Q.sub.m are methylene radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m−1 are independently selected from methylene, oxygen radical; and, m is an integer between 1 and 9.

6. The peptide nucleic acid derivative according to claim 5, or a pharmaceutically acceptable salt thereof: wherein, n is an integer between 11 and 16; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n are hydrido radical; X and Y independently selected from hydrido, substituted or non-substituted alkylacyl, or substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from adenine, thymine, guanine, cytosine, and unnatural nucleobases; at least five of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.2 , R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are hydrido radical; Q.sub.1 and Q.sub.m are methylene radical, and Q.sub.m is directly linked to the basic amino group; Q.sub.2, Q.sub.3, . . . , and Q.sub.m−1 are independently selected from methylene, and oxygen radical; and, m is an integer between 1 and 9.

7. The peptide nucleic acid derivative according to claim 6, or a pharmaceutically acceptable salt thereof: wherein, n is an integer between 11 and 16; the compound of Formula I possesses at least a 10-mer complementary overlap with the 14-mer pre-mRNA sequence of [(5′.fwdarw.3′) UGUACAGAUUGUCU] in the human tyrosinase pre-mRNA; the compound of Formula I is fully complementary to the human tyrosinase pre-mRNA; S.sub.1, S.sub.2, . . . , S.sub.n−1, S.sub.n, T.sub.1, T.sub.2, . . . , T.sub.n−1, and T.sub.n are hydrido radical; X is hydrido radical; Y represents substituted or non-substituted alkylacyl, or substituted or non-substituted alkyloxycarbonyl radical; Z represents substituted or non-substituted amino radical; B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from adenine, thymine, guanine, cytosine, and unnatural nucleobases; at least five of B.sub.1, B.sub.2, . . . , B.sub.n−1, and B.sub.n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are hydrido radical; L.sub.1 represents —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—, —CH.sub.2—O—(CH.sub.2).sub.2—, —CH.sub.2—O—(CH.sub.2).sub.3—, —CH.sub.2—O—(CH.sub.2).sub.4—, or —CH.sub.2—O—(CH.sub.2).sub.5— with the right end is directly linked to the basic amino group; and, L.sub.2 and L.sub.3 are independently selected from —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—, —(CH.sub.2).sub.3—O—(CH.sub.2).sub.2—, —(CH.sub.2).sub.2—O—(CH.sub.2).sub.3—, —(CH.sub.2).sub.2—, —(CH.sub.2).sub.3—, —(CH.sub.2).sub.4—, —(CH.sub.2).sub.5—, —(CH.sub.2).sub.6—, —(CH.sub.2).sub.7—, and —(CH.sub.2).sub.8— with the right end is directly linked to the basic amino group.

8. The peptide nucleic acid derivative according to claim 1, which is selected from the group of peptide nucleic acid derivatives provided below, or a pharmaceutically acceptable salt thereof: TABLE-US-00014 (N.fwdarw.C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Fethoc-AC(l/2)A(5)-GA(5)C-AA(5)T-CTG(6)-C(1/2)C-NH.sub.2; (N.fwdarw.C) Ac-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Benzoyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Piv-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Methyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) n-Propyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Fmoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) p-Toluenesulfonyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) H-Lys-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Fethoc-Lys-Gly-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-Lys-NH.sub.2; (N.fwdarw.C) Fethoc-Val-Gly-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Ac-Arg-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Benzyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH2; (N.fwdarw.C) Phenyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) [N-(2-Phenylethyl)amino]carbonyl-CA(5)G-ACA(5)-ATC(1O2)- TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Benzoyl-Leu-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Piv-Lys-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-Val-NH.sub.2; (N.fwdarw.C) Fethoc-CA(7)G-AC(2O2)A-A(4)TC(1O2)-TG(6)T-A-NH.sub.2; (N.fwdarw.C) Fethoc-CTG(6)-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Fethoc-CA(5)G-ATA(5)-ATC(1O2)-TG(5)T-A(5)-NH.sub.2; (N.fwdarw.C) Fethoc-TA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH.sub.2; (N.fwdarw.C) Fethoc-TCA(3)-GAC(1O5)-A(5)AT-C(1O2)TG(5)-TA(5)-NH.sub.2; (N.fwdarw.C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)C-NH.sub.2; (N.fwdarw.C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(6)TA(5)-C-NH.sub.2; (N.fwdarw.C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(6)TA(5)-CA(2O2)A-NH.sub.2; (N.fwdarw.C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C(1O2)-AA(6)-NH.sub.2; (N.fwdarw.C) Fethoc-AC(1O3)T-GA(6)C-TA(6)T-CTG(6)-TAC(1O5)-A(4)A-NH.sub.2; (N.fwdarw.C) Fethoc-GA(6)C-AA(6)T-CTG(6)-TA(6)C(1O2)-AA(6)-NH.sub.2; (N.fwdarw.C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C-A(5)AA(2O2)-A-NH.sub.2; (N.fwdarw.C) Fethoc-AA(6)T-CTG(6)-T A(6)C-A(5) AA(2O2)-A-NH.sub.2; (N.fwdarw.C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C-A(5)AA(2O3)-A-NH.sub.2; (N.fwdarw.C) Fethoc-GC(1O4)T-AC(1O2)A-GA(4)C-AAT-CTG(6)-TA(6)C-NH.sub.2; (N.fwdarw.C) Fethoc-GC(1O2)T-A(3)CA-GA(4)C-AAT-C(1O2)TG-NH, (N.fwdarw.C) Fethoc-GC(1O2)T-A(3)CA-G(2O2)AC-A(5)AT-C(1O2)TG-NH.sub.2;  and (N.fwdarw.C) Fethoc-GC(1O2)T-A()CA-GA(4)C-A(202)AT-C(1O2)TG-NH.sub.2: wherein, A, G, T, and C are PNA monomers with a natural nucleobase of adenine, guanine, thymine, and cytosine, respectively; C(pOq), A(p), A(pOq), G(p), and G(pOq) are PNA monomers with an unnatural nucleobase represented by Formula VI, Formula VII, Formula VIII, Formula IX, and Formula X, respectively; ##STR00017## wherein, p and q are integers; and, the abbreviations for the N- and C-terminus substituents are as specifically described as follows: “Fmoc-” is the abbreviation for “[(9-fluorenyl)methyloxy]carbonyl-”; “Fethoc-” for “[2-(9-fluorenyl)ethyl-1-oxy]carbonyl”; “Ac-” for “acetyl-”; “Benzoyl-” for “benzenecabonyl-”; “Piv-” for “pivalyl-”; “Methyl-” for “methyl-”; “n-Propyl-” for “1-(n-propyl)-”; “H-” for “hydrido-” group; “p-Toluenesulfonyl” for “(4-methylbenzene)-1-sulfonyl-”; “-Lys-” for amino acid residue “lysine”; “-Val-” for amino acid residue “valine”; “-Leu-” for amino acid residue “leucine”; “-Arg-” for amino acid residue “arginine”; “-Gly-” for amino acid residue “glycine”; “[N-(2-Phenylethyl)amino]carbonyl-” for “[N-1-(2-phenylethyl)amino]carbonyl-”; “Benzyl-” for “1-(phenyl)methyl-”; “Phenyl-” for “phenyl-”; “Me-” for “methyl-”; and “—NH.sub.2” for non-subsituted “-amino” group.

9. A pharmaceutical composition for treating diseases or conditions involving the expression of the human tyrosinase gene, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition for treating hyper-pigmentation, comprising the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

11. A method to treat diseases or conditions involving the expression of the human tyrosinase gene, comprising the administration of the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

12. A method to treat hyper-pigmentation, comprising the administration of the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof.

13. A use of the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treatment of diseases or conditions involving the expression of the human tyrosinase gene.

14. A use of the peptide nucleic acid derivative according to claim 1, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treatment of hyper-pigmentation.

Description

BRIEF EXPLANATION OF DRAWINGS

[0168] FIGS. 1a-1c. Examples of natural or unnatural (modified) nucleobases selectable for the peptide nucleic acid derivative of Formula I.

[0169] FIGS. 2a-2e. Examples of substituents selectable for the peptide nucleic acid derivative of Formula I.

[0170] FIG. 3. Chemical structures of PNA monomers with natural or modified nucleobase.

[0171] FIG. 4. Chemical structures for abbreviations of N- or C-terminus substituents.

[0172] FIG. 5a. Chemical structure of 13-mer PNA derivative “(N.fwdarw.C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)17-A(5)-NH.sub.2”.

[0173] FIG. 5b. Chemical structure of 15-mer PNA derivative “(N.fwdarw.C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(3O2)TA(5)-CA(2O2)A-NH.sub.2”.

[0174] FIG. 6. Chemical structures of Fmoc-PNA monomers used to synthesize the PNA derivatives of the present invention,

[0175] FIGS. 7a-7b. C.sub.18-reverse phase HPLC chromatograms of “ASO 1” before and after HPLC purification, respectively.

[0176] FIG. 8. ESI-TOF mass spectrum of “ASO 1” purified by C.sub.18-RP prep HPLC.

[0177] FIG. 9a. Electrophoretic analysis of the nested PCR products in B16F10 mouse melanoma cells treated with 0 (negative control), 1, 10 or 1,000 aM “ASO 1M”.

[0178] FIG. 9b. Sanger sequencing data for the PCR product assignable to the skipping of exons (2-3).

[0179] FIG. 10a. Changes in the full-length TYR mRNA level by qPCR B16F10 mouse melanoma cells treated with “ASO 1M” at 0 (negative control), 1, 10. 100 or 1,000 aM. (error bar by standard error)

[0180] FIG. 10b. TYR western blot data in B16F10 mouse melanonia cells treated for 24 hours with “ASO 1M” at 0 zM (negative control), 10 zM, 100 zM, 1 aM, or 10 aM (i.e. 10,000 zM).

[0181] FIG. 11. Changes in the melanin content in B16F10 mouse melanoma cells treated either with “ASO 1M” at 1, 10, 100 or 1,000 aM, or with 10 μg/mL, or 100 μg/mL arbutin. (error bar by standard error)

[0182] FIG. 12. Changes in the full-length TYR mRNA level by qPCR in human primary epithelial melanocytes treated with “ASO 1” at 0 zM (negative control), 1 zM, 100 zM or 10 aM. (error bar by standard error)

[0183] FIG. 13. Schematic Representation of Pre-mRNA Structure.

[0184] FIG. 14. Splicing of Pre-mRNA.

[0185] FIG. 15. Schematic representation of 3′ splice site and 5′ splice site.

BEST MODE FOR CARRYING OUT THE INVENTION

[0186] General Procedures for Preparation of PNA Oligomers

[0187] PNA oligomers were synthesized by solid phase peptide synthesis (SPPS) based on Fmoc-chemistry according to the method disclosed in the prior art [U.S. Pat. No. 6,133,444; WO96/40685] with minor but due modifications. The solid support employed in this study was H-Rink Amide-ChemMatrix purchased from PCAS BioMatrix Inc. (Quebec, Canada), Fmoc-PNA monomers with a modified nucleobase were synthesized as described in the prior art [PCT/KR 2009/001256] or with minor modifications. Such Fmoc-PNA monomers with a modified nucleobase and Fmoc-PNA monomers with a naturally occurring nucleobase were used to synthesize the PNA derivatives of the present invention. PNA oligomers were purified by C.sub.18-reverse phase HPLC (watre/acetonitrile or water/methanol with 0.1% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

[0188] Scheme 2 illustrates a typical monomer elongation cycle adopted in the SPPS of this study, and the synthetic details are provided as below. To a skilled person in the field, however, there are lots of minor variations obviously possible in effectively running such SPPS reactions on an automatic peptide synthesizer or manual peptide synthesizer. Each reaction step in Scheme 2 is briefly provided as follows.

##STR00012##

[0189] [Activation of H-Rink-ChemMatrix Resin] 0.01 mmol (ca 20 mg resin) of the ChemMatrix resin in 1.5 mL 20% piperidine/DMF was vortexed in a fibra tube for 20 min, and the DeFmoc solution was filtered off. The resin was washed for 30 sec each in series with 1.5 mL methylene chloride (MC), 1.5 mL dimethylformamide (DMF), 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC. The resulting free amines on the solid support were subjected to coupling either with an Fmoc-PNA monomer or with an Finoc-protected amino acid derivative.

[0190] [DeFmoc] The resin was vortexed in 1.5 mL 20% piperidine/DMF for 7 min, and the DeFmoc solution was filtered off. The resin was washed for 30 sec each in series with 1.5 mL MC, 1.5 mL DMF, 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC. The resulting free amines on the solid support were immediately subjected to coupling with an Fmoc-PNA monomer.

[0191] [Coupling with Fmoc-PNA Monomer] The free amines on the solid support were coupled with an Fmoc-PNA monomer as follows. 0.04 mmol of PNA monomer, 0.05 mmol HBTU, and 10 mmol DIEA were incubated for 2 min in 1 mL anhydrous DMF, and added to the resin with free amines. The resin solution was vortexed for 1 hour and the reaction medium was filtered off. Then the resin was washed for 30 sec each in series with 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC. The chemical structures of Fmoc-PNA monomers with a modified nucleobase used in the present invention are provided in FIG. 6. The Fmoc-PNA monomers with a modified nucleobase are provided in FIG. 6 should be taken as examples, and therefore should not be taken to limit the scope of the present invention. A skilled person in the field may easily figure out a number of variations in Fmoc-PNA monomers to synthesize the PNA derivative of Formula I.

[0192] [Capping] Following the coupling reaction, the unreacted free amines were capped by shaking for 5 min in 1.5 mL capping solution (5% acetic anhydride and 6% 2,6-leutidine in DMF). Then the capping solution was filtered off and washed for 30 sec each in series with 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC.

[0193] [Introduction of “Fethoc-” Radical in N-Terminus] “Fethoc-” radical was introduced to the N-terminus by reacting the free amine on the resin with “Fethoc-OSu” under basic coupling conditions. The chemical structure of “Fethoc-OSu” [CAS No. 179337-69-0, C.sub.20H.sub.17NO.sub.5, MW 351.36] is provided as follows.

##STR00013##

[0194] [Cleavage from Resin] PNA oligomers bound to the resin were cleaved from the resin by shaking for 3 hours in 1.5 mL cleavage solution (2.5% tri-isopropylsilane and 2.5% water in trifluoroacetic acid). The resin was filtered off and the filtrate was concentrated under reduced pressure. The resulting residue was triturated with diethylether and the resulting precipitate was collected by filtration for purification by reverse phase HPLC. [HPLC Analysis and Purification] Following a cleavage from resin, the crude product of a PNA derivative was purified by C.sub.18-reverse phase HPLC eluting water/acetonitrile or water/methanol (gradient method) containing 0.1% TFA. FIGS. 7a and 7b are exemplary HPLC chromatograms for “ASO 1” before and after HPLC purification, respectively. The oligomer sequence of “ASO 1” is as provided in Table 1.

[0195] Synthetic Examples for PNA Derivative of Formula I

[0196] In order to complementarily target the 3′ splice site of “exon 2” in the human TYR pre-mRNA, PNA derivatives of the present invention were prepared according to the synthetic procedures provided above or with minor modifications. Provision of such PNA derivatives targeting the human TYR pre-mRNA is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

TABLE-US-00005 TABLE 1 PXA derivatives complementarily targeting the 3′ splice site of “exon 2” in the human TYR pre-pRNA along with stuctural characterization data by mass speetometry PNA Exact Mass, m/z Example PSA sequence (N.fwdarw.C) theor..sup.a obs..sup.b ASO 1 Fathoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)- 4258.96 4260.99 NH.sub.2 ASO 2 Fethoc-AC(12)A-GA(5)C-AA(5)T-CTG(6)-TA)5)C 5532.55 5532.54 (1O2)-AA(5)NH.sub.2 ASO 3 Fethoc-AC(1O2)A(5)-GA(5)C-AA(5)T-CTG(6)-C 4592.11 4592.11 (1O2)C-NH.sub.2 .sup.a)theoretical exact mass, .sup.b)observed exact mass

[0197] Table 1 provides PNA derivatives complementarily targeting the 3′ splice site of “exon 2” in the human TYR pre-mRNA read out from the human TYR gene [NCBI Reference Sequence: NG_0008748] along with structural characterization data by mass spectrometry. Provision of the TYR ASOs in Table 1 is to exemplify the PNA derivatives of Formula I, and should not be interpreted to limit the scope of the present invention.

[0198] “ASO 1” has a 13-mer complementary overlap with the 13-mer sequence marked “bold” and “underlined” within the 30-mer RNA sequence of

TABLE-US-00006 [(5′.fwdarw.3′)ggguguuuuguacag | AUUGU-CUGUAGCCGA]
spanning the junction of “intron 1” and “exon 2” in the human TYR pre-mRNA. Thus “ASO 1” possesses a 5-mer overlap with “intron 1” and a 8-mer overlap with “exon 2” within the human TYR pre-mRNA.

[0199] Table 2 provides “ASO 1M” complementarily targeting the 3′ splice site of “exon 2” in the mouse TYR pre-mRNA read out from the mouse genomic DNA [accessed from NCBI Reference Sequence: NC_000073] along with structural characterization data by mass spectrometry. Provision of “ASO 1M” is to evaluate the antisense activity in cells of mouse origin.

TABLE-US-00007 TABLE 2 PXA derivatives complementanly targeting the 3′ splice site of “exon 2” in the mouse TYR pre-mRNA along with structural  characterization data by mass spectrometry. PNA Exact Mass, m/z Example PSA Sequence (N.fwdarw.C) theor..sup.a obs..sup.b ASO 1M Fethoc-CA(5)A-A(5)TG-A(5) 4289.95 4289.96 TC(1O2)-TG(6)T-G-NH.sub.2 .sup.a)theoretical exact mass, .sup.b)observed exact mass

[0200] A 30-mer RNA sequence of [(5′.fwdarw.3′) aauuguuuuucacag|AUCAUUUGUAGCAGA] spanning the junction of intron 1 and exon 2 in the mouse TYR pre-mRNA was read out from the mouse TYR gene [NCBI Gene Accession Number: NC_000073].

[0201] “ASO 1M” is equivalent to the DNA sequence of “(5′.fwdarw.3′) CAA-ATG-ATC-TGT-G” for complementary binding to the pre-mRNA. “ASO 1M” has a 13-mer complementary overlap with the 13-mer sequence marked “bold” and “underlined” within the 30-mer RNA sequence of

TABLE-US-00008 [(5′.fwdarw.3′) aauuguuuuucacag | AUCAUUUGUAGCAGA]
spanning the junction of “intron 1” and “exon 2” in the mouse TYR pre-mRNA.

[0202] Like “ASO 1” against the human TYR pre-mRNA, “ASO 1M” possesses a 5-mer overlap with “intron 1” and a 8-mer overlap with “exon 2” within the mouse TYR pre-mRNA. “ASO 1M” may serve as a good surrogate compound for the antisense activity of “ASO 1” in cells of human origin.

[0203] FIG. 7a is a HPLC chromatogram obtained with a crude product of “ASO 1”. The crude product was purified by C.sub.18-RP preparatory HPLC. FIG. 7b is a HPLC chromatogram for a purified product of “ASO 1”. The purity of “ASO 1” improved markedly following the preparatory HPLC purification. FIG. 8 provides a ESI-TOF mass spectrum obtained with the purified product of “ASO 1”. Provision of the analysis data for “ASO 1” is to illustrate how the PNA derivatives of Formula I were purified and identified in the present invention, and should not be interpreted to limit the scope of the present invention.

[0204] Binding Affinity of Model PNA Derivatives for Complementary DNA

[0205] The PNA derivatives in Tables 1 and 2 were evaluated for their binding affinity for 10-mer DNAs complementarily targeting either the N-terminal or C-terminal. The binding affinity was assessed by T.sub.m value for the duplex between PNA and 10-mer complementary DNA. The duplex between PNA derivatives and fully complementary DNAs show T.sub.m values too high to be reliably determined in aqueous buffer solution, since the buffer solution tends to boil during the T.sub.m measurement.

[0206] T.sub.m values were determined on a UV/Vis spectrometer as follows. A mixed solution of 4 μM PNA oligomer and 4 μM complementary 10-mer DNA in 4 mL aqueous buffer (pH 7,16, 10 mM sodium phosphate, 100 mM NaCl) in 15 mL polypropylene falcon tube was incubated at 90° C. for a minute and slowly cooled down to ambient temperature. Then the solution was transferred into a 3 mL quartz UV cuvette equipped with an air-tight cap, and subjected to a T.sub.m measurement at 260 nm on a UV/Visible spectrophotometer as described in the prior art [PCT/KR2009/001256] or with minor modifications. The 10-mer complementary DNAs for T.sub.m measurement were purchased from Bioneer (www.bioneer.com. Dajeon, Republic of Korea) and used without further purification.

[0207] Observed T.sub.m values of the PNA derivatives of Formula I are very high for a complementary binding to 10-mer DNA, and provided in Table 3. For example, “ASO 1” showed a T.sub.m value of 78.0° C. for the duplex with the 10-mer complementary DNA targeting the N-terminal 10-mer in the PNA as marked “bold” and “underlined” in

TABLE-US-00009 [(N.fwdarw.C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T- A(5)-NH.sub.2].
In the meantime, “ASO 1” showed a T.sub.m of 73.0° C. for the duplex with the 10-mer complementary DNA targeting the C-terminal 10-mer in the PNA as marked “bold” and “underlined” in

TABLE-US-00010 [(N.fwdarw.C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6) T-A(5)-NH.sub.2].

TABLE-US-00011 TABLE 3 T.sub.m values between PNAs and 10-mer complementary DNA targeting either the N-terminal or the C-terminal of PNA. T.sub.m Value, ° C. 10-mer DNA 10-mer DNA PNA against N-Terminal against C-Terminal ASO 1 78.0 73.0 ASO 1M 72.0 72.0

[0208] Examples for Biological Activities of PNA Derivatives of Formula I

[0209] PNA derivatives in the present invention were evaluated for in vitro TYR antisense activities in B16F10 mouse melanoma cells and human melanocytes. The biological examples were provided as examples to illustrate the biological profiles of the PNA derivatives of Formula I, and therefore should not be interpreted to limit the scope of the current invention.

EXAMPLE 1

Exon Skipping Induced by “ASO 1M”

[0210] “ASO 1M” specified in Table 2 has a 13-mer complementary overlap with the 13-mer sequence marked “bold” and “underlined” within the 30-mer RNA sequence of

TABLE-US-00012 [(5′.fwdarw.3′) aauuguuuuucacag | AUCAUUUGUAGCAGA]
spanning the junction of “intron 1” and “exon 2” in the mouse TYR pre-mRNA. In the meantime, “ASO 1M” possesses a 9-mer complementary along with 4 mismatches as marked “bold” and “underlined” within the 30-mer RNA sequence of [(5′.fwdarw.3′) ggguguuuug“u”acag|AU“UG”U“C”UGUAGCCGA] spanning the junction of “intron 1” and “exon 2” in the human TYR pre-mRNA. The 4 mismatches were marked with quote sign (“ ”).

[0211] “ASO 1M” was evaluated for its ability to induce the skipping of TYR “exon 2” in mouse B16F10 tnelanoma cells, even though “ASO 1M” possesses 4 mismatches with the 3′ splice site of “exon 2” in the human TYR pre-mRNA. “ASO 1M”, which is fully complementary to the mouse TYR pre-mRNA, may serve as a good surrogate compound for “ASO 1” which is fully complementary to the human TYR pre-mRNA.

[0212] [Cell Culture & ASO Treatment] B16F10 mouse melanoma cells (Cat. Number CRL-6475, ATCC) were maintained in DMEM (Dulbecco's modified Eagle's essential minimum medium) supplemented with 10% FBS, 1% streptomycin/penicillin, and 0.01 mg/ml bovine insulin. B16F10 cells grown in 60 mm culture dish containing 5 mL DMEM were incubated for 5 hours with “ASO 1M” at 0 (negative control), 1, 10,100 or 1000 aM.

[0213] [RNA Extraction & cDNA Synthesis by One-step PCR] Following an incubation for 5 hours, total RNA was extracted using “Universal RNA Extraction Kit” (Cat. Number 9767, Takara) according to the manufacturer's instructions. 200 ng of RNA template was used for a 25 μl reverse transcription reaction using Super Script® One-Step RT-PCR kit with Platinum® Taq polymerase (Cat. No. 10928-042, Invitrogen) against a set of exon-specific primers of [exon1_forward: (5′.fwdarw.3′) GTAAGTTTGGATTTGGGG; and exon 4_reverse: (5′.fwdarw.3′) AGAGC-GGTATGAAAGGAA] according to the following cycle conditions: 50° C. for 30 min and 94° C. for 2 min, followed by 15 cycles of 30 sec at 94° C. 30 sec at 52° C. and 40 sec at 72° C.

[0214] [Nested PCR Amplification] 1 μl of cDNA was further amplified in a 20 μl nested PCR reaction (Cat. No. K2612, Bioneer) against a set of primers of [exon 1n_forward: (5′.fwdarw.3′) GAGAACTAACTGGGGATGA; and exon 4n_reverse: (5′.fwdarw.3′) CGATAGGTGCATTGGCTT] according to the cycle conditions specified as follows: 95° C. for 5 min followed by 30 cycles of 30 sec at 95° C. 30 sec at 52° C. and 40 sec at 72° C.

[0215] [Identification of Exon Skipping Products] The PCR products were subjected to electrophoretic separation on a 2% agarose gel. The hands of target size were collected and analyzed by Sanger Sequencing.

[0216] FIG. 9a provides the electrophoresis data of the PCR products. The cells without “ASO 1M” treatment yielded two strong PCR bands, one for the full-length TYR mRNA and the other for the splice variant TYR mRNA lacking exons 2 and 3. Thus the skipping of exons 2-3 is considered to spontaneously occur. The cells treated with “ASO 1M” at 1 to 1,000 aM, however, yielded only the splice variant TYR mRNA lacking exons 2 and 3. Thus “ASO 1M” increases the propensity of the exons 2-3 skipping in B16F10 melanoma cells. The exon skipping PCR product was sequenced to he the skipping of exons 2-3 as shown in FIG. 9b.

EXAMPLE 2

qPCR for TYR mRNA in B16F10 Mouse Melanoma Cells Treated with “ASO 1M”

[0217] “ASO 1M” was evaluated by TYR nested qPCR for its ability to induce changes in the mouse TYR mRNA level in B16F10 cells as described below.

[0218] [Cell Culture & ASO Treatment] B16F10 cells grown in 60 mm culture dish containing 5 mL DMEM were treated with “ASO 1M” at 0 (negative control), 1, 10,100 or 1000 aM. (2 culture dishes per dose)

[0219] [RNA Extraction & cDNA Synthesis by One-step PCR] Following an incubation with “ASO 1M” for 5 hours, total RNA was extracted from cells using “Universal RNA Extraction Kit” (Cat. No. 9767, Takara) according to the manufacturer's instructions. 200 ng of RNA template were used for a 25 μl reverse transcription reaction using One Step RT-PCR kit (Invitrogen, USA) against a set of exon specific primers of [exon 1_forward: (5′.fwdarw.3′) GTAAGTTTGGATTTGGGG; and exon 4_reverse: (5′.fwdarw.3′) AGAGCGGTATGAAAGGAA] according to the following cycle conditions: 50° C. for 30 min and 94° C. for 2 min, followed by 15 cycles of 30 sec at 94° C., 30 sec at 52° C., and 40 sec at. 72° C.

[0220] [Nested qPCR Amplification] The cDNA solutions were diluted by 100 times, and 1 μl of each diluted PCR product was subjected to a 20 μl Real-Time PCR reaction with a Taqman probe set spanning the junction of exon 2 and exon 3 (Cat. No. Mm00495818_m1, Thermo Fisher Scientific) according to the following cycle conditions: 95° C. for 3 min followed by 30 cycles 10 sec at 95° C., and 30 sec at 60° C.

[0221] [Statistical Analysis] The nested gPCR experiment was repeated independently four times, and individual mRNA levels from each experiment were normalized against the mRNA level without “ASO 1M” treatment. The mRNA levels obtained from all the 4 separate experiments were pooled for statistical analysis by student's t-test. Thus the number of RNA samples is 8 per concentration.

[0222] FIG. 10a provides the pooled gPCR data, in which the full-length mRNA level significantly decreased by ca 40% in the cells treated with “ASO 1M” at 1 to 1,000 aM.

EXAMPLE 3

Inhibition of TYR Protein Expression in B16F10 Melanoma Cells by “ASO 1M”

[0223] “ASO 1M” was evaluated for its ability to down-regulate the TYR protein expression in B16F10 mouse melanoma cells as described below.

[0224] B16F10 cells grown in 60 mm culture dish containing 5 mL DMEM were treated with “ASO 1M” at 0 zM (negative control), 10 zM, 100 zM, 1 aM or 10 aM. 24 hours later, cells were washed 2 times with cold PBS (phosphate buffered saline), and then subjected to lysis with 200 μl 1× cell lysis butler (Cat. No. 9803, Cell Signaling Tech) supplemented with 1× protease inhibitors cocktail (Cat. No. P8340, Sigma). 200 μl of each lysate in 1.5 mL e-tube was mixed with 100 μl 5× sample buffer, and boiled at 100° C. for 5 min. 20 μl of the lysate was subjected to electrophoretic separation on a 4-15% gradient TGX gel (Cat No. 456-1086, Bio-Rad), and protein transfer on a 0.45 μm PVDF membrane. The membrane was probed with an anti-TYR antibody (Cat. No. 9319, Cell Signaling Tech) and anti-β-actin antibody (Cat. No. a3845, Sigma).

[0225] FIG. 10b provides the western blot data for the TYR protein expression in B16F10 cells. The band intensity of TYR expression was considerably weaker in the lysates with “ASO 1M” treatment than in the lysates without “ASO 1M” treatment, i.e. negative control. The TYR protein and mRNA level decreased comparably by the ASO treatment. (cf. “Example 2”)

EXAMPLE 4

Inhibition of Melanogenesis B16F10 Melanoma Cells by “ASO 1M”

[0226] “ASO 1M” was evaluated for its ability to inhibit the melanogenesis in B16F10 mouse melanoma cells as described below.

[0227] B16F10 cells sub-cultured in 60 mm culture dish containing 5 mL DMEM were treated with nothing (negative control), with “ASO 1M” at 1 to 1,000 aM, or with 10 or 100 μg/mL arbutin as the positive control. (2 culture dishes per dose) 24 hours later, cells were washed 2 times with cold PBS, and subjected to lysis with 200 μl 1N NaOH. Each lysate was collected in 1.5 mL e-tube, and kept overnight at room temperature. The melanin content in each lysate was determined by absorbance at 475 nm on an ELISA reader. The same experiment was repeated four times using cells at different passage. The four sets of melanin content data were pooled for statistical analysis by student's t-test against the melanin content without treatment (negative control).

[0228] FIG. 11 summarizes the changes of the melanin content in B16F 10 cells following a 24 hours incubation either with “ASO 1M” or with arbutin. The melanin content significantly decreased ca by 15% and 25% in the cells treated with 10 μg/mL and 100 μg/mL arbutin, respectively. In case of the cells treated with “ASO 1M”, the melanin content significantly decreased by ca 15% without much dose dependency. The inhibitory activity of “ASO 1M” was comparable to that of 10 μig/mL arbutin.

EXAMPLE 5

Preparation of Topical Cream Containing Compound of Formula I

[0229] A compound of Formula I, for example “ASO 2” was formulated as a cream for topical application to subjects. The topical cream was preparation as described below. Given that there are lots of variations of topical cream possible, this preparation should be taken as an example and should not be interpreted to limit the scope of the current invention.

[0230] [Preparation of Solution A] In a beaker, were mixed deionized water (196 g), EDTA disodium (0.06 g), glycerin (15 g), dipropylene glycol (30 g), phenoxyethanol (0.9 g), ethylhexylglycerin (0.9 g), hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer (2.4 g), and 1 nM aqueous solution of “ASO 2” (0.3 mL). The mixture was subjected to homogenization at 70˜75° C. using a homo mixer.

[0231] [Preparation of Solution B] In another beaker, were mixed at 70˜75° C. cetearyl ethylhexanoate (12 g), dimethicone (6 g), stearic acid/hydrogenated vegetable oil/behenyl alcohol/cetearyl alcohol (15 g), butyrospermum parkii (Shea) butter (9 g), and polyalyceryl-3-methylglucose distearate/glyceryl stearate SE/methyl glucose sesquistearate (6 g).

[0232] [Preparation of Topical Cream] “Solution A” and “Solution B” were mixed subjected to homogenization at 7,200 rpm and 70˜75° C. for 3 min using a homo mixer. The mixture was slowly cooled to room temperature to yield a topical cream containing 1 pM of “ASO 2”.

EXAMPLE 6

qPCR for TYR mRNA in Human Melanocytes Treated with “ASO 1”

[0233] “ASO 1” specified in Table 1 is a 13-mer TYR ASO fully complementary to the 3′ splice site spanning the junction of intron 1 and exon 2 in the human TYR as marked “bold” and “underlined” in the 30-mer human TYR pre-mRNA sequence of

TABLE-US-00013 [(5′.fwdarw.3′) ggguguuuuguacag | AUUGUCUGUAGCCGA]

[0234] “ASO 1” was evaluated by TYR nested qPCR for its ability to induce changes in the TYR mRNA level in human primary epidermal melanocytes as follows.

[0235] [Cell Culture & ASO Treatment] Primary epidermal melanocytes (Cat. Number PCS-200-013, ATCC) cells were maintained in Dermal Cell Basal Medium (Cat Number PCS-200-030, ATCC) supplemented with Adult Melanocyte Growth Kit Component (Cat. Number PCS-200-042, ATCC). Melanocytes grown in 60 mm culture dish containing 5 mL culture medium were treated with “ASO 1” at 0 zM (negative control), 1 zM, 100 zM, or 10 aM. (3 culture dishes per concentration)

[0236] [RNA Extraction & cDNA Synthesis by One-step PCR] Following an incubation with “ASO 1” for 5 hours, total RNA was extracted using “RNeasy Mini Kit” (Cat. Number 74106, Qiagen) according to the manufacturer's instructions. 200 ng of RNA template was subjected to a 25 μL reverse transcription reaction using Super Script® One-Step RT-PCR kit with Platinum® Taq polymerase (Cat. No. 10928-042, Invitrogen) against a set of exon-specific primers of [exon 1_forward(2): (5′.fwdarw.3′) CTCTTTGTCTGGATGCATT; and exon 5_reverse: (5′.fwdarw.3′) CTGTGGTAATCCTCTTTCT] according to the following cycle conditions specified: 50° C., for 30 min and 94° C. for 2 min, which was followed by 15 cycles of 30 sec at 94° C., 30 sec at 50° C., and 1 min at 72° C.

[0237] [Nested PCR Amplification] 1 μL of cDNA was further amplified in a 20 μL nested PCR reaction (Cat. No. K2612, Bioneer) against a set of exon-specific primers of [exon 2n_forward: (5′.fwdarw.3′) GATAAAGCTGCCAATTTC; and exon 3n_reverse: (5′.fwdarw.3′) TTGTGCATGCTGCTTTGA] against a Tagman probe [(5′.fwdarw.3′) 5,6-FAM-CACTGG-ZEN-AAGGATTTGCTAGTCCAC-3IABkFQ]. Cycle Conditions: 95° C. for 3 min followed by 40 cycles 10 sec at 95° C., and 30 sec at 60° C.

[0238] FIG. 12 provides the qPCR data, in which the full-length TYR mRNA level decreased by ca 30% in the human melanocytes treated with “ASO 1” at 1 zM to 10 aM. The observed decreases were significant (student's t-test) in the cells treated with “ASO 1” at 1 zM and 10 aM.