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US-12624068-B2 - Exon skipping by peptide nucleic acid derivatives

US12624068B2US 12624068 B2US12624068 B2US 12624068B2US-12624068-B2

Abstract

A peptide nucleic acid derivative of Formula I is provided to tightly bind to a splice site within a pre-mRNA in a sequence specific manner. Given with excellent cell membrane permeability and strong affinity for RNA, the peptide nucleic acid derivative induces exon skipping in cells treated with the peptide nucleic acid at sub-femtomolar concentration as “naked” oligonucleotide. The compound shows therapeutic activity in subjects upon systemic administration even at 1 μg/Kg or less, and therefore is useful to treat a disease or symptom at affordable treatment cost.

Inventors

  • Shin Chung
  • Daram Jung
  • Bongjun Cho
  • Kangwon Jang
  • Heungsik Yoon

Assignees

  • OLIPASS CORPORATION

Dates

Publication Date
20260512
Application Date
20171229

Claims (8)

  1. 1 . A peptide nucleic acid derivative represented by Formula I, or a pharmaceutically acceptable salt thereof, for inducing exon skipping by targeting intron/exon or exon/intron junction of pre-mRNA: wherein, n is an integer between 11 and 20; the compound of Formula I possesses at least a 10-mer complementary overlap with a 14-mer target splice site sequence that consists of 7-mer from intron and 7-mer from exon within a target pre-mRNA; the compound of Formula I is fully complementary to the target pre-mRNA sequence, or partially complementary to the target pre-mRNA sequence with one or two mismatches; S 1 , S 2 , . . . , S n-1 , S n , T 1 , T 2 , . . . , T n-1 , and T n are hydrido radical; X is hydrido radical; Y is substituted or non-substituted alkyloxycarbonyl radical; Z is amino radical; B 1 , B 2 , . . . , B n-1 , and B n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and at least five of B 1 , B 2 , . . . , B n-1 , and B n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV: wherein, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are hydrido radical; L 1 , L 2 and L 3 are a covalent linker represented by Formula V covalently linking the basic amino group to the nucleobase moiety: wherein, Q 1 and Q m are substituted or non-substituted methylene (—CH 2 —) radical, and Q m is directly linked to the basic amino group; Q 2 , Q 3 , . . . , and Q m-1 are independently selected from substituted or non-substituted methylene or oxygen radical; and m is an integer between 1 and 15.
  2. 2 . The peptide nucleic acid derivative according to claim 1 , or a pharmaceutical salt thereof: wherein, n is an integer between 11 and 19; the compound of Formula I possesses at least a 10-mer complementary overlap with a 14-mer target splice site sequence that consists of 7-mer from intron and 7-mer from exon within a target pre-mRNA; the compound of Formula I is fully complementary to the target pre-mRNA sequence; S 1 , S 2 , . . . , S n-1 , S n , T 1 , T 2 , . . . , T n-1 , and T n are hydrido radical; X is hydrido radical; Y is substituted or non-substituted alkyloxycarbonyl radical; Z is amino radical; B 1 , B 2 , . . . , B n-1 , and B n are independently selected from adenine, thymine, guanine, cytosine, and unnatural nucleobases; at least five of B 1 , B 2 , . . . , B n-1 , and B n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are hydrido radical; L 1 represents —(CH 2 ) 2 —O—(CH 2 ) 2 —, —CH 2 —O—(CH 2 ) 2 —, —CH 2 —O—(CH 2 ) 3 —, —CH 2 —O—(CH 2 ) 4 —, —CH 2 —O—(CH 2 ) 5 —, —CH 2 —O—(CH 2 ) 6 —, or —CH 2 —O—(CH 2 ) 7 — with the right end is directly linked to the basic amino group; and L 2 and L 3 are independently selected from —(CH 2 ) 2 —, —(CH 2 ) 3 —, —(CH 2 ) 4 —, —(CH 2 ) 5 —, —(CH 2 ) 6 —, —(CH 2 ) 7 —, —(CH 2 ) 8 —, —(CH 2 ) 2 —O—(CH 2 ) 2 —, —(CH 2 ) 3 —O—(CH 2 ) 2 —, and —(CH 2 ) 2 —O—(CH 2 ) 3 — with the right end is directly linked to the basic amino group.
  3. 3 . The peptide nucleic acid derivative of claim 1 , wherein the target splice site sequence is not [(5′→3′) UAAGUAGGAUAAGU (SEQ ID NO: 5)] within the human HIF-1α pre-mRNA.
  4. 4 . A method of inducing in cells the skipping of the target exon within the target pre-mRNA comprising contacting the cells with the peptide nucleic acid derivative of claim 1 .
  5. 5 . A method of inducing in a subject the skipping of the target exon within the target pre-mRNA comprising administering the peptide nucleic acid derivative of claim 1 .
  6. 6 . A method of treating a disease or condition involving the expression of the target gene comprising administering the peptide nucleic acid derivative of claim 1 .
  7. 7 . A method of modulating in cells the functional activity of the target gene comprising contacting the cells with the peptide nucleic acid derivative of claim 1 .
  8. 8 . A method of modulating in a subject the functional activity of the target gene comprising administering the peptide nucleic acid derivative of claim 1 .

Description

RELATED APPLICATIONS This application is a national-stage filing under 35 U.S.C. § 371 of International Application No. PCT/IB2017/001725, filed Dec. 29, 2017, which claims the benefit of priority to U.S. Provisional Application No. 62/440,929, filed Dec. 30, 2016, each of which is incorporated by reference herein in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 21, 2019, is named OSH-00501_(32567-00501)_SL.txt and is 41,305 bytes in size. BACKGROUND OF INVENTION Oligonucleotides have been used for diverse biological purposes including antisense inhibition of gene expression, PCR (polymerase chain reaction), diagnostic analysis by gene chip, and so on. Since oligonucleotides interact with nucleic acid including DNA and RNA in a sequence specific manner, they are useful to predictably modulate biological processes involving DNA or RNA within cell. Oligonucleotides with good cell permeability are able to modulate such biological processes within cell in a sequence predictable manner. Proteins as Drug Targets: Proteins mediate diverse cellular functions. It would not be not surprising to find that most of currently marketed drugs show therapeutic activity through modulating functions of protein(s). For example, non-steroidal anti-inflammatory drug aspirin inhibits enzymes called cyclooxygenases for its anti-inflammatory activity. Losartan binds to a trans-membrane receptor called angiotensin II receptor for its antihypertensive activity. Rosiglitazone selectively activates an intracellular receptor called peroxisome proliferator-activated receptor γ (PPARγ) to elicit its antidiabetic activity. Etanercept is a fusion protein which binds to a cytokine called tumor necrosis factor-α (TNF-α), and neutralizes the biological activity of TNF-α for its anti-rheumatic activity. Herceptin is a monoclonal antibody to treat breast cancer by selectively binding to erbB2 over-expressed in certain types of breast cancer cells. Pre-mRNA: Genetic information is carried on DNA (2-deoxyribose nucleic acid), which is transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. Mammalian pre-mRNA usually consists of exons and introns, and exon and intron are inter-connected to each other. Exons and introns are numbered as illustrated in FIG. 1A. Splicing of Pre-mRNA into mRNA: In the nucleus, pre-mRNA is processed into mRNA following deletion of introns and ligation of exons by a series of complex reactions collectively called “splicing” as schematically illustrated in FIG. 1B. [Ann. Rev. Biochem. 72(1), 291-336 (2003); Nature Rev. Mol. Cell Biol. 6(5), 386-398 (2005); Nature Rev. Mol. Cell Biol. 15(2), 108-121 (2014)] Splicing is initiated by forming “splicesome E complex” (i.e. early splicesome complex) between pre-mRNA and splicing adapter factors. In “splicesome E complex”, U1 binds to the junction of exon N and intron N, and U2AF35 binds to the junction of intron N and exon (N+1). Thus the junction of exon/intron or intron/exon is critical to the formation of the early splicesome complex. “Splicesome E complex” evolves into “splicesome A complex” upon additional complexation with U2. “Splicesome A complex” then undergoes a series of complex reactions to delete or splice out the intron to adjoin the neighboring exons. Alternative Splicing and Splice Variant: All the exons of pre-mRNA are not always retained to form the “full-length” mRNA during splicing. Certain exons are deleted, or spliced out to form variant mRNAs, i.e. “splice variants”. Thus pre-mRNA can be “alternatively spliced” to yield multiple splice variants. Alternative splicing in mammalian cells was first reported in 1981 with the gene encoding calcitonin. [Nature vol 290(5801), 63-65 (1981); Proc. Natl. Acad. Sci. USA vol 79(6), 1717-1721 (1982)] The gene consists of 6 exons, and the calcitonin mRNA is produced by the skipping of exon 5 and exon 6. In the meantime, the skipping of exon 4 yields an mRNA variant encoding calcitonin gene related peptide (CGRP). Alternative splicing appears to be completely up to cells and conditions that cells are exposed to. Due to alternative splicing, multiple proteins are produced from a single gene. Alternative splicing allows animals to generate more diversities of proteins for their genome size. In humans, 95% of multi-exonic genes are estimated to be alternatively spliced. [Nature Genetics vol 40(12), 1413-1415 (2008)] Splice Variants and Biological Functions: Splice variants are found as spontaneously occurring in a manner dependent on cell type or tissue, and encode proteins possessing biological profiles often different from the profiles of the full-length protein. Androgen receptor (AR) would be a good example of genes yielding multiple splice variants. [Int. J. Biol. Sci. vol 7(6), 815-822 (2011)] The AR pre-mRNA consis