JPWO2021028666A5

JPWO2021028666A5 -

Info

Publication number: JPWO2021028666A5
Application number: JP2022507761A
Authority: JP
Publication date: 2023-08-14

Claims

A conjugate comprising a peptide carrier covalently attached to a therapeutic molecule,
Said peptide carrier has a total length of 40 amino acid residues or less and two or more cationic domains each comprising at least 4 amino acid residues and a hydrophobic domain comprising at least 3 amino acid residues each. said peptide carrier does not contain artificial amino acid residues; and said therapeutic molecule comprises a nucleic acid, said nucleic acid comprising a plurality of trinucleotide repeats. A complex characterized by:

The complex of Claim 1, wherein the nucleic acid comprises a plurality of trinucleotide repeats selected from GTC, CAG, GCC, GGC, CTT, and CCG repeats.

3. The complex of claim 1 or 2, wherein the nucleic acid comprises multiple CAG repeats.

A complex according to any preceding claim, wherein the nucleic acid comprises 5 to 20 trinucleotide repeats, preferably 5 to 10 trinucleotide repeats, more preferably 7 trinucleotide repeats.

The complex of any one of claims 1-4, wherein the nucleic acid binds to a trinucleotide repeat extension.

The conjugate according to any one of claims 1 to 5, wherein the peptide carrier consists of natural amino acid residues.

A conjugate according to any preceding claim, wherein each cationic domain has a length of 4 to 12 amino acid residues, preferably 4 to 7 amino acid residues.

The conjugate of any of claims 1-7, wherein each cationic domain comprises at least 40%, at least 45%, or at least 50% cationic amino acids.

Each cationic domain comprises arginine, histidine, β-alanine, hydroxyproline and/or serine residues, preferably each cationic domain comprises arginine, histidine, β-alanine, hydroxyproline and / Or the conjugate according to any one of claims 1 to 8, which consists of a serine residue.

A conjugate according to any preceding claim, wherein the peptide carrier comprises two cationic domains.

Each cationic domain has the following sequence: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6). ), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13) , HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[ Hyp]RR[Hyp]R (SEQ ID NO: 19) or combinations thereof, preferably each cationic domain comprises the following sequences: RBRBRRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 1) 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9 ), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16) , BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19) or a combination thereof The composite according to any one of claims 1 to 10, comprising:

A complex according to any preceding claim, wherein each hydrophobic domain has a length of 3-6 amino acids, preferably each hydrophobic domain has a length of 5 amino acids.

Each hydrophobic domain comprises a number of hydrophobic amino acid residues, preferably each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, A conjugate according to any preceding claim comprising at least 95% or 100%.

Each hydrophobic domain comprises residues of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline and glutamine, preferably each hydrophobic domain comprises phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline and / Or the conjugate according to any one of claims 1 to 13, which consists of residues of glutamine.

A conjugate according to any preceding claim, wherein the peptide carrier comprises one hydrophobic domain.

The or each hydrophobic domain has the following sequences: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24). , WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26) or combinations thereof, preferably the or each hydrophobic domain comprises the following sequences: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO:21), ILFQY (SEQ ID NO:22), FQIY (SEQ ID NO:23), WWW, WWPWW (SEQ ID NO:24), WPWW (SEQ ID NO:25), WWPW (SEQ ID NO:26) or one of combinations thereof The complex according to any one of claims 1-15.

17. Any of claims 1-16, wherein the peptide carrier consists of two cationic domains and one hydrophobic domain, preferably the peptide consists of one hydrophobic core domain flanked by two cationic arm domains. Described complex.

The peptide carrier has the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO:27), RBRRBRFQILYRRBRBR (SEQ ID NO:29), RBRBRFQILYRBRRBRR (SEQ ID NO:30), RBRRBRRYQFLIRBRBR (SEQ ID NO:31), RBRRBRRILFQYRBRBR (SEQ ID NO:32), RBRRBRFQILYRBRBR (SEQ ID NO:33), RBRRBRFQILYBRBR (SEQ ID NO:35), RBRRBFQILYRBRBR (SEQ ID NO:36), RBRRBRRFQILYRBHBH (SEQ ID NO:37), RBRRBRRFQILYHBHBR (SEQ ID NO:38), RBRRBRRFQILYHBRBH (SEQ ID NO:39), RBRRBRRYQFLIRBHBH (SEQ ID NO:40), RBRRBRRILFQYRBHBH (SEQ ID NO:41), RBRHBHRF QILYRBRBR (SEQ ID NO:42), RBRRBRFQILYRBHBH (SEQ ID NO:44), RBRRBRRFQILYHBHBH (SEQ ID NO:52), RBRRBRWWWBRBR (SEQ ID NO:58), and RBRRBRWWPWWBRBR (SEQ ID NO:59). Complex.

The conjugate of any of claims 1-18, wherein the peptide carrier is covalently attached to the therapeutic molecule by a linker.

20. The conjugate of claim 19, wherein the linker is selected from G, BC, XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX, XB, E, GABA, and succinic acid.

A conjugate according to claim 19 or 20, wherein the linker is at the C-terminus of the peptide or at the N-terminus of the peptide.

(a) the peptide carrier RBRRBRFQILYBRBR (SEQ ID NO: 35) covalently linked by β-alanine (B) to an antisense oligonucleotide consisting of 7 CAG repeats;
(b) the peptide carrier RBRRBRFQILYBRBR (SEQ ID NO: 35) covalently linked by glutamic acid (E) to an antisense oligonucleotide consisting of 7 CAG repeats;
(c) the peptide carrier RBRRBRFQILYBRBR (SEQ ID NO: 35) covalently attached to an antisense oligonucleotide consisting of 7 CAG repeats by GABA (Ab);
(d) the peptide carrier RBRRBRRFQILYRBHBH (SEQ ID NO: 37) covalently linked by β-alanine (B) to an antisense oligonucleotide consisting of 7 CAG repeats;
(e) the peptide carrier RBRRBRRFQILYRBHBH (SEQ ID NO: 37) covalently linked by glutamic acid (E) to an antisense oligonucleotide consisting of 7 CAG repeats;
(f) the peptide carrier RBRRBRRFQILYRBHBH (SEQ ID NO: 37) covalently attached to an antisense oligonucleotide consisting of 7 CAG repeats by GABA (Ab);
(g) the peptide carrier RBRRBRFQILYRBHBH (SEQ ID NO: 44) covalently linked by β-alanine (B) to an antisense oligonucleotide consisting of 7 CAG repeats;
(h) a peptide carrier RBRRBRFQILYRBHBH (SEQ ID NO: 44) covalently attached to an antisense oligonucleotide consisting of 7 CAG repeats by glutamic acid (E); or (i) an antisense oligonucleotide consisting of 7 CAG repeats to which GABA ( Ab) covalently attached peptide carrier RBRRBRFQILYRBHBH (SEQ ID NO: 44)
wherein optionally the cell penetrating peptide of the conjugate is N-terminally acetylated.

The peptide is N-terminally modified, preferably the peptide is N-acetylated, N-methylated, N-trifluoroacetylated, N-trifluoromethylsulfonylated or N-methylsulfonylated. 23. The conjugate according to any one of 1 to 22.

The conjugate according to any one of claims 19-23, wherein the C-terminus of the peptide is attached to the antisense oligonucleotide via a linker.

The conjugate of any one of claims 1-24, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

26. The conjugate of claim 25, wherein the 3' end of the PMO is attached to the peptide carrier of the conjugate via a linker.

A conjugate according to any of claims 1-26 for use as a medicament.

A conjugate according to any one of claims 1 to 26 for use in the prevention or treatment of triplet repeat diseases.

A conjugate for use according to claim 28, wherein the triplet repeat disease is selected from polyglutamine disease or non-polyglutamine disease.

Triplet repeat diseases include DRPLA (dentate nucleus pallidus louis body atrophy), HD (Huntington's disease), HDL2 (Huntington's disease-like syndrome 2), SBMA (spinobulbar muscular atrophy), SCA1 (spinocerebellar Ataxia type 1), SCA2 (Spinocerebellar ataxia type 2), SCA3 (Spinocerebellar ataxia type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia type 6), SCA7 (Spinocerebellar ataxia type 73), SCA17 (Spinocerebellar ataxia type 17), HDL2 (Huntington's disease-like syndrome 2), FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome), FRAXE (Fragile XE mental retardation), FRDA (Friedreich) ataxia), DM1 (myotonic dystrophy type 1), SCA8 (spinocerebellar ataxia type 8) and SCA12 (spinocerebellar ataxia type 12). body.

The conjugate for use according to any one of claims 28 to 30, wherein the triplet repeat disease is myotonic dystrophy type 1 (DM1).