Cell penetrating peptides

Abstract

The present invention relates to peptides, in particular cell penetrating peptides, of 40 amino acid residues or less comprising at least one directly glycosylated amino residue and one or more arginine rich arm domains, and to conjugates of such cell penetrating peptides with a therapeutic molecule. The present invention further relates to the use of the peptides or conjugates in methods of treatment or as a medicament, especially in the treatment of genetic disorders of the central nervous system. page.

1 . A peptide comprising at least one directly glycosylated amino acid residue and one or more arginine-rich arm domains, wherein the total length of the peptide is 40 amino acid residues or less. 
     
     
         2 . A peptide according to  claim 1 , wherein the at least one directly glycosylated amino acid residue is O-linked glycosylated, N-linked glycosylated or S-linked glycosylated. 
     
     
         3 . A peptide according to  claim 1 , wherein at least one directly glycosylated amino acid residue is glycosylated at a functional group present in the amino acid side chain selected from an OH, NH 2 , NH 3  and SH. 
     
     
         4 . A peptide according to  claim 1 , wherein the at least one directly glycosylated amino acid residue is selected from a glycosylated serine, cysteine, threonine, asparagine, glutamine, aminoproline, hydroxyproline, tyrosine, lysine, and amino acid analogues thereof, preferably the at least one directly glycosylated amino acid residue is a glycosylated serine. 
     
     
         5 - 7 . (canceled) 
     
     
         8 . A peptide according to  claim 1 , wherein the at least one directly glycosylated amino acid residue is glycosylated with a sugar selected from: glucose, allose, altrose, idose, gulose, talose, xylose, lactose, mannose, galactose, mannoseamine, glucosamine, galactosamine, N-acetylgalactosamine, D-2-Acetylamino Glucose, N-acetylglucosamine, lactose, maltose, isomaltose, trehalose and sialic acid, preferably the at least one amino acid residue is directly glucosylated. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . A peptide according to  claim 8 , wherein the at least one amino acid residue is directly glucosylated with β-D glucose, preferably the at least one amino acid residue is a β-D glucosyl serine. 
     
     
         12 . (canceled) 
     
     
         13 . A peptide according to  claim 1 , wherein the arginine-rich arm domains comprise a combined total of between 5-10 Arginine residues. 
     
     
         14 . A peptide according to  claim 1 , wherein the arginine-rich arm domains comprise no more than 3 contiguous Arginine residues. 
     
     
         15 . A peptide according to  claim 1 , wherein the arginine-rich arm domains comprise a length of between 1-12 amino acid residues, preferably wherein the amino acid residues are selected from the group consisting of: arginine, alanine, beta-alanine, histidine, proline, glycine, cysteine, tryptophan, hydroxyproline, aminohexanoic acid, 3-azetidine-carboxylic acid (Az), 1-(amino)cyclohexanecarboxylic acid (Cy), amino acid analogues thereof, and any other non-natural amino acid. 
     
     
         16 . (canceled) 
     
     
         17 . A peptide according to  claim 1 , wherein each arginine-rich arm domain is selected from the following sequences: RXRRBRRXR (SEQ ID NO.81), RXRBRXR (SEQ ID NO.82), RXRRBRR (SEQ ID NO.83), RBRXR (SEQ ID NO.84), RBRRBRRBR (SEQ ID NO.85), RBRBRBR (SEQ ID NO.86), RGRRGRRGR (SEQ ID NO.87), RGRGRGR (SEQ ID NO.88), RPRRPRRPR (SEQ ID NO.89), RPRPRPR (SEQ ID NO.90), RHypRRHypRRHypR (SEQ ID NO.91), RHypRHypRHypR (SEQ ID NO.92), RARRARRAR (SEQ ID NO.93), RARARAR (SEQ ID NO.94), RCy*RRCy*RRCy*R (SEQ ID NO.95), RCy*RCy*RCy*R (SEQ ID NO.96), RRBRRBR (SEQ ID NO.97), RBRRBR (SEQ ID NO.98), RRBR (SEQ ID NO.99), RBR, R, RBRBR (SEQ ID NO.100), RBRBRR (SEQ ID NO.101), RBRRR (SEQ ID NO.102), RRRR (SEQ ID NO.103), RBRRBRRR (SEQ ID NO.104, RBRRRRR (SEQ ID NO.105), RRRRRR (SEQ ID NO.106), RRBRR (SEQ ID NO.107), RGRR (SEQ ID NO.108), GRRGR (SEQ ID NO.109), RGGRBRGGR (SEQ ID NO.110), RXRRBRRXRRXRBRXR (SEQ ID NO.113), RXRR (SEQ ID NO.114, RRXR (SEQ ID NO.115), RXR, RRBRBRXR (SEQ ID NO.117), RRBRRBRBRXR (SEQ ID NO.118), RXRRBRRBR (SEQ ID NO.119), RXRRBRRBRBR (SEQ ID NO.120), RXRRBR (SEQ ID NO.121), RXRBRR (SEQ ID NO.122), HXHRBRRXR (SEQ ID NO.123), RXHBHXR (SEQ ID NO.124), RR, RXRXR (SEQ ID NO.125), BRBRBR (SEQ ID NO.127), BRKBRKRBBR (SEQ ID NO.128), BRKBRKRBBRK (SEQ ID NO.129), RAzRRAzRR (SEQ ID NO.130), RAzRAzR (SEQ ID NO.131), and RXRBR (SEQ ID NO.132). 
     
     
         18 . (canceled) 
     
     
         19 . A peptide according to  claim 1 , wherein each of the arginine-rich arm domains present in the peptide is separated from any other arginine rich arm domain present in the peptide by a directly glycosylated amino acid residue. 
     
     
         20 . A peptide according to  claim 1 , wherein the peptide comprises a first arginine-rich arm domain selected from the following sequences: RXRRBRRXR (SEQ ID NO.81), RXRRBRR (SEQ ID NO.83), RBRBR (SEQ ID NO.100), and RXRXR (SEQ ID NO.125). 
     
     
         21 . A peptide according to  claim 20 , wherein the peptide comprises a second arginine-rich arm domain selected from the following sequences: RXRBRXR (SEQ ID NO.82), RBRXR (SEQ ID NO.84), RBRRBR (SEQ ID NO.98), RXRBR (SEQ ID NO.132), RBRBR (SEQ ID NO.100), and RXRXR (SEQ ID NO.125). 
     
     
         22 . A peptide according to  claim 1 , wherein the peptide comprises one or more hydrophobic core domains. 
     
     
         23 . A peptide according to  claim 22 , wherein the each hydrophobic core domain comprises between 1-4 hydrophobic amino acid residues, preferably wherein the amino acid residues are selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and amino acid analogues thereof. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . A peptide according to  claim 22 , wherein each hydrophobic core domain is contiguous with the at least one directly glycosylated amino acid residue, preferably wherein each hydrophobic core domain contiguous with the directly glycosylated amino acid residue is positioned between two flanking arginine-rich arm domains. 
     
     
         27 . (canceled) 
     
     
         28 . A peptide according to  claim 22 , wherein each hydrophobic core domain is selected from the following sequences: GFTGPL (SEQ ID NO.133), QFL, Z, ZL, F, FL, FQILY (SEQ ID NO.134), FQ, WF, QF, FQ, and YQFLI (SEQ ID NO.135). 
     
     
         29 - 34 . (canceled) 
     
     
         35 . A peptide according to  claim 1 , wherein the peptide is selected from the following sequences: RXRRBRRXRS*FLRXRBRXR (SEQ ID NO.14), RXRRBRRFS*RBRXR (SEQ ID NO.17), RXRRBRRZS*RBRXR (SEQ ID NO.73), RXRRBRRFS1*RBRXR (SEQ ID NO.74), RBRBRS*RBRBR (SEQ ID NO.70) and RXRXRS*RXRXR (SEQ ID NO.71); wherein Z represents 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and wherein S 1 * represents D-serine glycosylated with D-Glucose sugar. 
     
     
         36 . A conjugate comprising the peptide according to  claim 1  covalently linked to a therapeutic molecule. 
     
     
         37 . A conjugate according to  claim 36 , wherein the therapeutic molecule is selected from: a nucleic acid, peptide nucleic acid, antisense oligonucleotide (such as PNA, PMO), short interfering RNA, micro RNA, peptide, cyclic peptide, protein, pharmaceutical and drug, preferably the therapeutic molecule is an antisense oligonucleotide. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A method of treating a disease in a subject comprising administering a conjugate according to  claim 36  to the subject in a therapeutically effective amount. 
     
     
         41 . A method of treating a disease according to  claim 40 , wherein the disease is of the central nervous system. 
     
     
         42 . A method of treating a disease according to  claim 40 , wherein the disease is caused by splicing deficiencies. 
     
     
         43 . A method of treating a disease according to  claim 40 , wherein the disease is selected from: Duchenne Muscular Dystrophy (DMD), Bucher Muscular Dystrophy (BMD), Menkes disease, Beta-thalassemia, dementia, Parkinson's Disease, Spinal Muscular Atrophy (SMA), myotonic dystrophy (DM), Huntington's Disease, Hutchinson-Gilford Progeria Syndrome, Ataxia-telangiectasia, and cancer. 
     
     
         44 - 50 . (canceled)