MECP2 BASED THERAPY
MeCP2 based therapy. The present invention relates to synthetic polypeptides that are useful in the treatment of disorders associated with reduced MeCP2 activity, including Rett syndrome. The present invention provides synthetic polypeptides comprising: i) an MBD amino acid sequence showing at least 70% similarity with the amino acid sequence showing at least 70% similarity with the amino acid sequence as depicted in SEQ ID NO: 2, wherein the polypeptide has a deletion of at least 50 amino acids, when compared to the full length MeCP2 e1 and e2 sequences. The invention further provides nucleic acid constructs, expression vectors, virions, pharmaceutical compositions, and cells providing polynucleotides of the invention. The invention further provides methods of treating or preventing disease in an animal comprising administering to said animal a synthetic polypeptide according to the invention.
1 . A synthetic polypeptide comprising:
i) an MBD amino acid sequence showing at least 70% similarity with the amino acid sequence as depicted in SEQ ID NO: 1; and
ii) an NID amino acid sequence showing at least 70% similarity with the amino acid sequence as depicted in SEQ ID NO: 2,
wherein the polypeptide has a deletion of at least 50 amino acids, when compared to the full length MeCP2 e1 and e2 sequences (SEQ ID Nos 3 and 4).
2 . A synthetic polypeptide according to claim 1 wherein the polypeptide has less than 90% identity over the entire length of the amino acid sequences of MeCP2 as depicted in SEQ ID NO: 3 and SEQ ID NO: 4.
3 . A synthetic polypeptide according to claim 1 or claim 2 , having the structure:
A-B-C-D-E
wherein
portion B of the synthetic polypeptide is said MBD amino acid sequence, and
portion D of the synthetic polypeptide is said NID amino acid sequence,
and further wherein:
portion A of the synthetic polypeptide is less than 40 amino acids long and/or has less than 80% identity to the amino acid sequences as depicted in SEQ ID NOs:5 and 6, calculated over the entire length of the amino acid sequences as depicted in SEQ ID NOs: 5 and 6;
portion C of the synthetic polypeptide is less than 20 amino acids long and/or has less than 80% identity to the amino acid sequence as depicted in SEQ ID NO: 7, calculated over the entire length of the amino acid sequence as depicted in SEQ ID NO: 7; and/or
portion E of the synthetic polypeptide is absent, a protein tag, and/or has less than 80% identity to the amino acid sequence as depicted in SEQ ID NO: 8, calculated over the entire length of the amino acid sequence as depicted in SEQ ID NO: 8.
4 . A synthetic polypeptide according to any of claims 1 to 3 wherein said synthetic polypeptide is capable of recruiting a NCoR/SMRT co-repressor complex component, such as NCoR/SMRT, HDAC3, GPS2, TBL1X or TBLR1, preferably TBL1X or TBLR1, to methylated DNA.
5 . A synthetic polypeptide according to any preceding claim wherein said synthetic polypeptide consists of less than 430 amino acids, preferably less than 400, 350, 320, 270, or 200 amino acids, and further preferably less than 180 amino acids.
6 . A synthetic polypeptide according to any preceding claim wherein said polypeptide comprises a nuclear localization signal (NLS), preferably wherein said NLS is comprised within the amino acid sequence between the MBD and NID.
7 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence between the MBD and NID amino acid sequences has less than 75% identity to the amino acid sequence as depicted in SEQ ID NO: 7, calculated over the entire length of the amino acid sequence as depicted in SEQ ID NO: 7, preferably less than 50%, and further preferably less than 30% identity.
8 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence between the MBD and NID amino acid sequences is less than 50 amino acids long, preferably less than 30 amino acids long, and further preferably less than 20 amino acids long.
9 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence between the MBD and NID amino acid sequences has a substitution or deletion of at least 10 consecutive amino acids compared to the amino acid sequence from position 207 to position 271 of the full length human wild type MeCP2 polypeptide sequence (e2 isoform) as shown in SEQ ID NO: 4.
10 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence adjacent to the carboxy end of the NID amino acid sequence has less than 75% identity to the amino acid sequence as depicted in SEQ ID NO: 8, calculated over the entire length of the amino acid sequence as depicted in SEQ ID NO: 8, preferably less than 50%, and further preferably less than 30% identity.
11 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence adjacent to the carboxy end of the NID amino acid sequence is less than 50 amino acids long, preferably less than 30 amino acids long or less than 20 amino acids long, and further preferably wherein there is no amino acid sequence adjacent to the carboxy end of the NID amino acid sequence.
12 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence adjacent to the amino end of the MBD amino acid sequence has less than 75% identity to the amino acid sequences as depicted in SEQ ID NOs: 5 and 6, calculated over the entire length of the amino acid sequences as depicted in SEQ ID NOs: 5 and 6, preferably less than 50%, and further preferably less than 30% identity.
13 . A synthetic polypeptide according to any preceding claim wherein the amino acid sequence adjacent to the amino end of the MBD amino acid sequence is less than 50 amino acids long, preferably less than 30 amino acids long or less than 20 amino acids long, and further preferably less than 10 amino acids long.
14 . A synthetic polypeptide according to any preceding claim wherein the polypeptide has less than 90% identity over the entire length of the amino acid sequences of MeCP2 as depicted in SEQ ID NO: 3 and SEQ ID NO: 4, preferably less than 80% identity, less than 70% identity, or less than 60% identity, and further preferably less than 40% identity.
15 . A nucleic acid construct that encodes a polypeptide according to any preceding claim.
16 . An expression vector comprising a nucleotide sequence encoding a synthetic polypeptide according to any of claims 1 to 14 .
17 . An expression vector according to claim 16 further comprising one or more control elements selected from: a promoter for expression of the nucleotide sequence in neuronal cells, for example an Mecp2 or MECP2 promoter, one or more downstream miR binding sites from the MECP2 or Mecp2 3′UTR, and an AU-rich element.
18 . An expression vector according to claim 16 or claim 17 which is a viral vector, such as a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or an alphaviral vector.
19 . A virion comprising a vector according to claim 18 .
20 . A pharmaceutical composition comprising a synthetic polypeptide according to any of claims 1 to 14 , a nucleic acid construct according to claim 15 , an expression vector according to any of claims 16 to 18 and/or a virion according to claim 19 .
21 . A cell comprising a synthetic genetic construct adapted to express a polypeptide according to any of claims 1 to 14 .
22 . A cell according to claim 21 comprising a vector according to any of claims 16 to 18 .
23 . A cell according to claim 21 or 22 for producing a virion according to claim 19 .
24 . A method of treating or preventing disease in an animal comprising administering to said animal a synthetic polypeptide according to any of claims 1 to 14 .
25 . A method according to claim 24 wherein said disease is a neurological disorder associated with inactivating mutation of MeCP2, for example Rett syndrome.
26 . A method according to claim 24 or 25 , wherein said administering comprises administering a composition comprising a synthetic polypeptide according to any of claims 1 to 14 , a nucleic acid construct according to claim 15 , an expression vector according to any of claims 16 to 18 , a virion according to claim 19 and/or a pharmaceutical composition according to claim 20 .
27 . A synthetic polypeptide according to any of claims 1 to 14 , a nucleic acid construct according to claim 15 , an expression vector according to any of claims 16 to 18 , a virion according to claim 19 and/or a pharmaceutical composition according to claim 20 for the treatment or prevention of a neurological disorder associated with inactivating mutation of MeCP2, for example Rett syndrome.
28 . The use of a synthetic polypeptide according to any of claims 1 to 14 , a nucleic acid construct according to claim 15 , an expression vector according to any of claims 16 to 18 , a virion according to claim 19 and/or a pharmaceutical composition according to claim 20 in the manufacture of a medicament for the treatment or prevention of a neurological disorder associated with inactivating mutation of MeCP2, for example Rett syndrome.