MeCP2 expression cassettes

1. A nucleic acid molecule comprising a MeCP2 expression cassette, the expression cassette comprising, in operable linkage from 5′ to 3′:

(a) a 5′ transcriptional control region comprising a promoter capable of driving transcription in neural cells, wherein the promoter is a MeCP2 promoter comprising a MeCP2 silencer element and a CNS regulatory element;

(b) a Kozak sequence;

(c) an open reading frame encoding a MeCP2 protein;

(d) a 3′ untranslated region (3′UTR) comprising a MeCP2 polyadenylation signal

a binding site for miR-22,

a binding site for miR-19;

a binding site for miR-132,

a binding site for miR-124, and

one or more AU-rich element of the sequence AUUUA; and

(e) transcriptional termination signals; and

wherein the MeCP2 expression cassette is not more than about 5 kb in length.

2. The nucleic acid molecule of claim 1 , wherein the encoded MeCP2 protein comprises:

(i) a methyl-CpG binding domain (MBD) having a sequence

(SEQ ID NO: 5)

PAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYD

VYLINPQGKAFRSEVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQK

PP,

or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;

(ii) a NCoR/SMRT Interaction Domain (NID) having a sequence

(SEQ ID NO: 6)

PGSVVAAAAAEAKKAVKESSIRSVQETVLPIKKRKTRETV,

or a variant thereof having at least 70%%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and

(iii) a nuclear localisation signal (NLS).

3. The nucleic acid molecule of claim 2 , wherein the encoded MeCP2 protein comprises a sequence:

(SEQ ID NO: 9)

SEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKA

ETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRK

SGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGR

GSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRV

LEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQ

AIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETV

SIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPP

KKEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSV

CKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKYKHRGEGERKDIV

SSSMPRPNREEPVDSRTPVTERVSS;

or is a functional variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; or is a functional fragment thereof.

4. The nucleic acid molecule of claim 2 , wherein the encoded MeCP2 protein comprises a sequence:

(SEQ ID NO: 10)

PAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYD

VYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQK

PPKKPKSPKAPGTGRGRGRPKGSGTTRPKRATSEGVQVKRVLEKSPGKLL

VKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAIPKKRGRK

PGSVVAAAAAEAKKKAVKESSIRSVQETVLPTKKRKTRETV;

or is a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

5. The nucleic acid molecule of claim 2 , wherein the encoded MeCP2 protein comprises a sequence:

(SEQ ID NO: 11)

PAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYD

VYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQK

PPGSSGSSGPKKKRKVPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKK

RKTRETV.

6. The nucleic acid molecule of claim 2 , wherein the MeCP2 protein further comprises an N-terminal portion having a sequence MAAAAAAAPSGGGGGGEEERLEEK (SEQ ID NO: 12), MVAGMLGLREEK (SEQ ID NO: 13).

7. The nucleic acid molecule of claim 1 , wherein the 5′ transcription regulatory region comprises a core MeCP2 promoter, a MeCP2 silencer element, a CNS regulatory element, or any combination thereof.

8. The nucleic acid molecule of claim 1 , wherein the expression cassette is not more than about 4.9 kb, 4.8 kb, 4.7 kb, 4.6 kb, 4.5 kb or 4.4 kb in length.

9. The nucleic acid molecule of claim 1 , further comprising a 5′ ITR and a 3′ ITR, wherein the 5′ ITR and the 3′ ITR flank the expression cassette.

10. The nucleic acid molecule of claim 9 , wherein the nucleic acid molecule comprises a rAAV genome.

11. The nucleic acid molecule of claim 1 , wherein the expression cassette is not more than about 2.4 kb, not more than 2.3 kb, or not more than 2.2 kb in length.

12. The nucleic acid molecule of claim 11 , further comprising, from 5′ to 3′, a MeCP2 expression cassette and the reverse complement of said expression cassette.

13. The nucleic acid molecule of claim 12 , further comprising a 5′ ITR and a 3′ ITR.

14. The nucleic acid molecule of claim 13 , wherein the nucleic acid molecule is a scAAV vector genome.

15. An AAV virion particle comprising a nucleic acid molecule as defined by claim 14 or a nucleic acid molecule as defined by claim 14 .

16. A pharmaceutical composition comprising a nucleic acid molecule of claim 1 , a rAAV genome of claim 10 , a scAAV genome of claim 14 , or an AAV virion particle according to claim 15 , in combination with a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16 , formulated for intravenous or intrathecal administration.