EP4683677A1

EP4683677A1 - Gene therapy

Info

Publication number: EP4683677A1
Application number: EP24715739.9A
Authority: EP
Inventors: Mirko LUONI; Serena GIANNELLI; Vania Broccoli
Original assignee: Consiglio Nazionale delle Richerche CNR; Ospedale San Raffaele SRL
Current assignee: Consiglio Nazionale delle Richerche CNR; Ospedale San Raffaele SRL
Priority date: 2023-03-22
Filing date: 2024-03-22
Publication date: 2026-01-28
Also published as: MX2025010896A; CL2025002797A1; JP2026511500A; CN121219020A; IL323349A; WO2024194491A1; AU2024241563A1; KR20260016905A

Abstract

A polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and: (a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or (b) a nucleotide sequence encoding an inhibitor of MeCP2 expression, optionally a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15, 30 or 31.

Description

GENE THERAPY

FIELD OF THE INVENTION

The present invention relates to compounds for use in the treatment of neurological diseases. More specifically, the invention relates to polynucleotides comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and uses thereof in the treatment of Rett syndrome.

BACKGROUND TO THE INVENTION

Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disabilities in girls. RTT is characterised by a period of 6-12 months of overtly normal development followed by rapid regression with the loss of the purposeful motor skills and the onset of repetitive and autistic behaviours.

In the vast majority of cases RTT is caused by loss-of-function mutations in the MECP2 gene, which encodes the methyl-CpG binding protein 2 (MeCP2), a global chromatin regulator highly expressed in neurons (Bienvenu, T. et al. (2006) Nat Rev Genet 7: 415-426). Recent studies have revealed that the MECP2 loss significantly alters neuronal activity leading to a progressive imbalance of the excitatory-inhibitory synaptic activity across the brain with divergent modalities occurring between different circuits and regions of the brain (Banerjee, A. et al. (2016) PNAS 113: E7287-E7296).

One study demonstrated that the RTT pathological phenotype can be significantly reversed in mice by re-activating MECP2 even at advanced disease stages (Guy, J. et al. (2007) Science 315: 1143-1147). In fact, genetic reactivation of MECP2 in more than 70% of the neurons in adult mice normalised brain morphology and significantly improved several sensory-motor dysfunctions (Robinson, L. et al. (2012) Brain 135: 2699-2710).

These findings provide strong evidence that MeCP2 is a key factor in maintaining full neurological function during adulthood. Consistently, multiple pathological manifestations exhibited by adult mutant RTT mice can be fully recapitulated by deleting MeCP2 exclusively in adulthood.

Although MeCP2 is a ubiquitous neural epigenetic factor, its selective inactivation in the GABAergic neurons leads to several RTT distinctive phenotypes, suggesting that key neurological deficits in RTT are mediated by GABAergic neuronal dysfunctions. MECP2 genetic reconstitution exclusively in GABAergic neurons resulted in significant improvement of key motor and cognitive deficits in RTT mice (Ure, K. et al. (2016) eLife 5: 185). The inherent monogenic nature of RTT makes gene therapy a strong translational option for this disease. However, MECP2 gene duplication in humans is responsible for a serious and clinically distinguished neurodevelopmental disorder. Affected males present with early hypotonia, limb spasticity and severe intellectual disability. Thus, a successful gene therapy for RTT may require delivery of MeCP2 in a range comparable with endogenous levels.

Recent studies have suggested that intravenous administration of an AAV9 expressing wildtype (WT) MECP2 attenuated neurological dysfunctions and extended lifespan in RTT mice (Matagne, V. et al. (2017) Neurobiology of Disease 99: 1-11). However, the limited brain transduction obtained in these studies was not sufficient to determine a correlation between the viral dose, transduction efficiency and therapeutic benefits. Based on those studies, it was unclear whether a gene therapy approach is capable of rescuing molecular dysfunctions and transcriptional alterations caused by loss of MECP2 in the adult brain.

Other studies have provided encouraging results relating to intravenous administrations of adeno-associated viral (AVV) vectors expressing the wild-type (WT) Mecp2 (Luoni et al. (2020) ELife, 9; Gadalla et al. (2017) Molecular Therapy - Methods and Clinical Development 5: 180-190; Sinnett et al. (2017) Molecular Therapy - Methods and Clinical Development: 5: 106-115).

Endogenous MeCP2 expression is high in neurons and about 10-fold less in astroglial and oligoglial cells. Recapitulating the different MeCP2 expression levels in brain cells may further improve gene therapy efficacy and safety.

Accordingly, there remains a significant need for improved approaches for treating Rett syndrome.

SUMMARY OF THE INVENTION

The inventors have developed a therapeutic vector that enables a differentiated expression of the transgene between neural and glial cells. In addition, the inventors have further developed a vector that expresses an shRNA selectively silencing the endogenous MeCP2, but not the viral transgene, levelling the total of MeCP2 expression in the transduced cells independently of whether they are mutant or wild-type. The inventors’ vector approaches offer advantages with respect to previous vectors that further increase the efficacy and safety of gene therapy for RTT patients.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and: (a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or

(b) a nucleotide sequence encoding an inhibitor of MeCP2 expression.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and:

(a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or

(b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15.

(b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30.

(b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

(b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30; and/or (c) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and at least one miR-124 target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), at least one miR-124 target sequence, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and at least one miR-31 target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), at least one miR-31 target sequence, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and at least one miR-338-3p target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), at least one miR-338-3p target sequence, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), and at least one miR-124 target sequence and at least one miR-31 target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), at least one miR-124 target sequence and at least one miR-31 target sequence, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), and at least one miR-124 target sequence and at least one miR-338-3p target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), at least one miR-124 target sequence and at least one miR-338-3p target sequence, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), and at least one miR-124 target sequence, at least one miR-31 target sequence and at least one miR-338-3p target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), at least one miR-124 target sequence, at least one miR-31 target sequence and at least one miR-338-3p target sequence, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In some embodiments, the number of copies of each of the miRNA target sequences is independently selected from the group consisting of: one, two, three, and four.

In preferred embodiments, the polynucleotide comprises one miR-124 target sequence. In some embodiments, the polynucleotide comprises two miR-124 target sequences. In some embodiments, the polynucleotide comprises three miR-124 target sequences. In some embodiments, the polynucleotide comprises four miR-124 target sequences.

In some embodiments, the polynucleotide comprises one miR-31 target sequence. In some embodiments, the polynucleotide comprises two miR-31 target sequences. In some embodiments, the polynucleotide comprises three miR-31 target sequences. In preferred embodiments, the polynucleotide comprises four miR-31 target sequences.

In some embodiments, the polynucleotide comprises one miR-338-3p target sequence. In some embodiments, the polynucleotide comprises two miR-338-3p target sequences. In some embodiments, the polynucleotide comprises three miR-338-3p target sequences. In preferred embodiments, the polynucleotide comprises four miR-338-3p target sequences.

In preferred embodiments, the polynucleotide comprises one miR-124 target sequence, four miR-31 target sequences and four miR-338-3p target sequences.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), one miR-124 target sequence and four miR- 31 target sequences, four miR-338-3p target sequences. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), one miR-124 target sequence, four miR-31 target sequences, four miR-338-3p target sequences, and a nucleotide sequence encoding an inhibitor of MeCP2 expression. Preferably, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In some embodiments, the miR-124 target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 5.

In some embodiments, the miR-124 target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 5.

In some embodiments, the miR-124 target sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 5.

In some embodiments, the miR-31 target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 6.

In some embodiments, the miR-31 target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 6.

In some embodiments, the miR-31 target sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 6.

In some embodiments, the miR-338-3p target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 7.

In some embodiments, the miR-338-3p target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 7.

In some embodiments, the miR-338-3p target sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 7.

In some embodiments, the miRNA target sequence(s) is located after the nucleotide sequence encoding MeCP2 in the 5’ to 3’ direction.

In some embodiments, the miRNA target sequence(s) is located within the 3’-UTR of the nucleotide sequence encoding MeCP2.

In some embodiments, the miRNA target sequences or clusters of copies of the miRNA target sequences are, from 5’ to 3’, arranged in the order: miR-124 target sequence(s), miR-31 target sequence(s) and miR-338-3p target sequence(s). The target sequences, or clusters comprising one or more copy thereof, may be, for example, arranged from 5’ to 3’ such that they form groups according to their target specificity, for example, in some embodiments the polynucleotide comprises 5’ - [miR-124 target sequence^ - [miR-31 target sequence^ - [miR- 338-3p target sequence^ - 3’.

Both the individual target sequences and the clusters of target sequences may be contiguous with one another, separated by spacer sequences, or any combination thereof.

In some embodiments, the miRNA target sequences are separated by spacer sequence(s).

In some embodiments, the polynucleotide comprises a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 8.

In some embodiments, the polynucleotide comprises a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 8.

In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence encoding an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1 or 2.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence encoding an amino acid sequence that has at least 70% identity to SEQ ID NO: 1 or 2.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 2.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 3 or 4.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 3 or 4. In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of the nucleotide sequence of SEQ ID NO: 3 or 4.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence encoding an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 28.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence encoding an amino acid sequence that has at least 70% identity to SEQ ID NO: 28.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 29.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 29.

In some embodiments, the nucleotide sequence encoding MeCP2 comprises or consists of the nucleotide sequence of SEQ ID NO: 29.

In some embodiments, the nucleotide sequence encoding MeCP2 is operably linked to a promoter.

In some embodiments, the nucleotide sequence encoding MeCP2 is operably linked to a strong promoter. In some embodiments, the promoter is a neuron-, glial- or astrocyte-specific strong promoter.

In some embodiments, the promoter is selected from the group consisting of a chicken p-actin (CBA) promoter, a p-actin promoter, a CAG promoter, a cytomegalovirus (CMV) promoter, a human elongation factor-1 -alpha (HEF-1-alpha), a Chinese hamster elongation factor-1 -alpha (CHEF-1-alpha) promoter and a phosphoglycerate kinase (PGK) promoter.

In preferred embodiments, the promoter is a chicken p-actin (CBA) promoter.

In some embodiments, the nucleotide sequence encoding MeCP2 is operably linked to a 3’- UTR. In some embodiments, the 3’-UTR is less than or equal to about 1000 bp in length. In some embodiments, the 3’-UTR is less than or equal to about 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp or 200 bp in length.

In some embodiments, the 3’-UTR is less than or equal to about 500 bp in length. In preferred embodiments, the 3’-UTR is less than or equal to about 250 bp in length.

In some embodiments, the 3’-UTR is about 50-1000 bp, 50-900 bp, 50-800 bp, 50-700 bp, SO- GOO bp, 50-500 bp, 50-400 bp, 50-300 bp or 50-300 bp in length.

In some embodiments, the 3’-UTR is about 50-300 bp in length. In some embodiments, the 3’-UTR is about 50-250 bp in length. In some embodiments, the 3’-UTR is about 50-200 bp in length.

In some embodiments, the 3’-UTR is about 100-300 bp in length. In some embodiments, the 3’-UTR is about 100-250 bp in length. In some embodiments, the 3’-UTR is about 100-200 bp in length.

In some embodiments, the 3’-UTR is about 150-300 bp in length. In preferred embodiments, the 3’-UTR is about 150-250 bp in length.

In preferred embodiments, the 3’ UTR is derived from the MeCP2 3’-UTR. In preferred embodiments, the 3’-UTR is a truncated MeCP2 3’IITR.

In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of less than or equal to about 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp or 200 bp in length.

In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of less than or equal to about 500 bp in length. In preferred embodiments, the 3’-UTR is a MeCP2 3’-UTR of less than or equal to about 250 bp in length.

In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 50-1000 bp, 50-900 bp, 50- 800 bp, 50-700 bp, 50-600 bp, 50-500 bp, 50-400 bp, 50-300 bp or 50-300 bp in length.

In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 50-300 bp in length. In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 50-250 bp in length. In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 50-200 bp in length.

In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 100-300 bp in length. In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 100-250 bp in length. In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 100-200 bp in length. In some embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 150-300 bp in length. In preferred embodiments, the 3’-UTR is a MeCP2 3’-UTR of about 150-250 bp in length.

In some embodiments, the polynucleotide further comprises a polyadenylation sequence operably linked to the nucleotide sequence encoding MeCP2.

In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a tag (e.g. a V5 tag). In some embodiments, the polynucleotide does not comprise a nucleotide sequence encoding a tag (e.g. a V5 tag).

In some embodiments, the polynucleotide does not comprise a sequence encoding a V5 tag. For example, the invention may contemplate sequences that are the same as the sequences disclosed herein, but with the proviso that a sequence encoding a V5 tag is deleted.

In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding an inhibitor of MeCP2 expression.

In some embodiments, the inhibitor is an shRNA, siRNA, miRNA or antisense DNA/RNA. In some embodiments, the inhibitor is an shRNA.

In some embodiments, the nucleotide sequence encoding an shRNA has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 15 or 16, preferably SEQ ID NO: 15.

In some embodiments, the nucleotide sequence encoding an shRNA has at least 90% sequence identity to SEQ ID NO: 15 or 16, preferably SEQ ID NO: 15.

In some embodiments, the nucleotide sequence encoding an shRNA is SEQ ID NO: 15 or 16, preferably SEQ ID NO: 15.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 15.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and the nucleotide sequence of SEQ ID NO: 15.

In some embodiments, the nucleotide sequence encoding an shRNA has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 30 or 31.

In some embodiments, the nucleotide sequence encoding an shRNA has at least 90% sequence identity to SEQ ID NO: 30 or 31 .

In some embodiments, the nucleotide sequence encoding an shRNA is SEQ ID NO: 30 or 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 30.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31 .

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), a nucleotide sequence encoding an shRNA that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 30, and a nucleotide sequence encoding an shRNA that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30, and a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and the nucleotide sequence of SEQ ID NO:

30.

31.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), the nucleotide sequence of SEQ ID NO: 30, and the nucleotide sequence of SEQ ID NO: 31 .

In one aspect, the invention provides a vector comprising the polynucleotide of the invention.

In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV, retroviral, lentiviral or adenoviral vector. In preferred embodiments, the vector is an AAV vector.

In some embodiments, the vector is in the form of a viral vector particle. In preferred embodiments, the vector is in the form of an AAV vector particle.

In preferred embodiments, the viral vector particle is adapted for crossing the blood-brain barrier. In preferred embodiments, the AAV vector particle is adapted for crossing the bloodbrain barrier.

In some embodiments, the AAV vector particle comprises an artificial capsid amino acid sequence. In some embodiments, the artificial capsid amino acid sequence enables the vector particle to cross the blood-brain barrier.

In some embodiments, the AAV vector particle comprises a VP1 capsid protein comprising an amino acid sequence comprising at least four contiguous amino acids, such as at least five or 6, preferably seven amino acids, from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

In some embodiments, the AAV vector particle comprises a VP1 capsid protein comprising an amino acid sequence comprising the sequence DGTLAVPFKAQ (SEQ ID NO: 25). In some embodiments, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 70%, 75%, 80%, 85% or 90% identity to SEQ ID NO: 20, more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 20.

In some embodiments, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 70%, 75%, 80%, 85% or 90% identity to SEQ ID NO: 21 , more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 21.

In some embodiments, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 70%, 75%, 80%, 85% or 90% identity to SEQ ID NO: 22, more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 22.

In some embodiments, the vector is an AAV9 vector. In some embodiments, the vector particle is an AAV9 vector particle.

In some embodiments, the AAV vector particle has a serotype selected from the group consisting of AAV9; AAV9 PHP.B; AAV9 PHP.eB; and AAVrhIO. In some embodiments, the AAV vector particle has a AAV9 PHP.eB serotype.

In some embodiments, the AAV vector particle has a AAV-DJ serotype.

In some embodiments, the AAV vector particle comprises a capsid selected from the group consisting of an AAV9; AAV9 PHP.B; AAV9 PHP.eB; and AAVrhIO capsid. In some embodiments, the AAV vector particle comprises a AAV9 PHP.eB capsid.

In some embodiments, the vector is a cM2 AAV vector.

In some embodiments, the vector is in the form of a non-viral particle. In some embodiments, the vector is in the form of a nanoparticle.

In one aspect, the invention provides a cell comprising the polynucleotide or vector of the invention.

In one aspect, the invention provides a pharmaceutical composition comprising the polynucleotide, vector or cell of the invention and a pharmaceutically-acceptable carrier, diluent or excipient.

In some embodiments, the pharmaceutical composition is formulated for systemic or local delivery. In some embodiments, the pharmaceutical composition is formulated for intravascular, intravenous, intra-arterial, intracranial or intraparenchymal brain delivery. In one aspect, the invention provides the polynucleotide, vector, cell or pharmaceutical composition of the invention for use in therapy.

In one aspect, the invention provides the polynucleotide, vector, cell or pharmaceutical composition of the invention for use in treating or preventing Rett syndrome.

In one aspect the invention provides a method for treating or preventing Rett syndrome comprising administering the polynucleotide, vector, cell or pharmaceutical composition of the invention to a subject in need thereof.

In one aspect, the invention provides use of the polynucleotide, vector, cell or pharmaceutical composition of the invention for the manufacture of a medicament for treating or preventing Rett syndrome.

In some embodiments, the polynucleotide, vector or cell is administered to a subject systemically or locally.

In some embodiments, the polynucleotide, vector or cell is administered to a subject intracranially or intraparenchymally.

In some embodiments, the polynucleotide, vector or cell is administered simultaneously, sequentially or separately in combination with an immunosuppressant. In some embodiments, the immunosuppressant is cyclosporin A (CsA).

DESCRIPTION OF THE DRAWINGS

FIGURE 1

Increasing MeCP2 silencing in neuronal cultures transduced with AAV9 vectors containing additional number of miR-124 target sequences. A, AAV vector with the mouse Mecp2 isoform-1 sequence downstream of a CBA promoter and with 2 or 4 or none miR-124 target sequences (miR-124TS). B, V5 protein levels by Western blot on protein lysates of neuronal cultures transduced with an AAV9 containing the Mecp2 cassette with increasing number of miR-124TS.

FIGURE 2

Increasing MeCP2 silencing in astroglial cultures transduced with AAV9 vectors containing additional number of miR-31 target sequences. A, AAV vector with the mouse Mecp2 isoform-1 sequence downstream of a CBA promoter and with 2 or 4 or none miR-31 target sequences (miR-31TS). B, V5 protein levels by Western blot on protein lysates of neuronal cultures transduced with an AAV9 containing the Mecp2 cassette with increasing number of miR-31TS.

FIGURE 3

Increasing MeCP2 silencing in oligoglial cultures transduced with AAV9 vectors containing additional number of miR-338 target sequences. A, AAV vector with the mouse Mecp2 isoform-1 sequence downstream of a CBA promoter and with 2 or 4 or none miR-338 target sequences (miR-338TS). B, V5 protein levels by Western blot on protein lysates of neuronal cultures transduced with an AAV9 containing the Mecp2 cassette with increasing number of miR-338TS.

FIGURE 4

Increasing MeCP2 silencing in neuronal cultures transduced with AAV9 vectors containing additional number of miR-124 target sequences. A, Composition of the cM2- mR AAV vector containing 1 copy of the miR-124TS and 4 copies of both the miR-31TS and miR338TS. B, V5-Mecp2 protein levels by Western blot on protein lysates of neuronal, astroglial or oligoglial primary cultures transduced with an AAV9-cM2-mR showing significant lower levels of V5-Mecp2 in glial cells.

FIGURE S

Validation of the cM2-mR vector in vivo. A, The AAV9-cM2-mR vector was inoculated in the striatum of adult mice. B, High-magnification fluorescent images for the co-staining of neurons (NeuN) and astrocytes (GFAP) with V5-Mecp2. White arrows indicate the V5-Mecp2 staining in astrocytes that is significantly lower with respect to that found in neurons. Histogram represents quantification of the relative intensity of the v5-Mecp2 staining in neurons and astrocytes.

FIGURE S

Validation of the cM2-shU2 viral vector for the effective silencing of the endogenous Mecp2 protein. A, Top: Mecp2 genomic locus with highlighted in red the localisation on the 3'-UTR of the target sequences for the two shRNAs (U1 and U2); Bottom: structure of the cM2-shU2 vector which integrates the H1-shRNA-U2 cassette. B, Western blots for Mecp2 and viral V5-Mecp2 protein levels in primary neuronal cultures transduced with cM2-shU2 or the shU2 cassette only. H1-shRNA-U2 is highly efficient in downregulating endogenous, but not the viral Mecp2. FIGURE ?

Configuration of the AAV vector (cM2-ELO) which incorporates the H1-shRNA-U2 and the microRNA-TS cassettes.

FIGURE 8

A) Upper, scheme of the shRNA design to downregulate the endogenous human MECP2 gene. Lower, scheme of the experimental plan to test shRNA efficiency. In brief, HeLa cells were transfected with a plasmid carrying the shRNA (or a Scramble as a control) + GFP coding cassette under a constitutive promoter. B) RT-qPCR on transfected HeLa cells to analyze MEPC2 mRNA expression. Bar graphs depicting fold change differences of MECP2 mRNA in scramble and shRNA treated HeLa cells. * p<0.05 unpaired t-Test, n=3 biological replicates. C) Immunofluorescence analysis of transfected HeLa cells for GFP (green) and MeCP2 (red) to visualize the effect of the shRNA on MeCP2 protein levels. Scale bar: 100uM.

FIGURE 9

A) Upper, scheme of the protocol used to generate iPSC-derived human MECP2-KO neurons. Lower, scheme of the AAVs used to transduce V5-MECP2 coding sequence with or without the mirT cassette. B) Upper, immunofluorescence analysis of transduced human MECP2-KO neurons for V5 (green), MeCP2 (red) and MAP2 (magenta) to visualize the expression of the exogenous MeCP2 with or without the mirT. Scale bar: 100um. Lower, quantification of fluorescence intensity of exogenous MeCP2 with or without the mirT in human MECP2-KO neurons. * p<0.05 unpaired t-Test, n=30.

FIGURE 10

A) Upper, scheme of the protocol used to generate iPSC-derived human MECP2-KO astrocytes. Lower, scheme of the AAVs used to transduce V5-MECP2 coding sequence with or without the mirT cassette. B) Upper, immunofluorescence analysis of transduced human MECP2-KO astrocytes for V5 (green), and GFAP (red) to visualize the expression of the exogenous MeCP2 with or without the mirT. Scale bar: 100um. Lower, quantification of fluorescence intensity of exogenous MeCP2 with or without the mirT in human MECP2-KO astrocytes. * p<0.05 unpaired t-Test, n=30.

FIGURE 11

A) Scheme of the experimental protocol. In brief, neonatal /Wecp2-KO mice were treated with the AAV_V5-Mecp2-mirT + shRNA by intracerebroventricular injection. Treated animals were then sacrificed after 1 month from viral inoculation. B) Upper, immunofluorescence analysis of transduced cortexes for V5 (green) and in red a marker specific for neurons (NeuN) or astrocytes (Sox9). Scale bar: 200um. Lower, quantification of fluorescence intensity of exogenous Mecp2 (V5) in neurons (NeuN+) and astrocytes (Sox9+). ** p<0.01 unpaired t- Test, n=3 mice per group.

DETAILED DESCRIPTION OF THE INVENTION

The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including” or “includes”; or “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”.

RETT SYNDROME

In the vast majority of cases RTT is caused by loss-of-function mutations in the MECP2 gene, which encodes the methyl-CpG binding protein 2 (MeCP2) (Bienvenu, T. et al. (2006) Nat Rev Genet 7: 415-426).

Recent studies have revealed that the Mecp2 loss significantly alters neuronal activity leading to a progressive imbalance of the excitatory-inhibitory synaptic activity across the brain with divergent modalities occurring between different circuits and regions of the brain (Banerjee, A. et al. (2016) PNAS 113: E7287-E7296).

METHYL-CPG BINDING-PROTEIN 2 (MECP2)

Methyl-CpG binding protein 2 (MeCP2) is a global chromatin regulator highly expressed in neurons.

In some embodiments, the MeCP2 is human or mouse MeCP2. In preferred embodiments, the MeCP2 is human MeCP2.

An example MeCP2 sequence is:

MAAAAAAAPSGGGGGGEEERLEEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAE TSEGSGSAPAVPEASASPKQRRSI IRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVEL IAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRVL EKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAI PKKRGRKPGSWAAAAAEAKKKAV

KESSIRSVQETVLPIKKRKTRETVSIEVKEWKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPK

KEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQP

AVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS

( SEQ ID NO : 1 ; human )

A further example MeCP2 sequence is:

MAAAAATAAAAAAPSGGGGGGEEERLEEKSEDQDLQGLRDKPLKFKKAKKDKKEDKEGKHEPLQPSAHHSAEPAE

AGKAETSESSGSAPAVPEASASPKQRRSI IRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFR

SKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTGRPKAAASEGVQ

VKRVLEKSPGKLWKMPFQASPGGKGEGGGATTSAQVMVIKRPGRKRKAEADPQAI PKKRGRKPGSWAAAAAEA

KKKAVKESSIRSVHETVLPIKKRKTRETVSIEVKEWKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSA

SSPPKKEHHHHHHHSESTKAPMPLLPSPPPPEPESSEDPI SPPEPQDLSSSICKEEKMPRGGSLESDGCPKEPAK

TQPMVATTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS

( SEQ ID NO : 2 ; mouse )

A further example MeCP2 sequence is:

MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVP

EASASPKQRRSI IRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSL

DPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMP

FQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAI PKKRGRKPGSWAAAAAEAKKKAVKESSIRSVQETV

LPIKKRKTRETVSIEVKEWKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHHHHHSES

PKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKY KHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS

( SEQ ID NO : 28 ; human )

An example nucleotide sequence encoding MeCP2 is:

ATGGCCGCCGCCGCCGCCGCCGCGCCGAGCGGAGGAGGAGGAGGAGGCGAGGAGGAGAGACTGGAAGAAAAGTCA

GAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAG

AAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAG

ACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATC

CGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCT

GGCCGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTG

ATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGG

AGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGG

GGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGGCCACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTG

GAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGG

GCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCCGACCCTCAGGCC ATTCCCAAGAAACGGGGCCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTG

AAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGC

ATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACC

TGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAG

AAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCA

CCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGC

AAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCC

GCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCA

TCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCTGA

( SEQ ID NO : 3 ; human )

A further example nucleotide sequence encoding MeCP2 is:

ATGGGCAAGCCTATCCCTAACCCTCTGCTGGGCCTGGACTCCACAGGCAGCGGCACCGGTATGGCCGCCGCTGCC

GCCACCGCCGCCGCCGCCGCCGCGCCGAGCGGAGGAGGAGGAGGAGGCGAGGAGGAGAGACTGGAGGAAAAGTCA

GAAGACCAGGATCTCCAGGGCCTCAGAGACAAGCCACTGAAGTTTAAGAAGGCGAAGAAAGACAAGAAGGAGGAC

AAAGAAGGCAAGCATGAGCCACTACAACCTTCAGCCCACCATTCTGCAGAGCCAGCAGAGGCAGGCAAAGCAGAA

ACATCAGAAAGCTCAGGCTCTGCCCCAGCAGTGCCAGAAGCCTCGGCTTCCCCCAAACAGCGGCGCTCCATTATC

CGTGACCGGGGACCTATGTATGATGACCCCACCTTGCCTGAAGGTTGGACACGAAAGCTTAAACAAAGGAAGTCT

GGCCGATCTGCTGGAAAGTATGATGTATATTTGATCAATCCCCAGGGAAAAGCTTTTCGCTCTAAAGTAGAATTG

ATTGCATACTTTGAAAAGGTGGGAGACACCTCCTTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGG

AGCCCCTCCAGGAGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGGGGTCGG

GGACGCCCCAAAGGGAGCGGCACTGGGAGACCAAAGGCAGCAGCATCAGAAGGTGTTCAGGTGAAAAGGGTCCTG

GAGAAGAGCCCTGGGAAACTTGTTGTCAAGATGCCTTTCCAAGCATCGCCTGGGGGTAAGGGTGAGGGAGGTGGG

GCTACCACATCTGCCCAGGTCATGGTGATCAAACGCCCTGGCAGAAAGCGAAAAGCTGAAGCTGACCCCCAGGCC

ATTCCTAAGAAACGGGGTAGAAAGCCTGGGAGTGTGGTGGCAGCTGCTGCAGCTGAGGCCAAAAAGAAAGCCGTG

AAGGAGTCTTCCATACGGTCTGTGCATGAGACTGTGCTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGC

ATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTTGGTGAGAAAAGCGGGAAGGGACTGAAGACC

TGCAAGAGCCCTGGGCGTAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGTGCCTCCTCCCCACCTAAG

AAGGAGCACCATCATCACCACCATCACTCAGAGTCCACAAAGGCCCCCATGCCACTGCTCCCATCCCCACCCCCA

CCTGAGCCTGAGAGCTCTGAGGACCCCATCAGCCCCCCTGAGCCTCAGGACTTGAGCAGCAGCATCTGCAAAGAA

GAGAAGATGCCCCGAGGAGGCTCACTGGAAAGCGATGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCTATGGTC

GCCACCACTACCACAGTTGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCTTCC

ATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCTGA

( SEQ ID NO : 4 ; mouse )

A further example nucleotide sequence encoding MeCP2 is:

ATGGTAGCTGGGATGTTAGGGCTCAGGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCC

CTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCC

CACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCG GAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTG CCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCTGGCCGCTCTGCTGGGAAGTATGATGTGTATTTGATC AATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTG GACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAG CCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAG GCGGCCACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCT TTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGC CCCGGCAGGAAGCGAAAAGCTGAGGCCGACCCTCAGGCCATTCCCAAGAAACGGGGCCGAAAGCCGGGGAGTGTG GTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTA CTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTG TCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGC CCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCC CCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACC AGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAG AGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTAC AAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTG GACAGCCGGACGCCCGTGACCGAGAGAGTTAGCTAA

( SEQ ID NO : 29 ; human )

In some embodiments, the MeCP2 is encoded by a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% 99% or 100% identity to SEQ ID NO: 3 or 4 (preferably SEQ ID NO: 3), preferably wherein the protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by any one of SEQ ID NOs: 1 or 2.

In some embodiments, the MeCP2 is encoded by a nucleotide sequence that encodes an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% 99% or 100% identity to SEQ ID NO: 1 or 2 (preferably SEQ ID NO: 1), preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NOs: 1 or 2.

In some embodiments, the MeCP2 comprises or consists of an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% 99% or 100% identity to SEQ ID NO: 1 or 2 (preferably SEQ ID NO: 1), preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NOs: 1 or 2.

In some embodiments, the MeCP2 is encoded by a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% 99% or 100% identity to SEQ ID NO: 29, preferably wherein the protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented SEQ ID NO: 28. In some embodiments, the MeCP2 is encoded by a nucleotide sequence that encodes an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% 99% or 100% identity to SEQ ID NO: 28, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 28.

In some embodiments, the MeCP2 comprises or consists of an amino acid sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% 99% or 100% identity to SEQ ID NO: 28, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 28.

BLOOD-BRAIN BARRIER (BBB)

The term “blood brain barrier” (BBB) as used herein means the highly selective semi- permeable membrane barrier which separates the circulating blood from the brain and extracellular fluid in the central nervous system. It is formed by the selectivity of tight junctions between endothelial cells. The blood-brain barrier occurs along all capillaries of the brain and consists of tight junctions.

Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of most brain disorders.

Preferably, the vector particle (e.g. the AAV vector particle) is adapted for crossing an intact blood brain barrier. Preferably the vector particle (e.g. the AAV vector particle) does not impair blood-brain barrier integrity and/or selectivity and/or affect permeability.

In other words, the vector particle is adapted to cross a blood-brain barrier which has not been compromised or weakened, i.e. which maintains tight junctions between endothelial cells. Methods are known in the art which can determine whether or not a blood brain barrier is intact. For example, the permeability of the blood-brain barrier can be detected by perfusion of Evan’s blue dye. Alternatively a fluorescent-conjugated cadaverine dye can be used as a blood-brain barrier permeability marker, together with the AAV particle carrying a fluorescent marker.

Preferably, the vector particle (e.g. the AAV vector particle) does not cause microgliosis. Suitably, the vector particle (e.g. the AAV vector particle) does not cause sustained inflammation in the central nervous system.

The “central nervous system” as used herein means the nervous system consisting of the brain and spinal cord. The “peripheral nervous system” as used herein means the components of the nervous system outside of the central nervous system. The peripheral nervous system consists of the nerves and ganglia outside of the brain and spinal cord.

MICRORNA TARGET SEQUENCES

In some embodiments, the polynucleotide comprises one or more miRNA target sequence. Suitably, the nucleic acid sequence encoding MeCP2 is operably linked to the one or more miRNA target sequence.

MicroRNA (miRNA) genes are scattered across all human chromosomes, except for the Y chromosome. They can be either located in non-coding regions of the genome or within introns of protein-coding genes. Around 50% of miRNAs appear in clusters which are transcribed as polycistronic primary transcripts. Similar to protein-coding genes, miRNAs are usually transcribed from polymerase-ll promoters, generating a so-called primary miRNA transcript (pri-miRNA). This pri-miRNA is then processed through a series of endonucleolytic cleavage steps, performed by two enzymes belonging to the RNAse Type III family, Drosha and Dicer. From the pri-miRNA, a stem loop of about 60 nucleotides in length, called miRNA precursor (pre-miRNA), is excised by a specific nuclear complex, composed of Drosha and DiGeorge syndrome critical region gene (DGCR8), which crops both strands near the base of the primary stem loop and leaves a 5’ phosphate and a 2 bp long, 3’ overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by RAN-GTP and Exportin. Then, Dicer performs a double strand cut at the end of the stem loop not defined by the Drosha cut, generating a 19-24 bp duplex, which is composed of the mature miRNA and the opposite strand of the duplex, called miRNA*. In agreement with the thermodynamic asymmetry rule, only one strand of the duplex is selectively loaded into the RNA-induced silencing complex (RISC), and accumulates as the mature microRNA. This strand is usually the one whose 5’ end is less tightly paired to its complement, as was demonstrated by single-nucleotide mismatches introduced into the 5’ end of each strand of siRNA duplexes. However, there are some miRNAs that support accumulation of both duplex strands to similar extent.

MicroRNAs trigger RNAi, very much like small interfering RNAs (siRNA) which are extensively used for experimental gene knockdown. The main difference between miRNA and siRNA is their biogenesis. Once loaded into RISC, the guide strand of the small RNA molecule interacts with mRNA target sequences preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. It has been shown that nucleotides 2-8 counted from the 5' end of the miRNA, the so-called seed sequence, are essential for triggering RNAi. If the whole guide strand sequence is perfectly complementary to the mRNA target, as is usually the case for siRNAs and plant miRNAs, the mRNA is endonucleolytically cleaved by involvement of the Argonaute (Ago) protein, also called “slicer” of the small RNA duplex into the RNA-induced silencing complex (RISC). DGRC (DiGeorge syndrome critical region gene 8) and TRBP (TAR (HIV) RNA binding protein 2) are double-stranded RNA-binding proteins that facilitate mature miRNA biogenesis by Drosha and Dicer RNase III enzymes, respectively. The guide strand of the miRNA duplex gets incorporated into the effector complex RISC, which recognises specific targets through imperfect base-pairing and induces post-transcriptional gene silencing. Several mechanisms have been proposed for this mode of regulation: miRNAs can induce the repression of translation initiation, mark target mRNAs for degradation by deadenylation, or sequester targets into the cytoplasmic P-body.

On the other hand, if only the seed is perfectly complementary to the target mRNA but the remaining bases show incomplete pairing, RNAi acts through multiple mechanisms leading to translational repression. Eukaryotic mRNA degradation mainly occurs through the shortening of the polyA tail at the 3’ end of the mRNA, and de-capping at the 5’ end, followed by 5’-3’ exonuclease digestion and accumulation of the miRNA in discrete cytoplasmic areas, the so called P-bodies, enriched in components of the mRNA decay pathway.

Expression of the nucleic acid sequence encoding the transgene may be regulated by one or more endogenous miRNA using one or more corresponding miRNA target sequence. Using this method, one or more miRNA endogenously expressed in a cell prevents or reduces transgene expression in that cell by binding to its corresponding miRNA target sequence positioned in the polynucleotide or vector.

The target sequence may be fully or partially complementary to the miRNA. The term “fully complementary”, as used herein, may mean that the target sequence has a nucleic acid sequence which is 100% complementary to the sequence of the miRNA which recognises it. The term “partially complementary”, as used herein, may mean that the target sequence is only in part complementary to the sequence of the miRNA which recognises it, whereby the partially complementary sequence is still recognised by the miRNA. In other words, a partially complementary target sequence in the context of the present invention is effective in recognising the corresponding miRNA and effecting prevention or reduction of transgene expression in cells expressing that miRNA. Suitably, a partially complementary miRNA target sequence may be fully complementary to the miRNA seed sequence.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and at least one miR-124 target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and at least one miR-31 target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and at least one miR-338-3p target sequence.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), and at least one miR-124 target sequence and at least one miR-31 target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), and at least one miR-124 target sequence and at least one miR-338-3p target sequence. In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2), and at least one miR-124 target sequence, at least one miR-31 target sequence and at least one miR-338-3p target sequence.

Including more than one copy of a miRNA target sequence may increase the effectiveness of the system. Also, different miRNA target sequences can be included. For example, the proteincoding sequence may be operably linked to more than one miRNA target sequence, which may or may not be different. The miRNA target sequences may be in tandem, but other arrangements are envisaged. The polynucleotide may, for example, comprise 1 , 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequences.

Copies of miRNA target sequences may be separated by a spacer sequence. The spacer sequence may comprise, for example, at least one, at least two, at least three, at least four or at least five nucleotide bases.

An exemplary miR-124 target sequence is:

TATTGCCTTATTTC

( SEQ ID NO : 5 )

An exemplary miR-31 target sequence is:

CAGCTATGCCAGCATCTTGCC

( SEQ ID NO : 6 )

An exemplary miR-338-3p target sequence is:

C AACAAAAT CACT GATGCT GGA

( SEQ ID NO : 7 ) In some embodiments, the miR-338-3p target sequence comprises or consists of a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 7.

In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-124, miR-338-3p and miR-31.

In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-338-3p, miR-124 and miR-31.

In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-338-3p, miR-31 and miR-124.

In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-31 , miR-124 and miR-338-3p.

In some embodiments, the miRNA target sequences or clusters of copies of sequences are, from 5’ to 3’, arranged in the order: miR-31 , miR-338-3p and miR-124.

An exemplary triple miRNA target sequence combination is:

TATTGCCTTATTTCa cgcgtcaattg a CAGCTATGCCAGCATCTTGCCg 111 a a a c CAGCTATGCCAG CATCTTGCCa c t a g t CAGCTATGCCAGCATCTTGCCG ctagagaag g CAGCTATGCCAGCATCTTGCC actagtgtttaaaccctagagaaggcaattgtatcgataCAACAAAATCACTGATGCTGGAagcgctC AACAAAATCAC TGATGC TGGAg c t a g cCAACAAAATCACTGATGCTGGAg gccgcctcggc cCAACAA AATCACTGATGCTGGA ( SEQ ID NO : 8 ) miR-124 target sequence : Underline miR-31 target sequence : Itali c and underline miR-338-3p target sequence : Bold and underline

In some embodiments, the one or more miRNA target sequence suppresses transgene expression in neurons. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in astrocytes. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in oligodendrocytes.

The term “suppress expression” as used herein may refer to a reduction of expression in the relevant cell type(s) of a transgene to which the one or more miRNA target sequence is operably linked as compared to transgene expression in the absence of the one or more miRNA target sequence, but under otherwise substantially identical conditions. In some embodiments, transgene expression is suppressed by at least 50%. In some embodiments, transgene expression is suppressed by at least 60%, 70%, 80%, 90% or 95%. In some embodiments, transgene expression is substantially prevented.

Both the individual target sequences and the clusters of sequences may be contiguous with one another, separated by spacer sequences, or any combination thereof.

In some embodiments, the miRNA target sequences are separated by spacer sequences.

As used herein, a “spacer” may be a sequence (e.g. a nucleotide or amino acid sequence) that may be used to separate other sequence elements within a larger polymer.

Individual miRNA target sequences or groups of miRNA target sequences may be separated by one or more spacer sequence. In some embodiments, the miRNA target sequences are separated by one or more spacer sequence. The spacer sequence may comprise, for example, at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, or at least thirty nucleotide bases.

PROMOTERS AND REGULATORY SEQUENCES

The polynucleotides and vectors of the invention include elements allowing for the expression of MeCP2 in vitro or in vivo. These may be referred to as expression control sequences. Thus, the polynucleotides and vectors typically comprise expression control sequences (e.g. comprising a promoter sequence) operably linked to the nucleotide sequence encoding the transgene.

Any suitable promoter may be used, the selection of which may be readily made by the skilled person. The promoter sequence may be constitutively active (i.e. operational in any host cell background), or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the transgene in a particular cell type (e.g. a tissue-specific promoter such as an endothelial specific promoter). The promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the polynucleotide or vector is administered for therapy, it is preferred that the promoter should be functional in the target cell background.

In some embodiments, the promoter is neural-cell specific. In some embodiments, the promoter is astrocyte specific.

In some embodiments, the promoter is a chicken p-actin (CBA) promoter.

An example CBA promoter is:

CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGAC GTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA CTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC CATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTT ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCG GGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGC TGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCA CAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCCCGTTTAGTGAACCGTCAGATCGCCTGGA GACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGC CGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCC ACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTT CAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAA GGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTA ATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAG CTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGT GCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACA

( SEQ ID NO : 9 )

In some embodiments, the CBA promoter comprises or consists of a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 9.

The chicken beta-actin (CBA) promoter may optionally be used in combination with a cytomegalovirus (CMV) enhancer element.

The polynucleotide or vector of the invention comprise a 3’-UTR that is less than or equal to about 1000 bp in length.

In preferred embodiments, the 3’-UTR is derived from the MeCP2 3’-UTR (e.g. is a truncated form thereof). In preferred embodiments, the 3’-UTR is a truncated MeCP2 3’IITR.

Preferably, the 3’-UTR (e.g. the MeCP2 3’-UTR) is truncated at the 3’ end (e.g. retains its natural 5’ end).

In some embodiments, the 3’-UTR is a synthetic UTR assembled from regulatory elements in the wild type (8.6 kb long) 3’-UTR, as described in Matagne, V. et al. (2017) Neurobiol. Dis. 99: 1-11.

An example 3’-UTR sequence is:

CTTTACATAGAGCGGATTGCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGG CTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAACTTAGAGTTTCGTGGCT TCGGGGTGGGAGTAGTTGGAGCATTGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTAACCA

( SEQ ID NO : 10 ; mouse )

A further example 3’-UTR sequence is: CTTTACACGGAGCGGATTGCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGG

CTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAACTTAGAGTTTCGTGGCT TCAGGGTGGGAGTAGTTGGAGCATTGGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTAACCA

( SEQ ID NO : 11 ; human )

A further example 3’-UTR sequence is:

CTTTACACGGAGCGGATTGCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGG CTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA

( SEQ ID NO : 12 ; human )

In some embodiments, the 3’-UTR comprises or consists of a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 10, 11 or 12.

The polynucleotide or vector of the invention may also comprise one or more additional regulatory sequences which may act pre- or post-transcriptionally.

Regulatory sequences are any sequences which facilitate expression of the transgene, i.e. act to increase expression of a transcript, improve nuclear export of mRNA or enhance its stability. Such regulatory sequences include for example enhancer elements, post-transcriptional regulatory elements and polyadenylation sites. An example of a polyadenylation site is the Human or Bovine Growth Hormone poly-A signal.

An example human growth hormone poly-A sequence is:

TCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGT GCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGG GTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACC AAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCA GCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGG TTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCT GGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTT

( SEQ ID NO : 13 )

A further example poly-A sequence is:

GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTT GTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTG GTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCA GTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTG TTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTG GCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCG TGAACCACTGCTCCCTTCCCTGTCCTT

( SEQ ID NO : 14 )

In some embodiments, the poly-A sequence comprises or consists of a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 13 or 14.

An example of a post-transcriptional regulatory element for use in a polynucleotide or vector of the invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

Another regulatory sequence which may be used in a polynucleotide or vector of the invention is a scaffold-attachment region (SAR). Additional regulatory sequences may be readily selected by the skilled person.

INHIBITOR OF MECP2 EXPRESSION

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an inhibitor of MeCP2 expression (preferably endogenous MeCP2 expression).

The term “inhibitor”, as used herein in the context of inhibition of MeCP2 expression, may refer to an agent that reduces the expression of MeCP2 relative to the level of MeCP2 expression in the absence of the agent, but under otherwise substantially identical conditions. The inhibitor may, for example, reduce expression of MeCP2 (preferably endogenous MeCP2) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% relative to the level of MeCP2 expression in its absence. Preferably, the inhibitor reduces expression of MeCP2 by at least 70% relative to the level of MeCP2 expression in its absence. In some embodiments, the inhibitor prevents expression of MeCP2 (preferably endogenous MeCP2) entirely.

Expression levels of a protein, such as MeCP2, may be readily measured and quantified by the skilled person using techniques well known in the art, for example using Western blotting.

In some embodiments, the inhibitor is specific for endogenous MeCP2.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an inhibitor of endogenous MeCP2 expression (e.g. inhibits expression of MeCP2 that is endogenous to a cell into which the polynucleotide is introduced). In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding an inhibitor of endogenous MeCP2 expression (e.g. inhibits expression of MeCP2 that is endogenous to a cell into which the polynucleotide is introduced).

In some embodiments, the inhibitor does not inhibit expression of the MeCP2 encoded by the polynucleotide of the invention. In some embodiments, the inhibitor substantially does not inhibit expression of the MeCP2 encoded by the polynucleotide of the invention.

In some embodiments, the inhibitor inhibits expression of endogenous MeCP2 more than it inhibits expression of the MeCP2 encoded by the polynucleotide of the invention. For example, the inhibitor may inhibit expression of endogenous MeCP2 by at least 1 .5-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 1000-fold more than it inhibits expression of the MeCP2 encoded by the polynucleotide of the invention.

In some embodiments, the inhibitor targets the 3’-UTR of a gene (preferably an endogenous gene) encoding MeCP2. In some embodiments, the section of the 3’-UTR targeted by the inhibitor is not comprised in the polynucleotide of the invention.

In some embodiments, the inhibitor is an shRNA, siRNA, miRNA or antisense DNA/RNA. Preferably, the inhibitor is an shRNA.

In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG binding-protein 2 (MeCP2) and a nucleotide sequence encoding an shRNA that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 16.

An example nucleotide sequence encoding an shRNA that inhibits expression of MeCP2 is:

GGAGAAGACAGAAGAT AAA

( SEQ ID NO : 15 ) ( shRNA-U2 )

A further example nucleotide sequence encoding an shRNA that inhibits expression of MeCP2 is:

GAT T GT AGAT T CAGGT T AA ( SEQ ID NO : 16 ) ( shRNA-Ul )

In some embodiments, the polynucleotide further comprises a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 15.

In some embodiments, the polynucleotide further comprises a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16.

GAGACAGTTGGATTCTTTA

( SEQ ID NO : 30 )

TAAAGAAT CCAACT GT CT C

( SEQ ID NO : 31 )

In some embodiments, the polynucleotide further comprises a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 30.

In some embodiments, the polynucleotide further comprises a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 31.

The nucleotide sequence encoding the inhibitor of MeCP2 expression may be operably linked to a promoter, such as a H1 promoter.

An exemplary H1 promoter sequence is: GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTG

CGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTG GGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCAC

( SEQ ID NO : 17 )

In some embodiments, the H1 promoter comprises or consists of a nucleotide sequence that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 17. siRNAs, shRNAs, miRNAs and antisense DNAs/RNAs

Inhibition (e.g. of the MeCP2) may be achieved using post-transcriptional gene silencing (PTGS). Post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) is a conserved cellular defence mechanism for controlling the expression of foreign genes. It is thought that the random integration of elements such as transposons or viruses causes the expression of dsRNA which activates sequence-specific degradation of homologous singlestranded mRNA or viral genomic RNA. The silencing effect is known as RNA interference (RNAi) (Ralph et al. (2005) Nat. Medicine 11 : 429-433). The mechanism of RNAi involves the processing of long dsRNAs into duplexes of about 21-25 nucleotide (nt) RNAs. These products are called small interfering or silencing RNAs (siRNAs) which are the sequence-specific mediators of mRNA degradation. In differentiated mammalian cells, dsRNA >30 bp has been found to activate the interferon response leading to shut-down of protein synthesis and nonspecific mRNA degradation (Stark et al. (1998) Ann. Rev. Biochem. 67: 227-64). However, this response can be bypassed by using 21 nt siRNA duplexes (Elbashir et al. (2001) EMBO J. 20: 6877-88; Hutvagner et al. (2001) Science 293: 834-8) allowing gene function to be analysed in cultured mammalian cells. shRNAs consist of short inverted RNA repeats separated by a small loop sequence. These are rapidly processed by the cellular machinery into 19-22 nt siRNAs, thereby suppressing the target gene expression.

Micro-RNAs (miRNAs) are small (22-25 nucleotides in length) noncoding RNAs that can effectively reduce the translation of target mRNAs by binding to their 3’ untranslated region (UTR). Micro-RNAs are a very large group of small RNAs produced naturally in organisms, at least some of which regulate the expression of target genes. Founding members of the micro- RNA family are let-7 and lin-4. The let-7 gene encodes a small, highly conserved RNA species that regulates the expression of endogenous protein-coding genes during worm development. The active RNA species is transcribed initially as an ~70 nt precursor, which is post- transcriptionally processed into a mature ~21 nt form. Both let-7 and lin-4 are transcribed as hairpin RNA precursors which are processed to their mature forms by Dicer enzyme. The antisense concept is to selectively bind short, possibly modified, DNA or RNA molecules to messenger RNA in cells and prevent the synthesis of the encoded protein.

Methods for the design of siRNAs, shRNAs, miRNAs and antisense DNAs/RNAs to modulate the expression of a target protein are well known in the art.

VECTORS

A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid and/or facilitating the expression of the protein encoded by a segment of nucleic acid.

Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation.

Transfection may refer to a general process of incorporating a nucleic acid into a cell and includes a process using a non-viral vector to deliver a polynucleotide to a cell. Transduction may refer to a process of incorporating a nucleic acid into a cell using a viral vector.

Preferably, the vectors used to transduce cells in the invention are viral vectors. The vectors of the invention are preferably adeno-associated viral (AAV) vectors, although it is contemplated that other viral vectors may be used. The vector may be, for example, an adeno- associated viral (AAV) vector, a lentiviral vector, a retroviral vector or an adenoviral vector.

Preferably, the viral vector for use according to the present invention is in the form of a viral vector particle. In some embodiments, the viral vector particle is adapted for crossing the blood-brain barrier.

In some embodiments, the viral vector particle adapted for crossing the blood-brain barrier for use according to the invention is a retroviral, lentiviral, adeno-associated viral (AAV) or adenoviral vector particle. Preferably, the viral vector particle is a lentiviral or AAV vector particle, more preferably an AAV vector particle. Although, some embodiments of the invention have been described with respect to AAV vector particles, it will be appreciated that some embodiments may apply mutatis mutandis to other viral vectors disclosed herein.

Transfection of cells with mRNA vectors can be achieved, for example, using nanoparticles, such as liposomes.

In some embodiments, the vector is comprised in a nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle, inorganic nanoparticle or lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

The nanoparticle may be targeted to a specific cell type(s) using one or more ligand displayed on its surface.

In some embodiments, polynucleotide delivery is transposon mediated.

In some embodiments, the polynucleotide is an mRNA. The mRNA may be comprised in a nanoparticle.

Adeno-associated viral (AAV) vectors

In one aspect the invention provides a AAV vector particle, wherein the AAV vector particle comprises a polynucleotide comprising a nucleotide sequence encoding methyl-CpG bindingprotein 2 (MeCP2) and: (a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or (b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the AAV vector particle is adapted for crossing the blood-brain barrier.

In one aspect the invention provides a AAV vector particle, wherein the AAV vector particle comprises a polynucleotide comprising a nucleotide sequence encoding methyl-CpG bindingprotein 2 (MeCP2) and: (a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or (b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the AAV vector particle is adapted for crossing the blood-brain barrier.

In one aspect the invention provides a AAV vector particle, wherein the AAV vector particle comprises a polynucleotide comprising a nucleotide sequence encoding methyl-CpG bindingprotein 2 (MeCP2) and: (a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or (b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31. In some embodiments, the AAV vector particle is adapted for crossing the blood-brain barrier.

In one aspect the invention provides a AAV vector particle, wherein the AAV vector particle comprises a polynucleotide comprising a nucleotide sequence encoding methyl-CpG bindingprotein 2 (MeCP2) and: (a) at least one miR-124 target sequence, and/or at least one miR-31 target sequence, and/or at least one miR-338-3p target sequence; and/or (b) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 30; and/or (c) a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 31. In some embodiments, the AAV vector particle is adapted for crossing the blood-brain barrier.

Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

The AAV vector may comprise an AAV genome or a fragment or derivative thereof.

An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replication-deficient.

The AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.

The AAV genome may be from any naturally derived serotype, isolate or clade of AAV. Thus, the AAV genome may be the full genome of a naturally occurring AAV. As is known to the skilled person, AAVs occurring in nature may be classified according to various biological systems.

Commonly, AAVs are referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, a virus having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 , and also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain.

Several rAAV vectors have been reported to efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system (Zhang et al., Molecular Therapy 19: 1440-1448).

In some embodiments, the AAV is an AAV1 , AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 serotype. In some embodiments, the AAV vector particle comprises an AAV1 , AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 serotype capsid protein. In some embodiments, the AAV vector particle is an AAV1 , AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 vector particle.

In some embodiments, the AAV is an AAV9; AAV9 PHP.B; AAV9 PHP.eB; or AAVrhIO serotype. In some embodiments, the AAV vector particle comprises an AAV9; AAV9 PHP.B; AAV9 PHP.eB; or AAVrhIO serotype capsid protein.

In some embodiments, the AAV is an AAV-DJ serotype. In some embodiments, the AAV vector particle comprises a AAV-DJ capsid. AAV-DJ is described in Kondratov et al. (2021) Mol. Ther. 29: 2806-2820.

The capsid protein may be an artificial or mutant capsid protein.

The term “artificial capsid” as used herein means that the capsid particle comprises an amino acid sequence which does not occur in nature or which comprises an amino acid sequence which has been engineered (e.g. modified) from a naturally occurring capsid amino acid sequence.

In other words the artificial capsid protein comprises a mutation or a variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived where the artificial capsid amino acid sequence and the parent capsid amino acid sequences are aligned. Methods of sequence alignment are well known in the art and referenced herein.

The term “adapted for crossing the blood brain barrier” as used herein means that the vector particle has the ability to cross the blood brain barrier, for example the vector particle may comprise a mutation or modification relative to the wild type vector particle which improves the ability to cross the blood brain barrier relative to an unmodified or wild type viral particle. Improved ability to cross the blood brain barrier may be measured for example by measuring the expression of a transgene, e.g. GFP, carried by the vector particle, wherein expression of the transgene in the brain correlates with the ability of the viral particle to cross the blood brain barrier.

In some embodiments, the AAV vector particle comprises an artificial capsid amino acid sequence which enables the viral particle to cross the blood-brain barrier.

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising at least four contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising at least five contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising at least six contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

In some embodiments, the nucleic acid sequence encoding the at least four, at least five, at least six or all seven contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19) is inserted at a position corresponding to the position between a sequence encoding for amino acids 588 and 589 of AAV9 (SEQ ID NO: 20).

An example amino acid sequence of the (wild-type) AAV9 capsid is:

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEH DKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSS GNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMI P QYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKT INGSGQNQQTLKFSVAGPSNMAVQGRNYI PGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA SHKEGEDRFFPLSGSLI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQG ILPGMVWQDRDVYLQGPIWAKI PHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

( SEQ ID NO : 20 )

In some embodiments, the AAV vector particle comprises a AAV9 PHP.B capsid, preferably the AAV-PHP.B VP1 capsid protein.

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier is AAV9 PHP.B.

In some embodiments, the amino acid sequence of the AAV-PHP.B capsid VP1 protein is:

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEH DKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSS GNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMI P QYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT INGSGQNQQTLKFSVAGPSNMAVQGRNYI PGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA SHKEGEDRFFPLSGSLI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQAQT GWVQNQGILPGMVWQDRDVYLQGPIWAKI PHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLN SFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

( SEQ ID NO : 21 )

In some embodiments, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 70%, 75%, 80%, 85% or 90% identity to SEQ ID NO: 21 , more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 21 , wherein the AAV vector particle is capable of crossing the blood-brain barrier.

The AAV-PHP.B vector is described in Deverman et al. (2016) Nat Biotechnol 34: 204-209 and WO 2015/038958, which are incorporated herein by reference.

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising the sequence DGTLAVPFKAQ (SEQ ID NO: 25).

In some embodiments, the AAV vector particle capable of crossing the blood-brain barrier is AAV9 PHP.eB.

In some embodiments, the amino acid sequence of the AAV-PHP.eB capsid VP1 protein is: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEH DKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSS GNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMI P QYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKT INGSGQNQQTLKFSVAGPSNMAVQGRNYI PGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA SHKEGEDRFFPLSGSLI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSDGTLAVPFKAQAQT GWVQNQGILPGMVWQDRDVYLQGPIWAKI PHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLN SFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

( SEQ ID NO : 22 )

In some embodiments, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 70%, 75%, 80%, 85% or 90% identity to SEQ ID NO: 22, more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 22, wherein the AAV vector particle is capable of crossing the blood-brain barrier.

The AAV-PHP.eB vector is described in WO 2017/100671 , which is incorporated herein by reference.

Reviews of AAV serotypes may be found in Choi et al. (2005) Curr. Gene Ther. 5: 299-310 and Wu et al. (2006) Molecular Therapy 14: 316-27. The sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use in the invention may be derived from the following accession numbers for AAV whole genome sequences: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401 ; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno- associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno- associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAVs, and typically to a phylogenetic group of AAVs which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAVs which has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognisably distinct population at a genetic level. The skilled person can select an appropriate serotype, clade, clone or isolate of AAV for use in the invention on the basis of their common general knowledge.

The AAV serotype determines the tissue specificity of infection (or tropism) of an AAV virus.

Typically, the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. In preferred embodiments, one or more ITR sequences flank the nucleotide sequences encoding the MeCP2 nucleotide sequence. The AAV genome may also comprise packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1 , VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV particle.

A promoter will be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76: 5567-5571). For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene.

As discussed above, the AAV genome used in the AAV vector of the invention may therefore be the full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle in vitro. However, while such a vector may in principle be administered to patients, this will rarely be done in practice. Preferably the AAV genome will be derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatisation of the AAV genome and of the AAV capsid are reviewed in Coura and Nardi (2007) Virology Journal 4: 99, and in Choi et al. and Wu et al., referenced above.

Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell. Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.

In some embodiments, the AAV vector comprises at least one, such as two, AAV1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 ITRs. In some embodiments, the AAV vector comprises at least one AAV9 ITR.

In some embodiments, the AAV vector comprises two AAV9 ITRs.

The one or more ITRs will preferably flank the nucleotide sequence encoding MeCP2 at either end. The inclusion of one or more ITRs is preferred to aid concatamer formation of the vector of the invention in the nucleus of a host cell, for example following the conversion of singlestranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

In preferred embodiments, ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, a derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

The following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, in some embodiments, derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting. Where a derivative comprises capsid proteins i.e. VP1 , VP2 and/or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones or isolates of AAV within the same vector (i.e. a pseudotyped vector). Thus, in one embodiment the AAV vector is in the form of a pseudotyped AAV vector particle.

Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the AAV vector. Thus, these derivatives may display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting of a particular cell type compared to an AAV vector comprising a naturally occurring AAV genome, such as that of AAV2. Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to doublestranded form. Increased efficiency may also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed.

Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are co-transfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology. A library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.

The sequences of the capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.

The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population (e.g. to brain microvascular endothelial cells). The unrelated protein may also be one which assists purification of the viral particle as part of the production process, i.e. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common general knowledge.

The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

The AAV vector of the invention may take the form of a nucleotide sequence comprising an AAV genome or derivative thereof and a sequence encoding the MeCP2 transgene or derivatives thereof.

The AAV particles of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV particles of the invention also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid. The AAV particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor. Thus, for example, the AAV particles of the invention include those with an AAV2 genome and AAV9 capsid proteins (AAV2/9), or AAV9 PHP.B or PHP.eB capsid proteins.

The AAV vector may comprise multiple copies (e.g. 2, 3 etc.) of the nucleotide sequence referred to herein.

Retroviral and lentiviral vectors

A retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758- 63.

Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.

The basic structure of retrovirus and lentivirus genomes share many common features such as a 5’ LTR and a 3’ LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components - these are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.

In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.

The LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from the sequence unique to the 3’ end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5’ end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. In a defective retroviral vector genome gag, pol and env may be absent or not functional.

In a typical retroviral vector, at least part of one or more protein coding region essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and/or integrating its genome into a host genome.

Lentivirus vectors are part of the larger group of retroviral vectors. A detailed list of lentiviruses may be found in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis, P et al. (1992) EMBO J. 11 : 3053-8; Lewis, P.F. et al. (1994) J. Virol. 68: 510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.

The lentiviral vector may be a “primate” vector. The lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.

As examples of lentivirus-based vectors, HIV-1- and HIV-2-based vectors are described below.

The HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of Rev and/or RRE, full-length HIV-1 RNAs accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs.

Preferably, the viral vector used in the present invention has a minimal viral genome.

By “minimal viral genome” it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.

Preferably, the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell. Preferably, the vector lacks a functional gag-pol and/or env gene and/or other genes essential for replication.

However, the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).

The vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR. The vectors may be integration-defective. Integration defective lentiviral vectors (IDLVs) can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site; Naldini, L. et al. (1996) Science 272: 263-7; Naldini, L. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 11382-8; Leavitt, A.D. et al. (1996) J. Virol. 70: 721-8) or by modifying or deleting essential att sequences from the vector LTR (Nightingale, S.J. et al. (2006) Mol. Ther. 13: 1121-32), or by a combination of the above.

Adenoviral vectors

The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate. There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.

Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 10¹². Adenovirus is thus one of the best systems to study the expression of genes in primary non- replicative cells.

The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome.

Variants, derivatives, analogues, homologues and fragments

In addition to the specific proteins and nucleotides mentioned herein, the invention also encompasses variants, derivatives, analogues, homologues and fragments thereof.

In the context of the invention, a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.

The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.

The term “analogue” as used herein in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

Typically, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

The term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be equated with “identity”. In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology or identity between two or more sequences.

Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.

However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

“Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

Codon optimisation

The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.

EXEMPLARY VECTORS

In some embodiments, the vector of the invention comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 23 or a fragment thereof. Suitably, the vector of the invention comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 23 or a fragment thereof.

In some embodiments, the vector of the invention comprises or consists of the nucleotide sequence SEQ ID NO: 23 or a fragment thereof.

In some embodiments, the vector of the invention comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 24 or a fragment thereof. Suitably, the vector of the invention comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 24 or a fragment thereof.

In some embodiments, the vector of the invention comprises or consists of the nucleotide sequence SEQ ID NO: 24 or a fragment thereof.

Cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacc tttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactagggg ttcctgcggcctctagagaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcact aggcgggaacacccagcgcgcgtgcgccctggcaggaagatggctgtgagggacaggggagtggcgcc ctgcaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatgtctttggatttgg gaatcttataagttctgtatgagaccacagatccccGGAGAAGACAGAAGATAAAttcaagagaTTTA TCTTCTGTCTTCTCCtttttggaaaagcttatcgataacgcgctagttattaatagtaatcaattacg gggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtg tatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccca gtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtat ttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcgg ggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcg cgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcgg cgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccg ccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctg taattagcccgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatag aagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgcattggaacgcgg attccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttctt cttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaata atgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggc aatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatatt gctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattatt ctgagtccaagctaggcccttttgctaateatgtteatacctcttatcttcctcccacagctcctggg caacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacaccggtcgacga attcgttaacggatccgaacgccaccatgggcaagcctatccctaaccctctgctgggcctggactcc acaggcagcggcaccggtatggccgccgctgccgccaccgccgccgccgccgccgcgccgagcggagg aggaggaggaggcgaggaggagagactggaggaaaagtcagaagaccaggatctccagggcctcagag acaagccactgaagtttaagaaggcgaagaaagacaagaaggaggacaaagaaggcaagcatgagcca ctacaaccttcagcccaccattctgcagagccagcagaggcaggcaaagcagaaacatcagaaagctc aggctctgccccagcagtgccagaagcctcggcttcccccaaacagcggcgctccattatccgtgacc ggggacctatgtatgatgaccccaccttgcctgaaggttggacacgaaagcttaaacaaaggaagtct ggccgatctgctggaaagtatgatgtatatttgatcaatccccagggaaaagcttttcgctctaaagt agaattgattgcatactttgaaaaggtgggagacacctccttggaccctaatgattttgacttcacgg taactgggagagggagcccctccaggagagagcagaaaccacctaagaagcccaaatctcccaaagct ccaggaactggcaggggtcggggacgccccaaagggagcggcactgggagaccaaaggcagcagcatc agaaggtgttcaggtgaaaagggtcctggagaagagccctgggaaacttgttgtcaagatgcctttcc aagcatcgcctgggggtaagggtgagggaggtggggctaccacatctgcccaggtcatggtgatcaaa cgccctggcagaaagcgaaaagctgaagctgacccccaggccattcctaagaaacggggtagaaagcc tgggagtgtggtggcagctgctgcagctgaggccaaaaagaaagccgtgaaggagtcttccatacggt ctgtgcatgagactgtgctccccatcaagaagcgcaagacccgggagacggtcagcatcgaggtcaag gaagtggtgaagcccctgctggtgtccacccttggtgagaaaagcgggaagggactgaagacctgcaa gagccctgggcgtaaaagcaaggagagcagccccaaggggcgcagcagcagtgcctcctccccaccta agaaggagcaccatcatcaccaccatcactcagagtccacaaaggcccccatgccactgctcccatcc ccacccccacctgagcctgagagctctgaggaccccatcageccccctgagectcaggacttgagcag cagcatctgcaaagaagagaagatgccccgaggaggctcactggaaagcgatggctgccccaaggagc cagctaagactcagcctatggtcgccaccactaccacagttgcagaaaagtacaaacaccgaggggag ggagagcgcaaagacattgtttcatcttccatgccaaggccaaacagagaggagcctgtggacagccg gacgcccgtgaccgagagagttagctgacgataTATTGCCTTATTTCacgcgtcaattgaCAGCTATG CCAGCATCTTGCCgtttaaacCAGCTATGCCAGCATCTTGCCactagtCAGCTATGCCAGCATCTTGC CcctagagaaggCAGCTATGCCAGCATCTTGCCactagtgtttaaaccctagagaaggcaattgtatc gataCAACAAAATCACTGATGCTGGAagcgctCAACAAAATCACTGATGCTGGAgctagcCAACAAAA TCACTGATGCTGGAggccgcctcggccCAACAAAATCACTGATGCTGGAgctagcagcgctggccgcc tcggccatcgatttaattaactttacatagagcggattgcaaagcaaaccaacaagaataaaggcagc tgttgtctcttctccttatgggtagggctctgacaaagcttcccgattaactgaaataaaaaatattt ttttttctttcagtaaacttagagtttcgtggcttcggggtgggagtagttggagcattgggatgttt ttcttaccgacaagcacagtcaggttgaagacctaaccagatatctctagagatatcctegagagate tacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcc caccagecttgtcctaataaaattaagttgcateattttgtctgactaggtgtccttctataatatta tggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtc tattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttca agcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaat ttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcag gtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgt ccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggc cactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg ( SEQ ID NO : 23 )

Cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacc tttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactagggg ttcctgcggcctctagagaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcact aggcgggaacacccagcgcgcgtgcgccctggcaggaagatggctgtgagggacaggggagtggcgcc ctgcaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatgtctttggatttgg gaatcttataagttctgtatgagaccacagatccccGATTGTAGATTCAGGTTAAttcaagagaTTAA CCTGAATCTACAATCtttttggaaaagcttatcgataacgcgctagttattaatagtaatcaattacg gggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtg tatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccca gtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtat ttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcgg ggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcg cgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcgg cgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccg ccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctg taattagcccgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatag aagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgcattggaacgcgg attccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttctt cttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaata atgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggc aatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatatt gctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattatt ctgagtccaagctaggcccttttgctaateatgtteatacctcttatcttcctcccacagctcctggg caacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacaccggtcgacga attcgttaacggatccgaacgccaccatgggcaagcctatccctaaccctctgctgggcctggactcc acaggcagcggcaccggtatggccgccgctgccgccaccgccgccgccgccgccgcgccgagcggagg aggaggaggaggcgaggaggagagactggaggaaaagtcagaagaccaggatctccagggcctcagag acaagccactgaagtttaagaaggcgaagaaagacaagaaggaggacaaagaaggcaagcatgagcca ctacaaccttcagcccaccattctgcagagccagcagaggcaggcaaagcagaaacatcagaaagctc aggctctgccccagcagtgccagaagcctcggcttcccccaaacagcggcgctccattatccgtgacc ggggacctatgtatgatgaccccaccttgcctgaaggttggacacgaaagcttaaacaaaggaagtct ggccgatctgctggaaagtatgatgtatatttgatcaatccccagggaaaagcttttcgctctaaagt agaattgattgcatactttgaaaaggtgggagacacctccttggaccctaatgattttgacttcacgg taactgggagagggagcccctccaggagagagcagaaaccacctaagaagcccaaatctcccaaagct ccaggaactggcaggggtcggggacgccccaaagggagcggcactgggagaccaaaggcagcagcatc agaaggtgttcaggtgaaaagggtcctggagaagagccctgggaaacttgttgtcaagatgcctttcc aagcatcgcctgggggtaagggtgagggaggtggggctaccacatctgcccaggtcatggtgatcaaa cgccctggcagaaagcgaaaagctgaagctgacccccaggccattcctaagaaacggggtagaaagcc tgggagtgtggtggcagctgctgcagctgaggccaaaaagaaagccgtgaaggagtcttccatacggt ctgtgcatgagactgtgctccccatcaagaagcgcaagacccgggagacggtcagcatcgaggtcaag gaagtggtgaagcccctgctggtgtccacccttggtgagaaaagcgggaagggactgaagacctgcaa gagccctgggcgtaaaagcaaggagagcagccccaaggggcgcagcagcagtgcctcctccccaccta agaaggagcaccatcatcaccaccatcactcagagtccacaaaggcccccatgccactgctcccatcc ccacccccacctgagcctgagagctctgaggaccccatcagcccccctgagcctcaggacttgagcag cagcatctgcaaagaagagaagatgccccgaggaggctcactggaaagcgatggctgccccaaggagc cagctaagactcagcctatggtcgccaccactaccacagttgcagaaaagtacaaacaccgaggggag ggagagcgcaaagacattgtttcatcttccatgccaaggccaaacagagaggagcctgtggacagccg gacgcccgtgaccgagagagttagctgacgataTATTGCCTTATTTCacgcgtcaattgaCAGCTATG CCAGCATCTTGCCgtttaaacCAGCTATGCCAGCATCTTGCCactagtCAGCTATGCCAGCATCTTGC CcctagagaaggCAGCTATGCCAGCATCTTGCCactagtgtttaaaccctagagaaggcaattgtatc gataCAACAAAATCACTGATGCTGGAagcgctCAACAAAATCACTGATGCTGGAgctagcCAACAAAA

TCACTGATGCTGGAggccgcctcggccCAACAAAATCACTGATGCTGGAgctagcagcgctggccgcc tcggccatcgatttaattaactttacatagagcggattgcaaagcaaaccaacaagaataaaggcagc tgttgtctcttctccttatgggtagggctctgacaaagcttcccgattaactgaaataaaaaatattt ttttttctttcagtaaacttagagtttcgtggcttcggggtgggagtagttggagcattgggatgttt ttcttaccgacaagcacagtcaggttgaagacctaaccagatatctctagagatatcctcgagagatc tacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcc caccagecttgtcctaataaaattaagttgcateattttgtctgactaggtgtccttctataatatta tggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtc tattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttca agcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaat ttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcag gtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgt ccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggc cactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg

(SEQ ID NO: 24)

A furtherexemplaryvectorsequence(AAV_CBA_hMECP2_mirT)is: cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacc tttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactagggg ttcctggcggccgcaacgcgctagttattaatagtaatcaattacggggtcattagttcatagcccat atatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatg ggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccc ctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggacttt cctacttggcagtacatctacgtattagteatcgctattaccatggtcgaggtgagccccacgttctg cttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatttt gtgeagegatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcgg ggcggggcgaggcggagaggtgcggcggcagccaatcagagcggegegetccgaaagtttccttttat ggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggggagtcgctgcgacg ctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgt tactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcccgtttagtgaacc gtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagc ctccgcggattcgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacg taagtaccgcctatagagtctataggcccacaaaaaatgctttcttcttttaatatacttttttgttt atcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctc tttgcaccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatata aatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccag ctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttt tgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctg gcccatcactttggcaaagaattgggattcgaacaccggtcgacgaattcgttaacggatccgaacgc caccATGGTAGCTGGGATGTTAGGGCTCAGGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCA

AGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAG CCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGAAGG GTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTG ACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAA TCTGGCCGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAA AGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCA CGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAA GCTCCAGGAACTGGCAGAGGCCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGGCCAC GTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTT TTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATC AAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCCGACCCTCAGGCCATTCCCAAGAAACGGGGCCGAAA GCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCC GATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTC AAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTG

TAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCC

CCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCA CCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGA CTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCC CCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACAC CGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGT GGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCTAAaZAZZGCCZZAZZZCacgcgtcaattgaCAG CTATGCCAGCATCTTGCCg 111 a a a c CAGCTATGCCAGCATCTTGCCa c t a g t CAGCTATGCCAGCAT CTTGCCcctagagaaggCAGCTATGCCAGCATCTTGCCactagtgtttaaaccctagagaaggcaatt gtatcgat a CAACAAAATCACTGATGCTGGAa gcgc t CAACAAAATCACTGATGCTGGAg c t a g c CAA CAAAATCACTGATGCTGGAg gccgcct cggc c CAACAAAATCACTGATGCTGGAg ctagcagcgctgg ccgcctcggccat cgatttaatta aCTTTACATAGAGCGGATTGCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTG TCTCTTCTCCTTATGGGTAGGGCTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAACTT

AGAGTTTCGTGGCTTCGGGGTGGGAGTAGTTGGAGCATTGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTA

ACOAgatatctctagagatatcctcgagagatctac gggtggcatccctgtgacccctccccagtgcct ctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcat tttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaag ttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatct tggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttggg attccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatat tggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctggga ttacaggcgtgaaccactgctcccttccctgtcctt ctgattttgtaggtaaccacgtgcggaccgag cggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggc cgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgca gctgcctgcagg

( SEQ ID NO : 26 )

Bold: 3 ' and 5 ' ITR

BOLD : human ME CP 2 CDS

Capital letter : 3 ' UTR

Underline : p o 1 y- A

Underline : Chicken p -Actin promoter miR 124_target s ite : TATTGCCTTATTTC miR 31_target site : CAGCTATGCCAGCATCTTGCC miR 338_target site : CAACAAAATCACTGATGCTGGA

In some embodiments, the vector of the invention comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 26 or a fragment thereof. Suitably, the vector of the invention comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 26 or a fragment thereof.

In some embodiments, the vector of the invention comprises or consists of the nucleotide sequence SEQ ID NO: 26 or a fragment thereof.

A further exemplary vector sequence (AAV_shRNA_CBA_hMECP2_mirT) is: cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacc tttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactagggg ttcctgcggcctctagagaacgctgacgtcateaacccgctccaaggaatcgcgggcccagtgtcact a9.S^c9.S^^aacacccagcgcgcgtgogccctggcaggaagatggctgtgaggggcaggggagtggcgcc ctgcaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatgtctttggatttgg gaatcttataagttctgtatgagaccacagatccccGAGACAGTTGGATTCTTTAttcaagagaTAAA GAATCCAACTGTCTCtttttggaaaagcttatcgataacgcgctagttattaatagtaatcaattacg gggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtg tatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccca gtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtat ttatttattttttaattattttgtgcagegatgggggcggggggggggggggggcgcgcgccaggegg ggcggggcggggcgaggggcggggcggggcgaggeggagaggtgeggeggeagccaatcagageggeg cgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcgg cgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccg ccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctg taattagcccgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatag aagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgcattggaacgcgg attccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttctt cttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaata atgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggc aatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatatt getaatagcagctacaatccagetaccattctgcttttattttatggttgggataaggctggattatt ctgagtccaagctaggcccttttgetaatcatgttcatacctcttatcttcctcecacagctcctggg caacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacaccggtcgacga attcgttaacggatccgaacgccaccATGGTAGCTGGGATGTTAGGGCTCAGGGAAGAAAAGTCAGAA GACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGA

AGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAG

GCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAA

CAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGAC ACGGAAGCTTAAGCAAAGGAAATCTGGCCGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCC AGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTG GACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACC

TAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCCAAAGGGAGCGGCA

CCACGAGACCCAAGGCGGCCACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGG

AAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCAC

ATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCCGACCCTCAGGCCA

TTCCCAAGAAACGGGGCCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAA

GCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCG

GGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGA

GCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGC

AGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAA GGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCA CCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGC TCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCAC

GGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAA GGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCTAAaTATTGCCT

TATTTCacgcgtcaattgaCAGCTATGCCAGCATCTTGCCg111aaacCAGCTATGCCAGCATCTTGC CactagtCAGCTATGCCAGCATCTTGCCGctagagaaggCAGCTATGCCAGCATCTTGCCactagtgt ttaaaccctagagaaggcaattgtatcgataCAACAAAArCACrGArGCrGGAagcgctCAACAAAAG

CACTGATGCTGGAgctagcCAACAAAATCACTGATGCTGGAggccgcctcggccCAACAAAATCACTG

ATGCTGGAgctagcagcgctggccgcctcggccatcgatttaattaaCTTTACATAGAGCGGATTGCAAAGCA AACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGGCTCTGACAAAGCTTCCCGATTAACTGAAATAAAA

AATATTTTTTTTTCTTTCAGTAAACTTAGAGTTTCGTGGCTTCGGGGTGGGAGTAGTTGGAGCATTGGGATGTTTTTCTTACC

GACAAGCACAGTCAGGTTGAAGACCTAACCAgatatctctagagatatcctcgagagatctacgggtggcatcc ctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcc taataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggg gtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaag ctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgc ctcaqcctcccqaqttqttqqqattccaqqcatqcatqaccaqqctcaqctaatttttqtttttttqq taqaqacqqqqtttcaccatattqqccaqqctqqtctccaactcctaatctcaqqtqatctacccacc ttqqcctcccaaattqctqqqattacaqqcqtqaaccactqctcccttccctqtccttctqattttqt aqqtaaccacqtqcqqaccqaqcqqccqcaggaacccctagtgatggagttggccactccctctctgc gcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcc tcagtgagcgagcgagcgcgcagctgcctgcagg

( SEQ ID NO : 27 )

Bold : 3 ' and 5 ' ITR

Capital letter : shRNA hMECP2

Underline : Hl promoter

BOLD : human ME CP 2 CDS

Capital letter : 3 ' UTR

Underline : p o 1 y- A

Underline : Chicken p -Actin promoter miR 124_tarqet s ite : TATTGCCTTATTTC miR 31_tarqet s ite : CAGCTATGCCAGCATCTTGCC miR 338_tarqet s ite : CAACAAAATCACTGATGCTGGA

In some embodiments, the vector of the invention comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 27 or a fragment thereof. Suitably, the vector of the invention comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 27 or a fragment thereof.

In some embodiments, the vector of the invention comprises or consists of the nucleotide sequence SEQ ID NO: 27 or a fragment thereof.

METHOD OF TREATMENT

In some embodiments, the method of treatment provides MeCP2 to the central nervous system of a subject. In some embodiments, the method of treatment provides MeCP2 to the somatosensory cortex and/or striatum of a subject.

In some embodiments, the method of treatment provides MeCP2 to neuronal cells.

In some embodiments, the method of treatment provides an improvement in motor function in a subject. Methods for measuring motor function are known to those skilled in the art, for example, the beam balance test.

In some embodiments, the method of treatment provides an improvement in learning and/or cognitive function in a subject. Methods for measuring learning and/or cognitive function are known to those skilled in the art. For example, in humans the General Practitioner Assessment of Cognition (GPCOG) test may be used. Alternative cognitive tests include but are not limited to the Mini Mental State Examination (MMSE), The Six-item Cognitive Impairment Test (6CIT), Abbreviated Mental Test (AMT) and Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE).

Advantageously, the present invention provides a method for treatment by systemically administering the vector particle of the invention.

PHARMACEUTICAL COMPOSITIONS AND INJECTED SOLUTIONS

Although the agents for use in the invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.

The medicaments, for example vector particles, of the invention may be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the medicament, a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabiliser or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, e.g. intravenous or intra-arterial.

The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001 % may be used. In some cases, serum albumin may be used in the composition. For injection, the active ingredient may be in the form of an aqueous solution which is pyrogen- free, and has suitable pH, isotonicity and stability. The skilled person is well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included as required.

For delayed release, the medicament may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.

ADMINISTRATION

In some embodiments, the polynucleotide, vector, cell or pharmaceutical composition is administered to a subject systemically.

In some embodiments, the polynucleotide, vector, cell or pharmaceutical composition is administered to a subject locally.

In some embodiments, the polynucleotide, vector, cell or pharmaceutical composition is administered to a subject intracranially, intracerebrally or intraparenchymally.

The term “systemic delivery” or “systemic administration” as used herein means that the agent of the invention is administered into the circulatory system, for example to achieve broad distribution of the agent. In contrast, topical or local administration restricts the delivery of the agent to a localised area e.g. intracerebral administration entails direct injection into the brain.

In some embodiments, the polynucleotide, vector, cell or pharmaceutical composition is administered intravascularly, intravenously or intra-arterially.

Suitably, in some embodiments the polynucleotide, vector, cell or pharmaceutical composition is administered to the internal carotid artery.

As used herein, the term “agent” may refer to the polynucleotide, vector, cell or pharmaceutical composition of the invention.

In some embodiments, the polynucleotide, vector, cell or pharmaceutical composition is administered simultaneously, sequentially or separately in combination with an immunosuppressant. In some embodiments, the immunosuppressant is cyclosporin A (CsA).

The term “combination”, or terms “in combination”, “used in combination with” or “combined preparation” as used herein may refer to the combined administration of two or more agents simultaneously, sequentially or separately.

The term “simultaneous” as used herein means that the agents are administered concurrently, i.e. at the same time.

The term “sequential” as used herein means that the agents are administered one after the other.

The term “separate” as used herein means that the agents are administered independently of each other but within a time interval that allows the agents to show a combined, preferably synergistic, effect. Thus, administration “separately” may permit one agent to be administered, for example, within 1 minute, 5 minutes or 10 minutes after the other.

Dosage

The skilled person can readily determine an appropriate dose of an agent of the invention to administer to a subject. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.

SUBJECT

The term “subject” as used herein refers to either a human or non-human animal.

Examples of non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats. The non-human animal may be a companion animal.

Preferably, the subject is human.

In one embodiment the subject is a mouse model of Rett disease. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

Preferred features and embodiments of the invention will now be described by way of nonlimiting examples.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

EXAMPLES

EXAMPLE 1

RESULTS

Given the highly differentiate expression of Mecp2 between neuronal and glial cells in the brain, we sought to implement a system to differentiate the expression of the therapeutic gene and be compatible with the strict packaging size of AAV vectors (<4.8kb). For this goal, we conceived to put the viral therapeutic Mecp2 gene under the control of cell-type specific microRNAs. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules that may contain 21 to 23 nucleotides which are involved in RNA silencing. miRNAs basepair to complementary sequences in mRNA molecules leading to their gene silencing through mRNA cleavage, destabilisation and translation arrest. We reasoned that incorporating in the viral vector target sequences for miRNAs selectively expressed in glial cells will modulate transgene expression in these cells, but not in neurons. Moreover, we postulated that increasing number of targets for a specific miRNA will increment its silencing efficiency, therefore, providing a way to finely regulate expression levels of the transgene by choosing the right number of miRNA target sequences in the vector. To test this hypothesis, we inserted in the cM2 AAV viral vector, containing a strong CBA promoter upstream to the V5-Mecp2 isoform-1 gene, 2, 4 or none copies of the target sequences for the well-known miR-124 (miR- 124TS) which is specifically expressed in neurons (Figure 1). Next, we produced AAV9 particles and transduced mouse primary neuronal cultures in the dish. 7 days after transduction, protein lysates were prepared from the neuronal cultures and Western blots were performed to test the levels of V5 which is a surrogate of the Mecp2 protein to which the tag is fused at the 5’ terminal. Interestingly, V5-Mecp2 protein levels were strongly diminished in the vectors incorporating the miR-124TS and its silencing levels were highest when 4 copies of the miR-124TS were included (Figure 1). These results suggest that the inclusion of miR target sites in AAV vector can repress the expression of the viral gene and its silencing levels are proportionate to the number of miR target site copies integrated in the vector. To take advantage of this system, we generated new cM2 vectors where we cloned 2 or 4 copies of the target sites for miR-31 and miR-338, specifically expressed in astrocytes or oligodendrocytes, respectively (Figures 2,3). cM2 vectors with miR-31 TS were produced in AAV9 particles to transduced primary mouse astrocyte cultures. 7 days after transduction, astrocytes were collected and protein lysates were analysed through Western blotting. Interestingly, V5-Mecp2 protein levels were significantly reduced when miR-31TS were incorporated in the vector with the strongest silencing obtained with 4 miR-31 TS copies (Figure 2). A very similar trend of V5-Mecp2 protein silencing was observed in mouse primary oligodendrocyte cultures transduced with the cM2 vectors incorporating increasing number of target sites for the miR-338 (Figure 3).

Next, we decided to assemble the miR target sequences together in a unique viral vector. Given the very low levels of MeCP2 in glial cells, we decided to integrate 4 copies of the miR- 31 TS and miR-338TS (Figure 4). Although, Mecp2 is highly expressed in neurons, the expression of viral Mecp2 is arguably higher given the multiple copies of AAV vector entering in the neurons and the use of a strong CBA promoter. Thus, we decided to add one single miR-124TS copy in order to only partially reduce total gene levels in the transduced neuronal cells (Figure 4A). The resulting viral vector, named cM2-mR, was produced in AAV9 particles to transduced either mouse primary neuronal, astroglial or oligodendrocyte cultures in the dish. Transduced cell cultures were lysed and Western blots were performed for protein quantification. V5-Mecp2 levels were high in neuronal cell lysates, but significantly lower in astroglial and oligodendrocyte cultures (Figure 4B). Remarkably, this protein profile is highly consistent with the endogenous Mecp2 protein levels. Thus, the cM2-mR viral vector with the integration of a defined number of target sequences for cell type specific miRNAs is able to differentiate viral Mecp2 expression levels in different cell types achieving a pattern comparable with the expression profile of the endogenous gene. Next, cM2-mR AAV9 particles were inoculated by stereotaxic-guided injections in the striatum of adult wild-type mice (Figure 5A). 3 weeks post-transduction, the brains were isolated and processed for immunofluorescence analysis. V5-Mecp2 was co-stained with NeuN and GFAP to analyze V5-Mecp2 protein levels in neurons or astrocytes, respectively (Figure 5B). Remarkably, V5- Mecp2 immune signal was significantly lower in GFAP⁺ astrocytes compared to NeuN⁺ neurons as assessed by unbiased intensity signal quantification (Figure 5B). These findings validate the efficiency of the miR-TS cassettes to differentially reduce the expression of the viral Mecp2 transgene in brain cells in vivo.

Another overlooked challenge to establish a safe gene therapy for RTT is the brain somatic mosaicism with both intermingled wild-type and mutated MECP2 cells. Thus, transduction of wild-type cells with a standard Mecp2 expressing viral vector will boost its total expression to supra-physiological levels that might lead to undesired side-effects. To solve this problem, we conceived to add in the vector a shRNA cassette capable to silence the endogenous, but not the viral Mecp2 expression. Thus, silencing only endogenous MeCp2, the vector will ensure homogenous levels of the viral MeCp2 in both wild-type and mutant brain cells. Moreover, this silencing approach could be also helpful in case of repressing mutant Mecp2 forms that can compete with the functional Mecp2 though a gain-of-function pathological mechanism. Thus, we designed two shRNAs (shRNA-U1 (SEQ ID NO: 16) and shRNA-U2 (SEQ ID NO: 15)) on the 3’-UTR sequence of the mouse Mecp2 and cloned them downstream to the Poll 11 promoter H1 in a viral vector alone or in cM2 (Figure 6A). Next, these vectors were produced in AAV9 particles to transduce mouse primary neuronal cultures. 7 days after transduction, qPCRs and Western blots with neuronal cell lysates were performed for mRNA and protein analysis, respectively. Both shRNAs were able to strongly downregulate endogenous Mecp2 mRNA levels (Figure 6B). Moreover, shRNA-U2 (shU2) was able to robustly downregulate endogenous Mecp2 protein levels as assessed by Western blotting (Figure 60). However, in neurons transduced with the CM2-shU2 Mecp2 protein was readily detectable by Western blot corresponding to the V5-Mecp2 form expressed by the viral vector. These results confirm that the shRNA-U2 is efficiently silencing the endogenous mouse Mecp2, but not the viral Mecp2 form.

At this point, we integrated the H1-shRNA-U2 cassette in cM2-mR generating the final vector coined as CM2-ELO (endogenous level optimised). CM2-ELO contains both the miR-TS cassettes for Mecp2 regulated expression in brain cells and the Mecp2-shRNA sequence for silencing the endogenous Mecp2 gene while expressing a functional copy of the gene under the strong CBA promoter with an engineered MeCP2 3’-UTR sequence. The final composition of the CM2-ELO vector is depicted in Figure 7. MATERIALS AND METHODS

Generation of gene transfer vectors

The Mecp2 CDS including 3’-UTR (223bp) was PCR amplified in order to add the V5 tag at the 5’ of the coding sequence and inserted in the CBA-CreNLS vector described in Morabito (2017) Mol Ther. 25: 2727-2742 to generate the cM2 vector. Target sequences for the selected micro-RNAs were designed using the miRbase software (https://www.mirbase.org) and cloned between the Mecp2 cDNA and the 3’-UTR sequence. shRNA sequences for Mecp2 silencing were designed using the TargetScan software (https://www.targetscan.org/vert_80/).

AAV vectors production

AAV replication-incompetent recombinant viral particles were produced 293T cells, cultured in Dulbecco Modified Eagle Medium high glucose (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 1% non-essential amino acids (Gibco), 1% sodium pyruvate (Sigma- Aldrich), 1% glutamine (Sigma-Aldrich) and 1 % penicillin/streptomycin (Sigma-Aldrich). Cells were split every 3-4 days using Trypsin 0.25% (Sigma-Aldrich). Replication-incompetent, recombinant viral particles were produced in 293T cells by polyethylenimine (PEI, Polyscience) co-transfection of three different plasmids: transgene-containing plasmid, packaging plasmid for rep and cap genes and pHelper (Agilent) for the three adenoviral helper genes. The cells and supernatant were harvested at 120 hrs. Cells were lysed in hypertonic buffer (40mM Tris, 500mM NaCI, 2mM MgCh, pH=8) containing 100U/ml Salt Active Nuclease (SAN, Arcticzymes) for 1 h at 37°C, whereas the viral particles present in the supernatant were concentrated by precipitation with 8% PEG8000 (Polyethylene glycol 8000, Sigma-Aldrich) and then added to supernatant for an additional incubation of 30min at 37°C. In order to clarify the lysate, cellular debris were separated by centrifugation (4000g, 30min). The viral phase was isolated by iodixanol step gradient (15%, 25%, 40%, 60% Optiprep, Sigma-Aldrich) in the 40% fraction and concentrated in PBS (Phosphate Buffer Saline) with 100K cut-off concentrator (Amicon Ultra15, MERCK-Millipore). Virus titres were determined using AAVpro© Titration Kit Ver2 (TaKaRa).

Primary mouse neuronal cultures

Primary neuronal cultures were prepared at embryonic day 18.5 (E18.5) from male mouse embryos. Cortices were individually dissected, sequentially incubated in trypsin (0,005%, 15 min at 37°C, Sigma-Aldrich) and DNAse (0,1mg/mL, 3 min at room temperature, Sigma- Aldrich) in HBSS (Hank’s buffered salt solution without Ca²⁺ and Mg²⁺, Euroclone). Cells were plated on poly-L-lysine (Sigma-Aldrich) coated dishes (2.0 x 10⁵ cells/cm²) in Neurobasal medium (ThermoFisher Scientific) enriched with 0,6% glucose (Sigma-Aldrich), 0,2% penicillin/streptomycin (Sigma-Aldrich), 0,25% L-glutamine (Sigma-Aldrich) and 1% B27 (ThermoFisher Scientific).

Primary mouse astroglial cultures

Astrocytic cultures were established from P1 mouse pups. After dissection, hippocampi and cortices were dissociated by treatment with trypsin (0.25%, Gibco, Thermo Fisher) and DNase-l (Sigma-Aldrich) for 15 min at 37°C, followed by fragmentation with a pipette. Dissociated cells were plated on poly-L-lysine-coated (Sigma Aldrich) T75 flasks in minimal essential medium (MEM, Invitrogen, Life Technologies) supplemented with 20% fetal bovine serum (FBS) (Gibco, Life Technologies) and glucose (5.5 g/L, Sigma Aldrich). To obtain a pure astrocyte monolayer, microglial cells were harvested from 10-14-day-old cultures by orbital shaking for 30 min at 200 rpm.

Primary mouse oligoglial cultures

P3 mouse brains were collected for cell dissociation and 04-positive cell enrichment using a magnetic coupled antibody (MACS, Miltenyi Biotec). Pooled brains (n = 4) were dissociated using the Papain Neural Tissue Dissociation Kit. Brain homogenate cells were first incubated with the anti-04 microBeads antibody and O4⁺ cells were isolated. O4⁺ cells (approximately 60.000 OPCs from each pup) were cultured on poly-d-ornithine (Sigma Aldrich) coated 24- well plates (30.000 cells/well) in OPC medium containing Neurobasal (Life Technologies), 2% B27 (Life Technologies), 1% L-glutamine (Euroclone), 1 % penicillin/streptomycin (Euroclone), 10 ng/mL PDGF-AA (Sigma Aldrich), and 10 ng/mL FGF2 (Space Import Export, Milan, Italy). After 2 days, cells were either fixed or switched to oligodendrocyte differentiation medium containing DMEM (Euroclone), 1% N-2 supplement (Life Technologies), 2% B27, 0.01% BSA (Sigma Aldrich), 1% l-glutamine, 1% penicillin/streptomycin, and 10 ng/mL triiodothyronine (T3) (Sigma Aldrich). Cells were differentiated for 3 or 4 days and fixed for immunocytochemistry.

Western blots

Protein extracts were prepared in RIPA buffer (10 mM Tris-HCI pH7.4, 150 mM NaCI, 1 mM EGTA, 0.5% Triton and complete 1% protease and phosphatase inhibitor mixture, Roche Diagnostics). Primary neurons, brain and liver lysate samples (50 pg protein lysates) were separated using 8% polyacrylamide gel and then transferred to PVDF membranes. Membranes were incubated overnight at 4°C with the following primary antibodies in 1X PBST with 5% w/v nonfat dry: mouse anti-V5 (1 :1000; ThermoFisher Scientific), rabbit anti-Calnexin (1 :50000, Sigma), mouse anti-p-Actin (1 :50000; Sigma). Subsequently, membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1 :10000; Dako). The signal was then revealed with a chemiluminescence solution (ECL reagent, RPN2232; GE Healthcare) and detected with the ChemiDoc imaging system (Bio-Rad).

Immunofluorescence

Cells (neurons, astrocytes and oligodendrocytes) were fixed with ice-cold 4% paraformaldehyde (PFA) for 30 min at 4°C, washed with PBS (3x) and incubated for 1 h at room temperature with 10% donkey serum, which saturates the unspecific binding site, and 0.1 % Triton X-100 to create pores on the cell membrane facilitating antibody binding to intracellular antigens. Subsequently cells are incubated overnight at 4°C with the primary antibody. Upon wash with PBS (3x), cells were incubated for 1 h at room temperature in blocking solution with DAPI (1 :1000, Sigma-Aldrich) and with Alexa Fluor-488 and Alexa Fluor- 594 anti-rabbit or anti-mouse secondary antibodies (1 :1000, ThermoFisher Scientific). After PBS washes (3x), cells were mounted with fluorescent mounting medium (Dako). Images were captured with a Nikon Eclipse 600 fluorescent microscope.

Brain tissues were sectioned using cryostat after optimal cutting temperature compound (OCT) embedding in dry ice. Free-floating 50pm-thick coronal sections were rinsed in PBS and were incubated with 10% donkey serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma- Aldrich) for 1 h at room temperature before the overnight incubation at 4°C with the primary antibody (diluted in the blocking solution). Upon wash with PBS (3x), sections were incubated for 1 h at room temperature in blocking solution with DAPI (1 :1000, Sigma-Aldrich) and with Alexa Fluor-488 and Alexa Fluor-594 anti-rabbit or anti-mouse secondary antibodies (1 :1000, ThermoFisher Scientific). After PBS washes (3x), sections were mounted with fluorescent mounting medium (Dako). Confocal images were captured at X 125 magnification with Leica TCS SP5 Laser Scanning Confocal microscope (Leica Microsystems Ltd). Cell and tissue were stained with the following primary antibody: rabbit anti-MeCP2 (1 :500; Cell Signaling Technology) and anti-chicken_MAP2 (1 :500, Abeam).

Animal surgery procedure

Following the anaesthesia with isoflurane and the positioning of the animal on the stereotaxic support, two holes were made in the skull to allow the entry of the neuro-needle containing the virus to be injected into the mouse striatum according to the coordinates: Anteroposterior (AP) -2.0, mediolateral (ML) +1.5 mm, dorsoventral (DV) -2.0 mm. Throughout the surgical procedure, oxygen and isoflurane levels were adjusted according to the health state of the mice. The animals were then sacrificed two weeks after viral transduction, then the brains were collected.

Statistics

Values are expressed as mean ± standard deviation as indicated. All statistical analysis was carried out in GraphPad Prism 8.0, using one-way ANOVA, two-way ANOVA and unpaired t- test. P-values below 0.05 were considered significant. In multi-group comparisons, multiple testing correction for pairwise tests among groups was applied using Tukey’s post hoc analysis.

EXAMPLE 2

RESULTS

Downregulation of the endogenous human MECP2 gene

T o generate a transgene cassette applicable to human cells where MECP2 expression is finely regulated as in the case of mouse model, we started by selecting an shRNA to downregulate the endogenous MECP2 gene. As described in the mouse model, this shRNA was designed to target a region of the MECP2 3'UTR that is not contained in the transgene cassette but is present in the endogenous locus (Fig. 8A). We then evaluated its efficiency in human HeLa cells using qRT-PCR and immunofluorescence, transfecting a plasmid expressing the selected shRNA (or a scramble control) and GFP as a marker of transfected cells (Fig. 8A). Two days after transfection, we performed qRT-PCR and confirmed a downregulation of the MECP2 gene of about 70% compared to the control (Fig. 8B). Finally, we verified the actual decrease of the MeCP2 protein by immunofluorescence, evaluating the intensity of the MeCP2 signal in the treated transfected cells (GFP+) compared to the scramble control (Fig. 8C)

Validation of MECP2 cell-type specific expression in Rett-iPSC derived neurons and astrocytes

Having validated the shRNA, we confirmed the efficiency of our cassette of miRNA target sequences (mirT: mir-124 1 copy, mir-31 4 copies, mir-338 4 copies) in regulating MeCP2 expression directly in a human cellular context. To do this, we generated neurons from human neuronal progenitors (NPCs) in turn derived from hIPSCs. After 4 weeks of differentiation, human neurons were transduced with the AAV expressing the MECP2 CDS with or without the mirT cassette (Fig. 9A). 10 days after infection, cells were fixed and exogenous MeCP2 expression was evaluated using the V5 tag (Fig. 9B). In this way, we verified that the presence of one copy of the mir-124 target sequence is able to moderate the expression of transgene MeCP2 in neurons while maintaining a high level of expression (Fig. 9B).

We then repeated the same experiment in human astrocytes also derived from neuronal progenitors (Fig. 10A). In this context as well, we analyzed MeCP2 expression in astrocytes with or without the mirT cassette using immunofluorescence, exploiting the V5 tag (Fig. 10B). Analysis of the V5 signal intensity confirmed a marked reduction in expression in GFAP+ cells in the presence of mirT, confirming the ability of this cassette to differentially control MeCP2 expression also in human cells (Fig. 10B).

Validation of AAV9_V5-Mecp2-mirT + shRNA vector in mutant Mecp2-K0 animals.

We validated the cassette (mirT + shRNA) with the murine Mecp2 CDS in vivo in the Mecp2- KO mouse model. Mice were treated by intracerebroventricular injection in the neonatal period, injecting the regulated Mecp2 cassette by using the AAV9 as vehicle to target brain parenchyma, injecting 2 * 1O¹⁰ vg per mouse (Fig. 11 A). After 4 weeks, animals were sacrificed and brains were fixed to study transduction and expression by immunofluorescence using the V5 tag (Fig. 11 B). In this way, we verified that the presence of mirT is able to differentiate the expression of exogenous Mecp2 between neurons and astrocytes, mimicking the endogenous profile. In fact, the V5 fluorescence intensity was found to be significantly higher in NeuN+ neurons than in Sox9+ astrocytes (Fig. 11 B).

MATERIALS AND METHODS

HeLa cell culture

HeLa cells were maintained in Dulbecco Modified Eagle Medium - high glucose containing 10% fetal bovine serum, 1 % non-essential amino acids, 1% sodium pyruvate, 1 % glutamine, and 1% penicillin/streptomycin. Cells were split every 3-4 days using Trypsin 0.25%. For shRNA validation, cells were plated (2 x 10⁵ cells/cm2) and transfected at the first day in vitro (DIV1) with a plasmid vector expressing the shRNA (or a scramble sequence as a control) and the GFP under a constitutive promoter. At DIV3, cells were fixed for immunofluorescence staining (4% PFA, 4°C, 30’) or lysate for total RNA extraction.

RT-qPCR

Total RNA was isolated from HeLa cells using TRI reagent (sigma). Then, MECP2 mRNA levels were evaluated through qRT-PCR using the following primers: 5’- GATCAATCCCCAGGGAAAAGC-3’and 5’- TCTCCCAGTTACCGTGAAGTC-3’. The RNA levels were normalized against 18S using the following primers: 5'- GTAACCCGTTGAACCCCATT-3' and 5 -CCATCCAATCGGTAGTAGCG-3'. The results were reported as the fold change (2^-AACt) of MECP2 expression in shRNA treated HeLa cells relative to scramble control.

Immunofluorescence

Cells and tissues were fixed in PFA 4% at 4°C for 10 minutes. Brains were cut with a cryostat to obtain slice of 50um. Cells and slices were then washed with PBS (3x) and incubated with 10% donkey serum and Triton X-100 (Sigma) for 1 hr at RT to saturate the unspecific binding site before the overnight incubation at 4°C with the primary antibody. Upon wash with PBS (3x), cells were incubated for 1 h at RT in blocking solution with DAPI and with Alexa Fluor- 488 and Alexa Fluor-594 anti-rabbit or anti-mouse secondary antibodies (1 :1000, ThermoFisher Scientific). After PBS washes (3x), cells and slices were mounted with fluorescent mounting medium (Dako). Images were captured with a Nikon Eclipse 600 fluorescent microscope or with Leica TCS SP5 Laser Scanning Confocal microscope. Cell and slices were stained with the following primary antibody: rabbit anti-MeCP2 (1 :500; Cell Signaling), rabbit anti-NeuN (1 :500; Merck), rabbit-Sox9 (1 :500; Merck), chicken-GFP (1 :500; ThermoFisher), mouse-V5 (1 :500, ThermoFisher), chicken-MAP2 (1 :500; Abeam) and chicken-GFAP (1 :500, Abeam). The quantification of signal fluorescence intensity was performed using Imaged software (NIH, US). iPSCs-derived neurons differentiation iPSCs were initially differentiated in Neural Progenitors Cells (NPCs). NPCs were, then, dissociated with Accutase and plated on matrigel-coated 6-well plates (3 x 10⁵ cells per well) in NPC medium. Two days after, the medium was changed with the differentiation medium containing Neurobasal, 1 % Pen/Strep, 1% Glutamine, 1 :50 B27, 10 p.M SU5402, 8 p.M PD0325901 , and 10 p.M DAPT was added and kept for 3 days. After 3 days, the cells were dissociated with Accutase and plated on poly-L-lysine/laminin/fibronectin (100 pg/ml, 2 pg/ml, 2 pg/ml)-coated 12-well plates (2 x 10⁵ cells per well) and 24-well plates (1 x 10⁵ cells per well) in neuronal maturation medium supplemented with ROCK inhibitor Y27632 (10 pM) for the first 24 h. Neuronal maturation medium was composed by Neurobasal, 1% Pen/Strep, 1 % Glutamine, 1 :50 B27, 20 ng/ml human BDNF, 200 pM Ascorbic Acid, 250 pM Dibutyryl cAMP, 10 pM DAPT, 1 pg/pl Laminin. The culture medium was replaced the next day to remove the ROCK inhibitor, and at this stage half of the medium was changed every 2-3 days. Viral particles were directly added to cultured neurons after six weeks of differentiation, with a final concentration 5 * 10¹⁰ vg/ml. All the analysis was conducted one week after the infection upon fixation of the specimen (4% PFA, 4°C, 30’). NPC-derived astrocytes differentiation

Neural Progenitors Cells (NPCs) were differentiated in astrocytes. In brief, at differentiation day -1 , 90-95% confluent NPC cultures were washed with PBS and incubated for 5 min at 37 °C with accutase. Accutase reaction was then stopped by resuspending NPCs in NPC medium followed by centrifugation at 300* g for 5 min. The supernatant was removed, and cells were resuspended by gently up and down pipetting to obtain a single-cell suspension. NPCs were then seeded in low-attachment 35-mm dishes and incubated shaking at 90 rpm for 24 h at 37 °C to obtain NPC spheres. The day after, NPC medium was replaced with DMEM-F12 supplemented with 1 :100 B-27, 1 :200 N-2, and 5 pM ROCK inhibitor. After 48 h, medium was changed to the astrocyte growth medium (AGM Bullet Kit, Lonza, #CC-3186) for 15 days, shaking at 90 rpm at 37 °C, changing medium every third day. After 2 weeks, the thus-obtained spheres were plated on poly-ornithine and laminin-coated dishes in astrocyte growth medium. When cells reach -95% confluence, spheres were aspirated with a tip and adhered cells were passaged to a new dish. The culture was considered pure astrocytic after the third passage. Viral particles were directly added to cultured astrocytes with a final concentration 5 * 10¹⁰ vg/ml. All the analysis was conducted one week after the infection upon fixation of the specimen (4% PFA, 4°C, 30’).

Animals

Mice were maintained at San Raffaele Scientific Institute Institutional mouse facility (Milan, Italy) in micro-isolators under sterile conditions and supplied with autoclaved food and water. The Mecp2-KO mice (The Jackson Laboratory stock #003890) were maintained on C57BL/6J background. All procedures were performed according to protocols approved by the internal IACUC and reported to the Italian Ministry of Health according to the European Communities Council Directive 2010/63/EU.

AAV vector injection and tissue collection

Mice were genotyped at P0 using the primers provide from the Jackson Laboratory: https://www.jax.org/Protocol?stockNumber=003890&protocollD=2082. At P1 Mecp2-KO mice were intracerebroventricularly injected with 5ul of AAV9 carrying the CBA-V5-Mecp2-mirT + shRNA at starting concentration of 4*10¹² vg/ml. Following injection, all mice were weighed twice a week and after 1 month of age they were sacrificed to isolate the brains. Brains were then post-fixed in 4% PFA for two days and then soaked in cryoprotective solution (30% sucrose in PBS) for immunofluorescence analysis.

Statistics Values are expressed as mean ± standard deviation as indicated. All statistical analysis was carried out in Prism 8.0 (GraphPad), using unpaired t-Test. P-values below 0.05 were considered significant.

Various features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras):

1. A polynucleotide comprising a nucleotide sequence encoding methyl-CpG bindingprotein 2 (MeCP2) and:

(b) a nucleotide sequence encoding an inhibitor of MeCP2 expression, optionally a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15.

2. The polynucleotide of para 1 , wherein the polynucleotide comprises one miR-124 target sequence, four miR-31 target sequences and four miR-338-3p target sequences.

3. The polynucleotide of para 1 or 2, wherein:

(a) the miR-124 target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 5;

(b) the miR-31 target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 6; and/or;

(c) the miR-338-3p target sequence comprises or consists of a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 7.

4. The polynucleotide of any preceding para, wherein the polynucleotide comprises a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 8.

5. The polynucleotide of any preceding para, wherein the nucleotide sequence encoding MeCP2 comprises a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding an amino acid sequence that has at least 70% identity to SEQ ID NO: 1 or 2; (b) a nucleotide sequence that has at least 70% identity to SEQ ID NO: 3 or 4; and

(c) the nucleotide sequence of SEQ ID NO: 3 or 4.

6. The polynucleotide of any preceding para, wherein the nucleotide sequence encoding MeCP2 is operably linked to a strong promoter and/or a 3’-UTR, wherein the 3’-UTR is less than or equal to about 1000 bp in length.

7. The polynucleotide of any preceding para, wherein the polynucleotide further comprises a nucleotide sequence encoding an inhibitor of MeCP2 expression.

8. A vector comprising the polynucleotide of any preceding para.

9. The vector of para 8, wherein the vector comprises a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 23 or 24.

10. The vector of para 8 or 9, wherein the vector is an AAV, retroviral, lentiviral or adenoviral vector.

11 . The vector of any one of paras 8-10, wherein the vector is in the form of a viral vector particle.

12. The vector of para 11 , wherein the AAV vector particle comprises a capsid selected from the group consisting of an AAV9; AAV9 PHP.B; AAV9 PHP.eB; and AAVrhIO capsid.

13. A cell comprising the polynucleotide or vector of any preceding para.

14. A pharmaceutical composition comprising the polynucleotide, vector or cell of any preceding para and a pharmaceutically-acceptable carrier, diluent or excipient.

15. The polynucleotide, vector, cell or pharmaceutical composition of any preceding para for use in therapy.

16. The polynucleotide, vector, cell or pharmaceutical composition of any one of paras 1- 14 for use in treating or preventing Rett syndrome.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed polynucleotides, vectors, cells, compositions, uses and methods of the invention will be apparent to the skilled person without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to the skilled person are intended to be within the scope of the following claims.

Claims

(b) a nucleotide sequence encoding an inhibitor of MeCP2 expression, optionally a nucleotide sequence encoding an shRNA that has at least 90% sequence identity to SEQ ID NO: 15, 30 or 31.

2. The polynucleotide of claim 1 , wherein the polynucleotide comprises one miR-124 target sequence, four miR-31 target sequences and four miR-338-3p target sequences.

3. The polynucleotide of claim 1 or 2, wherein:

4. The polynucleotide of any preceding claim, wherein the polynucleotide comprises a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 8.

5. The polynucleotide of any preceding claim, wherein the nucleotide sequence encoding MeCP2 comprises a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding an amino acid sequence that has at least 70% identity to SEQ ID NO: 1 , 2 or 28;

(b) a nucleotide sequence that has at least 70% identity to SEQ ID NO: 3, 4 or 29; and

(c) the nucleotide sequence of SEQ ID NO: 3, 4 or 29.

6. The polynucleotide of any preceding claim, wherein the nucleotide sequence encoding MeCP2 is operably linked to a strong promoter and/or a 3’-UTR, wherein the 3’-UTR is less than or equal to about 1000 bp in length.

7. The polynucleotide of any preceding claim, wherein the polynucleotide further comprises a nucleotide sequence encoding an inhibitor of MeCP2 expression.

8. A vector comprising the polynucleotide of any preceding claim.

9. The vector of claim 8, wherein the vector comprises a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 23, 24, 26 or 27.

10. The vector of claim 8 or 9, wherein the vector is an AAV, retroviral, lentiviral or adenoviral vector.

11. The vector of any one of claims 8-10, wherein the vector is in the form of a viral vector particle.

12. The vector of claim 11 , wherein the AAV vector particle comprises a capsid selected from the group consisting of an AAV9; AAV9 PHP.B; AAV9 PHP.eB; AAVrhIO; and AAV-DJ capsid.

13. A cell comprising the polynucleotide or vector of any preceding claim.

14. A pharmaceutical composition comprising the polynucleotide, vector or cell of any preceding claim and a pharmaceutically-acceptable carrier, diluent or excipient.

15. The polynucleotide, vector, cell or pharmaceutical composition of any preceding claim for use in therapy.

16. The polynucleotide, vector, cell or pharmaceutical composition of any one of claims 1- 14 for use in treating or preventing Rett syndrome.