gene therapy

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, and optionally a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NOs: 15, 30, or 31.

Description

[Technical Field]

[0001] The present invention relates to compounds for use in the treatment of neurological disorders. More specifically, the present invention relates to polynucleotides comprising a nucleotide sequence encoding methyl-CpG binding protein 2 (MeCP2), and their use in the treatment of Rett syndrome. [Background technology]

[0002] Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in girls. RTT is characterized by overtly normal development during the 6–12-month period, followed by rapid regression with intentional loss of motor skills, and the onset of repetitive and autistic behaviors.

[0003] In the majority of cases, RTT is caused by loss-of-function mutations in the MECP2 gene, which encodes methyl-CpG-binding protein 2 (MeCP2), a comprehensive chromatin regulator highly expressed in neurons (Bienvenu, T. et al. (2006) Nat Rev Genet 7:415-426). Recent studies have shown that loss of MECP2 significantly alters neuronal activity, leading to a progressive imbalance of excitatory-inhibitory synaptic activity across the brain, with diverse modalities occurring between different circuits and regions of the brain (Banerjee, A. et al. (2016) PNAS 113:E7287-E7296).

[0004] One study demonstrated that the pathological phenotype of RTT in mice could be significantly restored by reactivating MECP2, even in advanced disease stages (Guy, J. et al. (2007) Science 315:1143-1147). In fact, reactivation of the MECP2 gene in over 70% of neurons in adult mice normalized brain morphology and significantly improved several sensorimotor disorders (Robinson, L. et al. (2012) Brain 135:2699-2710).

[0005] These findings provide strong evidence that MeCP2 is a crucial factor in maintaining complete neurological function during adulthood. Consistently, multiple pathological expressions observed in adult mutant RTT mice can be fully reproduced by exclusively deleting MeCP2 during adulthood.

[0006] MeCP2 is a ubiquitous neuronal epigenetic factor, but its selective inactivation in GABAergic neurons results in several RTT-specific phenotypes, suggesting that key neurological deficits in RTT are mediated by GABAergic neuron dysfunction. Rearrangement of the MECP2 gene in GABAergic neurons alone resulted in significant improvements in key motor and cognitive impairments in RTT mice (Ure, K. et al. (2016) eLife 5:185).

[0007] Due to the monogenic nature of RTT, gene therapy is a powerful clinical option for this disease. However, MECP2 gene duplication in humans is a cause of severe, clinically distinct neurodevelopmental disorders. Affected males exhibit early hypotonia, limb spasticity, and severe intellectual disability. Therefore, successful gene therapy for RTT is thought to require delivery of MeCP2 at levels comparable to endogenous levels.

[0008] Recent studies suggest that intravenous administration of AAV9 expressing wild-type (WT) MECP2 attenuated neurological dysfunction 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 insufficient to determine the correlation between viral dose, transduction efficiency, and therapeutic benefit. Based on these studies, it remained unclear whether gene therapy approaches could rescue the molecular dysfunction and transcriptional changes caused by MECP2 loss in the adult brain.

[0009] Other studies have provided promising results regarding the intravenous administration of adeno-associated virus (AVV) vectors expressing 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).

[0010] Endogenous MeCP2 expression is high in neurons and about one-tenth as low in astroglial and oligoglial cells. Recreating these different MeCP2 expression levels in brain cells could further improve the efficacy and safety of gene therapy.

[0011] Therefore, there remains a strong need for improved approaches to treating Rett syndrome. [Overview of the Initiative]

[0012] The inventors have developed a therapeutic vector that enables altered expression of transgenes between nerve cells and glial cells. In addition, the inventors have further developed a vector that expresses shRNA that selectively silences endogenous MeCP2 but does not silence viral transgenes, and that normalizes the total MeCP2 expression in transduced cells, regardless of whether they are mutant or wild-type. The inventors' vector approach offers advantages over previous vectors in that it further enhances the efficacy and safety of gene therapy for RTT patients.

[0013] In one embodiment, the present invention relates to a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2), (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) Provides a polynucleotide comprising a nucleotide sequence encoding an inhibitor of MeCP2 expression.

[0014] In one embodiment, the present invention relates to a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2), (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 polynucleotide comprising a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 15.

[0015] In one embodiment, the present invention relates to a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2), (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 polynucleotide comprising a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 30.

[0016] In one embodiment, the present invention relates to a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2), (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 polynucleotide comprising a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0017] In one aspect, the present 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 having at least 90% sequence identity with SEQ ID NO: 30, and / or (c) a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0018] In one aspect, the present 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 present 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 having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0019] In one aspect, the present 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 present 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 having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0020] In one aspect, the present 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 present 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 having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an inhibitor of MeCP2 expression is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0021] In one embodiment, the present 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. In one embodiment, the present 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 the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0022] In one embodiment, the present 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. In one embodiment, the present 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 the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0023] In one embodiment, the present 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. In one embodiment, the present 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 the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding the MeCP2 expression inhibitor is a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0024] In some embodiments, the copy number of each miRNA target sequence is independently selected from the group consisting of 1, 2, 3, and 4.

[0025] 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.

[0026] In some embodiments, the polynucleotide contains one miR-31 target sequence. In some embodiments, the polynucleotide contains two miR-31 target sequences. In some embodiments, the polynucleotide contains three miR-31 target sequences. In preferred embodiments, the polynucleotide contains four miR-31 target sequences.

[0027] In some embodiments, the polynucleotide contains one miR-338-3p target sequence. In some embodiments, the polynucleotide contains two miR-338-3p target sequences. In some embodiments, the polynucleotide contains three miR-338-3p target sequences. In preferred embodiments, the polynucleotide contains four miR-338-3p target sequences.

[0028] In a preferred embodiment, the polynucleotide comprises one miR-124 target sequence, four miR-31 target sequences, and four miR-338-3p target sequences.

[0029] In one embodiment, the present 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, and four miR-338-3p target sequences. In one embodiment, the present 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 the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding the inhibitor of MeCP2 expression is a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0030] In some embodiments, the miR-124 target sequence comprises or consists of a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5.

[0031] In some embodiments, the miR-124 target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 5.

[0032] In some embodiments, the miR-124 target sequence includes or consists of the nucleotide sequence of SEQ ID NO: 5.

[0033] In some embodiments, the miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0034] In some embodiments, the miR-31 target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 6.

[0035] In some embodiments, the miR-31 target sequence includes or consists of the nucleotide sequence of SEQ ID NO: 6.

[0036] In some embodiments, the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0037] In some embodiments, the miR-338-3p target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7.

[0038] In some embodiments, the miR-338-3p target sequence includes or consists of the nucleotide sequence of SEQ ID NO: 7.

[0039] In some embodiments, the miRNA target sequence is located in the 5' to 3' direction, after the nucleotide sequence encoding MeCP2.

[0040] In some embodiments, the miRNA target sequence is located within the 3'-UTR of the nucleotide sequence encoding MeCP2.

[0041] In some embodiments, clusters of miRNA target sequences or copies of miRNA target sequences are arranged from 5' to 3' in the order of miR-124 target sequence, miR-31 target sequence, and miR-338-3p target sequence. Clusters containing target sequences, or one or more copies thereof, may be arranged, for example, from 5' to 3', such that they form groups according to their target specificity. For example, in some embodiments, a polynucleotide comprises 5'-[miR-124 target sequence]1-[miR-31 target sequence]4-[miR-338-3p target sequence]4-3'.

[0042] Both individual target sequences and clusters of target sequences may be contiguous with each other, separated by spacer sequences, or any combination thereof.

[0043] In some embodiments, the miRNA target sequence is separated by a spacer sequence.

[0044] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.

[0045] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8.

[0046] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

[0047] In some embodiments, the nucleotide sequence encoding MeCP2 includes or comprises a nucleotide sequence encoding an amino acid sequence having 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 with SEQ ID NO: 1 or 2.

[0048] In some embodiments, the nucleotide sequence encoding MeCP2 includes, or consists of, a nucleotide sequence encoding an amino acid sequence having at least 70% identity with SEQ ID NO: 1 or 2.

[0049] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 2.

[0050] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of a nucleotide sequence having 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 with SEQ ID NO: 3 or 4.

[0051] In some embodiments, the nucleotide sequence encoding MeCP2 includes, or consists of, a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 3 or 4.

[0052] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of the nucleotide sequence of SEQ ID NO: 3 or 4.

[0053] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of a nucleotide sequence encoding an amino acid sequence having 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 with SEQ ID NO: 28.

[0054] In some embodiments, the nucleotide sequence encoding MeCP2 includes, or consists of, a nucleotide sequence encoding an amino acid sequence having at least 70% identity with SEQ ID NO: 28.

[0055] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28.

[0056] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of a nucleotide sequence having 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 with SEQ ID NO: 29.

[0057] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 29.

[0058] In some embodiments, the nucleotide sequence encoding MeCP2 includes or consists of the nucleotide sequence of SEQ ID NO: 29.

[0059] In some embodiments, the nucleotide sequence encoding MeCP2 is operably ligated to a promoter.

[0060] 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.

[0061] In some embodiments, the promoter is selected from the group consisting of chicken β-actin (CBA) promoter, β-actin promoter, CAG promoter, cytomegalovirus (CMV) promoter, human elongation factor-1-alpha (HEF-1-alpha), Chinese hamster elongation factor-1-alpha (CHEF-1-alpha) promoter, and phosphoglycerate kinase (PGK) promoter.

[0062] In a preferred embodiment, the promoter is a chicken β-actin (CBA) promoter.

[0063] In some embodiments, the nucleotide sequence encoding MeCP2 is operably ligated to the 3'-UTR. In some embodiments, the 3'-UTR is approximately 1000 bp or less in length.

[0064] In some embodiments, the 3'-UTR is approximately 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, or 200 bp in length or less.

[0065] In some embodiments, the 3'-UTR is about 500 bp or less in length. In preferred embodiments, the 3'-UTR is about 250 bp or less in length.

[0066] In some embodiments, the 3'-UTR is approximately 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.

[0067] In some embodiments, the 3'-UTR is approximately 50–300 bp long. In some embodiments, the 3'-UTR is approximately 50–250 bp long. In some embodiments, the 3'-UTR is approximately 50–200 bp long.

[0068] In some embodiments, the 3'-UTR is approximately 100–300 bp long. In some embodiments, the 3'-UTR is approximately 100–250 bp long. In some embodiments, the 3'-UTR is approximately 100–200 bp long.

[0069] In some embodiments, the 3'-UTR is about 150–300 bp long. In preferred embodiments, the 3'-UTR is about 150–250 bp long.

[0070] In a preferred embodiment, the 3'UTR is derived from the MeCP2 3'-UTR. In a preferred embodiment, the 3'-UTR is a cleaved MeCP2 3'UTR.

[0071] In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, or 200bp or less.

[0072] In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 500 bp or less. In preferred embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 250 bp or less.

[0073] In some embodiments, the 3'-UTR is a MeCP2 3'-UTR of approximately 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.

[0074] In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 50 to 300 bp. In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 50 to 250 bp. In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 50 to 200 bp.

[0075] In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 100-300 bp. In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 100-250 bp. In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 100-200 bp.

[0076] In some embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 150–300 bp. In preferred embodiments, the 3'-UTR is a MeCP2 3'-UTR with a length of approximately 150–250 bp.

[0077] In some embodiments, the polynucleotide further comprises a polyadenylated sequence operably linked to a nucleotide sequence encoding MeCP2.

[0078] 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 include a nucleotide sequence encoding a tag (e.g., a V5 tag).

[0079] In some embodiments, the polynucleotide does not include a sequence encoding the V5 tag. For example, the present invention may involve sequences that are the same as those disclosed herein, but with the V5 tag encoding sequence deleted.

[0080] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding an inhibitor of MeCP2 expression.

[0081] In some embodiments, the inhibitor is shRNA, siRNA, miRNA, or antisense DNA / RNA. In some embodiments, the inhibitor is shRNA.

[0082] In some embodiments, the nucleotide sequence encoding the shRNA has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15 or 16, preferably SEQ ID NO: 15.

[0083] In some embodiments, the nucleotide sequence encoding the shRNA has at least 90% sequence identity with SEQ ID NO: 15 or 16, preferably SEQ ID NO: 15.

[0084] In some embodiments, the nucleotide sequence encoding shRNA is SEQ ID NO: 15 or 16, preferably SEQ ID NO: 15.

[0085] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15.

[0086] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 15.

[0087] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and the nucleotide sequence of Sequence ID No. 15.

[0088] In some embodiments, the nucleotide sequence encoding the shRNA has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30 or 31.

[0089] In some embodiments, the nucleotide sequence encoding the shRNA has at least 90% sequence identity with SEQ ID NO: 30 or 31.

[0090] In some embodiments, the nucleotide sequence encoding the shRNA is SEQ ID NO: 30 or 31.

[0091] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30.

[0092] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 30.

[0093] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31.

[0094] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0095] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2), a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30, and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31.

[0096] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2), a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 30, and a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31.

[0097] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and the nucleotide sequence of Sequence ID No. 30.

[0098] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and the nucleotide sequence of Sequence ID No. 31.

[0099] In one embodiment, the present 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.

[0100] In one embodiment, the present invention provides a vector comprising the polynucleotide of the present invention.

[0101] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV, retrovirus, or adenovirus vector. In preferred embodiments, the vector is an AAV vector.

[0102] 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.

[0103] In a preferred embodiment, the viral vector particles are configured to pass through the blood-brain barrier. In a preferred embodiment, the AAV vector particles are configured to pass through the blood-brain barrier.

[0104] In some embodiments, the AAV vector particles include an artificial capsid amino acid sequence. In some embodiments, the artificial capsid amino acid sequence allows the vector particles to cross the blood-brain barrier.

[0105] In some embodiments, the AAV vector particles include a VP1 capsid protein comprising an amino acid sequence containing at least four consecutive amino acids, for example, at least five or six, preferably seven amino acids, from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

[0106] In some embodiments, the AAV vector particles include a VP1 capsid protein containing an amino acid sequence including the sequence DGTLAVPFKAQ (SEQ ID NO: 25).

[0107] In some embodiments, the AAV vector particle includes a capsid containing an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO: 20, more preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 20.

[0108] In some embodiments, the AAV vector particle includes a capsid having an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO: 21, more preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 21.

[0109] In some embodiments, the AAV vector particle includes a capsid having an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO: 22, more preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 22.

[0110] In some embodiments, the vector is an AAV9 vector. In some embodiments, the vector particle is an AAV9 vector particle.

[0111] In some embodiments, the AAV vector particles have a serotype selected from the group consisting of AAV9, AAV9 PHP.B, AAV9 PHP.eB, and AAVrh10. In some embodiments, the AAV vector particles have the AAV9 PHP.eB serotype.

[0112] In some embodiments, the AAV vector particles have the AAV-DJ serotype.

[0113] In some embodiments, the AAV vector particles include a capsid selected from the group consisting of AAV9, AAV9 PHP.B, AAV9 PHP.eB, and AAVrh10 capsids. In some embodiments, the AAV vector particles include the AAV9 PHP.eB capsid.

[0114] In some embodiments, the vector is a cM2 AAV vector.

[0115] 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.

[0116] In one embodiment, the present invention provides cells comprising the polynucleotide or vector of the present invention.

[0117] In one embodiment, the present invention provides a pharmaceutical composition comprising a polynucleotide, vector, or cell of the present invention and a pharmaceutically acceptable carrier, diluent, or excipient.

[0118] In some embodiments, the pharmaceutical composition is formulated for systemic or topical delivery. In some embodiments, the pharmaceutical composition is formulated for intravascular, intravenous, intraarterial, intracranial, or intraparenchymal delivery.

[0119] In one embodiment, the present invention provides polynucleotides, vectors, cells, or pharmaceutical compositions for use in therapy.

[0120] In one embodiment, the present invention provides polynucleotides, vectors, cells, or pharmaceutical compositions for use in the treatment or prevention of Rett syndrome.

[0121] In one embodiment, the present invention provides a method for treating or preventing Rett syndrome, comprising administering a polynucleotide, vector, cell, or pharmaceutical composition of the present invention to a subject in need thereof.

[0122] In one embodiment, the present invention provides the use of the polynucleotide, vector, cell, or pharmaceutical composition of the present invention for the manufacture of a pharmacopoeia for treating or preventing Rett syndrome.

[0123] In some embodiments, polynucleotides, vectors, or cells are administered systemically or topically to a subject.

[0124] In some embodiments, polynucleotides, vectors, or cells are administered to the subject intracranially or intraparally.

[0125] In some embodiments, polynucleotides, vectors, or cells are administered simultaneously, sequentially, or separately in combination with an immunosuppressant. In some embodiments, the immunosuppressant is cyclosporine A (CsA). [Brief explanation of the drawing]

[0126] [Figure 1] Increased MeCP2 silencing in neuronal cultures transduced with an AAV9 vector containing an additional number of miR-124 target sequences. A. AAV vector having a mouse Mecp2 isoform-1 sequence downstream of the CBA promoter and containing or lacking two or four miR-124 target sequences (miR-124TS). B. Western blot of V5 protein levels in protein lysates of neuronal cultures transduced with AAV9 containing a Mecp2 cassette with an increased number of miR-124TS. [Figure 2] Increased MeCP2 silencing in astroglial cultures transduced with an AAV9 vector containing an additional number of miR-31 target sequences. A. AAV vector having a mouse Mecp2 isoform-1 sequence downstream of the CBA promoter and containing or lacking two or four miR-31 target sequences (miR-31TS). B. Western blot V5 protein levels of protein lysates of neuronal cultures transduced with AAV9 containing a Mecp2 cassette with an increased number of miR-31TS. [Figure 3] Increased MeCP2 silencing in oligoglia cultures transduced with an AAV9 vector containing an additional number of miR-338 target sequences. A. AAV vector having a mouse Mecp2 isoform-1 sequence downstream of the CBA promoter and containing or lacking two or four miR-338 target sequences (miR-338TS). B. Western blot of V5 protein levels in protein lysates of neuronal cultures transduced with AAV9 containing a Mecp2 cassette with an increased number of miR-338TS. [Figure 4]Increased MeCP2 silencing in neuronal cultures transduced with an AAV9 vector containing an additional number of miR-124 target sequences. A. Composition of the cM2-mR AAV vector containing 1 copy of miR-124TS, 4 copies of miR-31TS, and 4 copies of miR338TS. B. V5-Mecp2 protein levels by Western blot on protein lysates of primary cultures of neurons, astroglia, or oligoglia transduced with AAV9-cM2-mR, showing significantly lower levels of V5-Mecp2 in glial cells. [Figure 5-1] In vivo validation of the cM2-mR vector. A. The AAV9-cM2-mR vector was inoculated into the striatum of adult mice. B. High-magnification fluorescence images of co-staining of neurons (NeuN) and astrocytes (GFAP) with V5-Mecp2. White arrows indicate V5-Mecp2 staining in astrocytes, which is significantly lower than that found in neurons. The histogram shows the quantification of the relative intensity of V5-Mecp2 staining in neurons and astrocytes. [Figure 5-2] In vivo validation of the cM2-mR vector. A. The AAV9-cM2-mR vector was inoculated into the striatum of adult mice. B. High-magnification fluorescence images of co-staining of neurons (NeuN) and astrocytes (GFAP) with V5-Mecp2. White arrows indicate V5-Mecp2 staining in astrocytes, which is significantly lower than that found in neurons. The histogram shows the quantification of the relative intensity of V5-Mecp2 staining in neurons and astrocytes. [Figure 6]Verification of the cM2-shU2 viral vector for effective silencing of endogenous Mecp2 protein. A. Top: Mecp2 genomic locus with localization of target sequences for two shRNAs (U1 and U2) on the 3'-UTR highlighted in red. Bottom: Structure of the cM2-shU2 vector incorporating the H1-shRNA-U2 cassette. B. Western blots of Mecp2 and viral V5-Mecp2 protein levels in primary neuron cultures transduced with cM2-shU2 or the shU2 cassette alone. H1-shRNA-U2 is highly efficient at downregulating endogenous Mecp2 but not efficient at downregulating viral Mecp2. [Figure 7] The structure of the AAV vector (cM2-ELO) incorporating H1-shRNA-U2 and a microRNA-TS cassette. [Figure 8-1] A) Top. Scheme for designing shRNA to downregulate the endogenous human MECP2 gene. Bottom. Scheme for experimental design to test shRNA efficiency. Briefly, HeLa cells were transfected with a plasmid containing shRNA (or scrambled as a control) + GFP coding cassette under a constitutive promoter. B) RT-qPCR on transfected HeLa cells to analyze MECP2 mRNA expression. Bar graph showing the difference in fold change of MECP2 mRNA in scrambled and shRNA-treated HeLa cells. *p<0.05, unpaired t-test, n=3 biological replicates. C) Immunofluorescence analysis for GFP (green) and MeCP2 (red) in transfected HeLa cells to visualize the effect of shRNA on MeCP2 protein levels. Scale bar: 100uM. [Figure 8-2]A) Top. Scheme for designing shRNA to downregulate the endogenous human MECP2 gene. Bottom. Scheme for experimental design to test shRNA efficiency. Briefly, HeLa cells were transfected with a plasmid containing shRNA (or scrambled as a control) + GFP coding cassette under a constitutive promoter. B) RT-qPCR on transfected HeLa cells to analyze MECP2 mRNA expression. Bar graph showing the difference in fold change of MECP2 mRNA in scrambled and shRNA-treated HeLa cells. *p<0.05, unpaired t-test, n=3 biological replicates. C) Immunofluorescence analysis for GFP (green) and MeCP2 (red) in transfected HeLa cells to visualize the effect of shRNA on MeCP2 protein levels. Scale bar: 100uM. [Figure 9-1] A) Top. Scheme of the protocol used to generate iPSC-derived human MECP2-KO neurons. Bottom. Scheme of the AAV used to transduce V5-MECP2 coding sequences with or without mirT cassettes. B) Top. Immunofluorescence analysis of V5 (green), MeCP2 (red), and MAP2 (magenta) in transduced human MECP2-KO neurons to visualize the expression of exogenous MeCP2 with or without mirT. Scale bar: 100um. Bottom. Quantification of fluorescence intensity of exogenous MECP2 with or without mirT in human MECP2-KO neurons. *p<0.05, unpaired t-test, n=30. [Figure 9-2]A) Top. Scheme of the protocol used to generate iPSC-derived human MECP2-KO neurons. Bottom. Scheme of the AAV used to transduce V5-MECP2 coding sequences with or without mirT cassettes. B) Top. Immunofluorescence analysis of V5 (green), MeCP2 (red), and MAP2 (magenta) in transduced human MECP2-KO neurons to visualize the expression of exogenous MeCP2 with or without mirT. Scale bar: 100um. Bottom. Quantification of fluorescence intensity of exogenous MECP2 with or without mirT in human MECP2-KO neurons. *p<0.05, unpaired t-test, n=30. [Figure 10-1] A) Top. Scheme of the protocol used to generate iPSC-derived human MECP2-KO astrocytes. Bottom. Scheme of the AAV used to transduce V5-MECP2 coding sequences with or without mirT cassettes. B) Top. Immunofluorescence analysis of V5 (green) and GFAP (red) in transduced human MECP2-KO astrocytes to visualize the expression of exogenous MeCP2 with or without mirT. Scale bar: 100um. Bottom. Quantification of fluorescence intensity of exogenous MECP2 with or without mirT in human MECP2-KO astrocytes. *p<0.05, unpaired t-test, n=30. [Figure 10-2] A) Top. Scheme of the protocol used to generate iPSC-derived human MECP2-KO astrocytes. Bottom. Scheme of the AAV used to transduce V5-MECP2 coding sequences with or without mirT cassettes. B) Top. Immunofluorescence analysis of V5 (green) and GFAP (red) in transduced human MECP2-KO astrocytes to visualize the expression of exogenous MeCP2 with or without mirT. Scale bar: 100um. Bottom. Quantification of fluorescence intensity of exogenous MECP2 with or without mirT in human MECP2-KO astrocytes. *p<0.05, unpaired t-test, n=30. [Figure 11-1]A) Experimental protocol scheme. Briefly, neonatal Mecp2-KO mice were treated with AAV_V5-Mecp2-mirT+shRNA by intracerebroventricular injection. The treated animals were then sacrificed one month after viral inoculation. B) Top. Immunofluorescence analysis of V5 (green) and red neuronal (NeuN) or astrocyte (Sox9) specific markers in the transduced cortex. Scale bar: 200um. Bottom. Quantification of exogenous Mecp2 (V5) fluorescence intensity in neurons (NeuN+) and astrocytes (Sox9+). **p<0.01, unpaired t-test, n=3 mice / group. [Figure 11-2] A) Experimental protocol scheme. Briefly, neonatal Mecp2-KO mice were treated with AAV_V5-Mecp2-mirT+shRNA by intracerebroventricular injection. The treated animals were then sacrificed one month after viral inoculation. B) Top. Immunofluorescence analysis of V5 (green) and red neuronal (NeuN) or astrocyte (Sox9) specific markers in the transduced cortex. Scale bar: 200um. Bottom. Quantification of exogenous Mecp2 (V5) fluorescence intensity in neurons (NeuN+) and astrocytes (Sox9+). **p<0.01, unpaired t-test, n=3 mice / group. [Modes for carrying out the invention]

[0127] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including” or “includes,” or “containing” or “contains,” and are inclusive or non-exclusive, and do not exclude additional, unlisted members, elements, or steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0128] Rett syndrome Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in girls. RTT is characterized by overtly normal development during the 6–12-month period, followed by rapid regression with intentional loss of motor skills, and the onset of repetitive and autistic behaviors.

[0129] In the majority of cases, RTT is caused by loss-of-function mutations in the MECP2 gene, which encodes methyl-CpG binding protein 2 (MeCP2) (Bienvenu, T. et al. (2006) Nat Rev Genet 7:415-426).

[0130] Recent studies have shown that Mecp2 loss significantly alters neuronal activity, leading to a progressive imbalance of excitatory-inhibitory synaptic activity across the brain, involving diverse modalities between different circuits and regions (Banerjee, A. et al. (2016) PNAS 113:E7287-E7296).

[0131] Methyl-CpG binding protein 2 (MECP2) Methyl-CpG-binding protein 2 (MeCP2) is a comprehensive chromatin regulator that is highly expressed in neurons.

[0132] In some embodiments, MeCP2 is human or mouse MeCP2. In preferred embodiments, MeCP2 is human MeCP2.

[0133] An example of a MeCP2 sequence is as follows: MAAAAAAAPSGGGGGGEEERLEEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRK SGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEG GGATTSTQVMVIKRPGRKRKAEADPQAIPKKRGRKPGSWAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVKEWKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPP KKEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS (Sequence ID 1, Human)

[0134] Further examples of MeCP2 sequences are as follows: MAAAAATAAAAAAPSGGGGGGEEERLEEKSEDQDLQGLRDKPLKFKKAKKDKKEDKEGKHEPLQPSAHHSAEPAEAGKAETSESSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRK LKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTGRPKAAASEGVQVKRVLEKSPGKLWKMPFQASPGGK GEGGGATTSAQVMVIKRPGRKRKAEADPQAIPKKRGRKPGSWAAAAAEAKKKAVKESSIRSVHETVLPIKKRKTRETVSIEVKEWKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSAS SPPKKEHHHHHHHSESTKAPMPLLPSPPPPEPESSEDPISPPEPQDLSSSICKEEKMPRGGSLESDGCPKEPAKTQPMVATTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS (Sequence ID 2, mouse)

[0135] Further examples of MeCP2 sequences are as follows: MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYD VYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTS TQVMVIKRPGRKRKAEADPQAIPKKRGRGKPGSWAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVKEWKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKE HHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS (Sequence ID 28, Human)

[0136] An example of a nucleotide sequence encoding MeCP2 is as follows: (Sequence ID 3, Human)

[0137] Further examples of nucleotide sequences encoding MeCP2 are as follows: (Sequence ID 4, mouse)

[0138] Further examples of nucleotide sequences encoding MeCP2 are as follows: (Sequence ID 29, Human)

[0139] In some embodiments, MeCP2 is encoded by a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3 or 4 (preferably SEQ ID NO: 3), and preferably the protein encoded by the nucleotide sequence substantially retains the intrinsic function of the protein represented by either SEQ ID NO: 1 or 2.

[0140] In some embodiments, MeCP2 is encoded by a nucleotide sequence that encodes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1 or 2 (preferably SEQ ID NO: 1), preferably the amino acid sequence substantially retains the intrinsic function of the protein represented by SEQ ID NO: 1 or 2.

[0141] In some embodiments, MeCP2 comprises or consists of an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1 or 2 (preferably SEQ ID NO: 1), preferably the amino acid sequence substantially retains the intrinsic function of the protein represented by SEQ ID NO: 1 or 2.

[0142] In some embodiments, MeCP2 is encoded by a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 29, and preferably the protein encoded by the nucleotide sequence substantially retains the intrinsic function of the protein represented by SEQ ID NO: 28.

[0143] In some embodiments, MeCP2 is encoded by a nucleotide sequence that encodes an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 28, preferably the amino acid sequence substantially retains the intrinsic function of the protein represented by SEQ ID NO: 28.

[0144] In some embodiments, MeCP2 comprises or consists of an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 28, preferably the amino acid sequence substantially retains the intrinsic function of the protein represented by SEQ ID NO: 28.

[0145] Blood-brain barrier (BBB) As used herein, the term “blood-brain barrier” (BBB) ​​refers to a highly selective semipermeable barrier in the central nervous system that separates circulating blood from the brain and extracellular fluid. It is formed by the selectivity of tight junctions between endothelial cells. The blood-brain barrier is present along all capillaries in the brain and consists of tight junctions.

[0146] Overcoming the difficulty of delivering therapeutic agents to specific areas of the brain presents a major challenge in treating most brain disorders.

[0147] Preferably, the vector particles (e.g., AAV vector particles) are configured to pass through the intact blood-brain barrier. Preferably, the vector particles (e.g., AAV vector particles) do not impair the integrity and / or selectivity of the blood-brain barrier, and / or affect its permeability.

[0148] In other words, the vector particles are configured to pass through the blood-brain barrier without being damaged or weakened, i.e., maintaining the tight junctions between endothelial cells. Methods for determining whether the blood-brain barrier is intact are known in the art. For example, blood-brain barrier permeability can be detected by perfusion with Evans blue dye. Alternatively, a fluorescently bound cadaverine dye can be used as a blood-brain barrier permeability marker, along with AAV particles possessing a fluorescent marker.

[0149] Preferably, the vector particles (e.g., AAV vector particles) do not cause microgliosis. Preferably, the vector particles (e.g., AAV vector particles) do not cause persistent inflammation in the central nervous system.

[0150] As used herein, the term "central nervous system" refers to the nervous system consisting of the brain and spinal cord.

[0151] As used herein, “peripheral nervous system” refers to the components of the nervous system outside the central nervous system. The peripheral nervous system consists of nerves and ganglia outside the brain and spinal cord.

[0152] MicroRNA target sequences In some embodiments, the polynucleotide comprises one or more miRNA target sequences. Preferably, the nucleic acid sequence encoding MeCP2 is operably ligated to one or more miRNA target sequences.

[0153] MicroRNA (miRNA) genes are scattered across all human chromosomes except the Y chromosome. They can be located either in non-coding regions of the genome or within introns of protein-coding genes. Approximately 50% of miRNAs appear in clusters that are transcribed as polycistronic primary transcripts. Like protein-coding genes, miRNAs are typically transcribed from the polymerase II promoter, producing a so-called primary miRNA transcript (pri-miRNA). This pri-miRNA is then processed through a series of endonuclease cleavage steps carried out by two enzymes belonging to the RNAse type III family, Drosha and Dicer. From the pri-miRNA, a stem-loop approximately 60 nucleotides long, called a pre-miRNA, is cleaved by a specific nuclear complex consisting of the Drosha and DiGeorge syndrome key region gene (DGCR8), which cleaves both strands near the bases of the primary stem-loop, leaving a 5' phosphate and a 2 bp long 3' overhang. Next, pre-miRNA is actively transported from the nucleus to the cytoplasm by RAN-GTP and Exportin. Then, Dicer performs double-strand breaks at the stem-loop ends not defined by dross cleavage, separating the mature miRNA and miRNA. * This generates a 19-24 bp double helix, consisting of a double helix called the opposite strand. Consistent with the law of thermodynamic asymmetry, only one strand of the double helix is ​​selectively loaded into the RNA-induced silencing complex (RISC) and accumulated as a mature microRNA. This strand is typically one whose 5' end is not tightly paired with its complement, as demonstrated by the single nucleotide mismatch introduced at the 5' end of each strand of the siRNA double helix. However, there are some miRNAs that support the accumulation of both strands of the double helix to an equal degree.

[0154] MicroRNAs induce RNAi very similarly to small interfering RNAs (siRNAs), which are widely used for experimental gene knockdown. The main difference between miRNAs and siRNAs lies in their biosynthesis. When loaded into RISC, the guide strand of the small RNA molecule interacts with the mRNA target sequence, which is preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. The 2nd to 8th nucleotides from the 5' end of the miRNA, the so-called seed sequence, have been shown to be essential for inducing RNAi. If the entire guide strand sequence is perfectly complementary to the mRNA target, as is typically seen in siRNAs and plant miRNAs, the mRNA is endonuclease-likely cleaved by the involvement of the Argonaut (Ago) protein, also known as the "slicer" of the small RNA double helix incorporated into the RNA-induced silencing complex (RISC). DGRC (DiGeorge syndrome key region gene 8) and TRBP (TAR(HIV)RNA-binding protein 2) are double-stranded RNA-binding proteins that promote mature miRNA biosynthesis by Drosha and Dicer RNase III enzymes, respectively. The guide strand of the miRNA double helix is ​​incorporated into the effector complex RISC, which recognizes specific targets through incomplete base pairing and induces post-transcriptional gene silencing. Several mechanisms have been proposed for this regulatory mechanism, including the ability of miRNAs to induce repression of translation initiation, characterize target mRNAs for degradation by deadenylation, or sequester targets in the cytoplasmic P-isomer.

[0155] 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 that result in translational repression. Eukaryotic mRNA degradation occurs primarily through shortening of the poly(A) tail at the 3' end of the mRNA, decapsulation at the 5' end, subsequent digestion by 5'-3' exonucleases, and accumulation of miRNAs in a separate cytoplasmic region rich in components of the mRNA degradation pathway, the so-called P-form.

[0156] The expression of nucleic acid sequences encoding a transgene can be regulated by one or more endogenous miRNAs using one or more corresponding miRNA target sequences. Using this method, one or more miRNAs endogenously expressed in a cell prevent or reduce transgene expression in that cell by binding to their corresponding miRNA target sequences located on polynucleotides or vectors.

[0157] The target sequence may be fully or partially complementary to the miRNA. As used herein, the term “fully complementary” may mean that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. As used herein, the term “partially complementary” may mean that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, thereby the partially complementary sequence is still recognized by the miRNA. In other words, a partially complementary target sequence in the context of the present invention is effective in recognizing the corresponding miRNA and resulting in the prevention or reduction of transgene expression in cells expressing that miRNA. Preferably, a partially complementary miRNA target sequence may be fully complementary to the miRNA seed sequence.

[0158] In one embodiment, the present 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 embodiment, the present 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 embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and at least one miR-338-3p target sequence.

[0159] In one embodiment, the present 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. In one embodiment, the present 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. In one embodiment, the present 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.

[0160] Including more than one copy of the miRNA target sequence can increase the effectiveness of the system. It is also possible to include different miRNA target sequences. For example, a protein-coding sequence can be operably ligated 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 also conceivable. A polynucleotide may contain, for example, one, two, three, four, five, six, seven, or eight copies of the same or different miRNA target sequences.

[0161] Copies of the miRNA target sequence can be separated by a spacer sequence. The spacer sequence may contain, for example, at least one, at least two, at least three, at least four, or at least five nucleotide bases.

[0162] In some embodiments, the copy number of each miRNA target sequence is independently selected from the group consisting of 1, 2, 3, and 4.

[0163] 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.

[0164] In some embodiments, the polynucleotide contains one miR-31 target sequence. In some embodiments, the polynucleotide contains two miR-31 target sequences. In some embodiments, the polynucleotide contains three miR-31 target sequences. In preferred embodiments, the polynucleotide contains four miR-31 target sequences.

[0165] In some embodiments, the polynucleotide contains one miR-338-3p target sequence. In some embodiments, the polynucleotide contains two miR-338-3p target sequences. In some embodiments, the polynucleotide contains three miR-338-3p target sequences. In preferred embodiments, the polynucleotide contains four miR-338-3p target sequences.

[0166] In a preferred embodiment, the polynucleotide comprises one miR-124 target sequence, four miR-31 target sequences, and four miR-338-3p target sequences.

[0167] An example of a miR-124 target sequence is as follows: TATTGCCTTATTTC (Sequence ID 5)

[0168] In some embodiments, the miR-124 target sequence comprises or consists of a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5.

[0169] In some embodiments, the miR-124 target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 5.

[0170] In some embodiments, the miR-124 target sequence includes or consists of the nucleotide sequence of SEQ ID NO: 5.

[0171] An example of a miR-31 target sequence is as follows: CAGCTATGCCAGCATCTTGCC (Sequence ID 6)

[0172] In some embodiments, the miR-31 target sequence comprises or consists of a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0173] In some embodiments, the miR-31 target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 6.

[0174] In some embodiments, the miR-31 target sequence includes or consists of the nucleotide sequence of SEQ ID NO: 6.

[0175] An example miR-338-3p target sequence is as follows: CAACAAAATCACTGATGCTGGA (Sequence ID 7)

[0176] In some embodiments, the miR-338-3p target sequence comprises or consists of a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0177] In some embodiments, the miR-338-3p target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7.

[0178] In some embodiments, the miR-338-3p target sequence includes or consists of the nucleotide sequence of SEQ ID NO: 7.

[0179] In some embodiments, clusters of miRNA target sequences or copies of miRNA target sequences are arranged from 5' to 3' in the order of miR-124 target sequence, miR-31 target sequence, and miR-338-3p target sequence. Clusters containing target sequences, or one or more copies thereof, may be arranged, for example, from 5' to 3', such that they form groups according to their target specificity. For example, in some embodiments, a polynucleotide comprises 5'-[miR-124 target sequence]1-[miR-31 target sequence]4-[miR-338-3p target sequence]4-3'.

[0180] In some embodiments, the cluster of miRNA target sequences or sequences is arranged from 5' to 3' in the order of miR-124, miR-338-3p, and miR-31.

[0181] In some embodiments, the cluster of miRNA target sequences or sequences is arranged from 5' to 3' in the order of miR-338-3p, miR-124, and miR-31.

[0182] In some embodiments, the cluster of miRNA target sequences or sequences is arranged from 5' to 3' in the order of miR-338-3p, miR-31, and miR-124.

[0183] In some embodiments, the cluster of miRNA target sequences or sequences is arranged in the order of miR-31, miR-124, and miR-338-3p from 5' to 3'.

[0184] In some embodiments, the cluster of miRNA target sequences or sequences is arranged from 5' to 3' in the order of miR-31, miR-338-3p, and miR-124.

[0185] An example of a triple miRNA target sequence combination is as follows:

[0186] [Table 1] (Sequence 8) miR-124 target sequence: underlined miR-31 target sequence: italics and underlined miR-338-3p target sequence: bold and underlined

[0187] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.

[0188] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8.

[0189] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

[0190] In some embodiments, one or more miRNA target sequences suppress transgene expression in neurons. In some embodiments, one or more miRNA target sequences suppress transgene expression in astrocytes. In some embodiments, one or more miRNA target sequences suppress transgene expression in oligodendrocytes.

[0191] As used herein, the term “suppress expression” may refer to a reduction in the expression of a transgene in a relevant cell type in which one or more miRNA target sequences are operably ligated, compared to transgene expression under conditions where one or more miRNA target sequences are absent but otherwise substantially identical. 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.

[0192] Both individual target sequences and sequence clusters may be contiguous, separated by spacer sequences, or any combination thereof.

[0193] In some embodiments, the miRNA target sequence is separated by a spacer sequence.

[0194] As used herein, “spacer” may be a sequence (e.g., a nucleotide sequence or an amino acid sequence) that can be used to separate other sequence elements within a larger polymer.

[0195] Individual miRNA target sequences or groups of miRNA target sequences may be separated by one or more spacer sequences. In some embodiments, miRNA target sequences are separated by one or more spacer sequences. A spacer sequence may, for example, contain 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.

[0196] Promoter and regulatory elements The polynucleotides and vectors of the present invention include elements that enable the expression of MeCP2 in vitro or in vivo. These may be referred to as expression regulatory sequences. Thus, the polynucleotides and vectors typically include expression regulatory sequences (e.g., including promoter sequences) operably ligated to a nucleotide sequence encoding a transgene.

[0197] Any suitable promoter may be used, and its selection can be easily made by those skilled in the art. The promoter sequence may be constitutively active (i.e., operational in any host cell background), or alternatively, active only in a specific host cell environment, thereby enabling targeted expression of the transgene in a particular cell type (e.g., tissue-specific promoters such as endothelial cell-specific promoters). The promoter may also exhibit inducible expression in response to the presence of another factor, such as a factor present in the host cell. In any case, when a polynucleotide vector is administered for therapeutic purposes, it is preferable that the promoter is functional in the target cell background.

[0198] In some embodiments, the promoter is neuronal cell-specific. In some embodiments, the promoter is astrocyte-specific.

[0199] In some embodiments, the promoter is selected from the group consisting of chicken β-actin (CBA) promoter, β-actin promoter, CAG promoter, cytomegalovirus (CMV) promoter, human elongation factor-1-alpha (HEF-1-alpha), Chinese hamster elongation factor-1-alpha (CHEF-1-alpha) promoter, and phosphoglycerate kinase (PGK) promoter.

[0200] In some embodiments, the promoter is a chicken β-actin (CBA) promoter.

[0201] An example of a CBA promoter is as follows: (Sequence ID 9)

[0202] In some embodiments, the CBA promoter includes or comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 9.

[0203] The chicken beta-actin (CBA) promoter may optionally be used in combination with the cytomegalovirus (CMV) enhancer element.

[0204] The polynucleotide or vector of the present invention contains a 3'-UTR of about 1000 bp or less in length.

[0205] In a preferred embodiment, the 3'-UTR is derived from MeCP2 3'-UTR (e.g., its cleaved form). In a preferred embodiment, the 3'-UTR is a cleaved MeCP2 3'UTR.

[0206] Preferably, the 3'-UTR (e.g., MeCP2 3'-UTR) is cleaved at its 3' end (e.g., retaining its native 5' end).

[0207] In some embodiments, the 3'-UTR is a synthetic UTR constructed from the regulatory element of a wild-type (8.6 kb long) 3'-UTR, as described in Matagne, V. et al. (2017) Neurobiol. Dis. 99: 1-11.

[0208] An example of a 3'-UTR sequence is as follows: CTTTACATAGAGCGGATTGCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGGCTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAACTTAGAGTTTCGTGGCTTCGGGGTGGGAGTAGTTGGAGCATTGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTAACCA (Sequence ID 10, mouse)

[0209] Further examples of 3'-UTR sequences are as follows: CTTTACACGGAGCGGATTGCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGGCTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAACTTAGAGTTTCGTGGCTTCAGGGTGGGAGTAGTTGGAGCATTGGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTAACCA (Sequence ID 11, Human)

[0210] Further examples of 3'-UTR sequences are as follows: CTTTACACGGAGCGGATTGCAAAGCAAACCAAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGGCTCTGACAAAGCTTCCCGATTAACTGAAATAAAAAATATTTTTTTTTCTTTCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (Sequence ID 12, Human)

[0211] In some embodiments, the 3'-UTR comprises or consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of sequence numbers 10, 11, or 12.

[0212] The polynucleotide or vector of the present invention may also include one or more further regulatory sequences that can act before or after transcription.

[0213] A regulatory sequence is any sequence that promotes the expression of a transgene, that is, increases the expression of the transcript, improves the nuclear export of mRNA, or enhances its stability. Examples of such regulatory sequences include enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. An example of a polyadenylation site is the human or bovine growth hormone poly(A) signaling pathway.

[0214] An example of a human growth hormone polyA sequence is as follows: TCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTG ACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCAC AATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAG AGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTT (Sequence ID 13)

[0215] Further examples of polyA sequences are as follows: GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTT GGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGCGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTT (Sequence ID 14)

[0216] In some embodiments, the polyA sequence includes or consists of a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with sequence number 13 or 14.

[0217] An example of a post-transcriptional regulatory element for use in the polynucleotide or vector of the present invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

[0218] Another regulatory sequence that may be used in the polynucleotide or vector of the present invention is a scaffold-attachment region (SAR). Further regulatory sequences can be readily selected by those skilled in the art.

[0219] MECP2 expression inhibitors In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an inhibitor of MeCP2 expression (preferably endogenous MeCP2 expression).

[0220] When used herein in the context of inhibiting MeCP2 expression, the term "inhibitor" may refer to a drug that reduces MeCP2 expression compared to the level of MeCP2 expression under otherwise substantially identical conditions, in the absence of the drug. An inhibitor may, for example, reduce MeCP2 expression (preferably endogenous MeCP2) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the level of MeCP2 expression in its absence. Preferably, the inhibitor reduces MeCP2 expression by at least 70% compared to the level of MeCP2 expression in its absence. In some embodiments, the inhibitor completely prevents the expression of MeCP2 (preferably endogenous MeCP2).

[0221] The expression levels of proteins such as MeCP2 can be easily measured and quantified by those skilled in the art using techniques well known in the art, for example, by Western blotting.

[0222] In some embodiments, the inhibitor is specific to endogenous MeCP2.

[0223] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an inhibitor of endogenous MeCP2 expression (e.g., inhibiting the expression of endogenous MeCP2 in cells 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., inhibiting the expression of endogenous MeCP2 in cells into which the polynucleotide is introduced).

[0224] In some embodiments, the inhibitor does not inhibit the expression of MeCP2 encoded by the polynucleotide of the present invention. In some embodiments, the inhibitor does not substantially inhibit the expression of MeCP2 encoded by the polynucleotide of the present invention.

[0225] In some embodiments, the inhibitor inhibits the expression of endogenous MeCP2 more than the expression of MeCP2 encoded by the polynucleotide of the present invention. For example, the inhibitor can inhibit the expression of endogenous MeCP2 at least 1.5 times, 2 times, 2.5 times, 5 times, 10 times, 20 times, 50 times, 100 times, or 1000 times more than the expression of MeCP2 encoded by the polynucleotide of the present invention.

[0226] In some embodiments, the inhibitor targets the 3'-UTR of the gene encoding MeCP2 (preferably an endogenous gene). In some embodiments, the section of the 3'-UTR targeted by the inhibitor is not included in the polynucleotide of the present invention.

[0227] In some embodiments, the inhibitor is shRNA, siRNA, miRNA, or antisense DNA / RNA. Preferably, the inhibitor is shRNA.

[0228] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15.

[0229] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16.

[0230] An example of a nucleotide sequence encoding an shRNA that inhibits MeCP2 expression is as follows: GGAGAAGACAGAAGATAAA (Sequence ID 15)(shRNA-U2)

[0231] Further examples of nucleotide sequences encoding shRNAs that inhibit MeCP2 expression are as follows: GATTGTAGATTCAGGTTAA (Sequence ID 16)(shRNA-U1)

[0232] In some embodiments, the polynucleotide further comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15.

[0233] In some embodiments, the polynucleotide further comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16.

[0234] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30.

[0235] In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence encoding methyl-CpG-binding protein 2 (MeCP2) and a nucleotide sequence encoding shRNA having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31.

[0236] Further examples of nucleotide sequences encoding shRNAs that inhibit MeCP2 expression are as follows: GAGACAGTTGGATTCTTTA (Sequence ID 30)

[0237] Further examples of nucleotide sequences encoding shRNAs that inhibit MeCP2 expression are as follows: TAAAGAATCCAACTGTCTC (Sequence ID 31)

[0238] In some embodiments, the polynucleotide further comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 30.

[0239] In some embodiments, the polynucleotide further comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 31.

[0240] Nucleotide sequences encoding MeCP2 expression inhibitors can be operably ligated to promoters such as the H1 promoter.

[0241] An example H1 promoter sequence is as follows: GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCAC (Sequence ID 17)

[0242] In some embodiments, the H1 promoter includes or comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17.

[0243] siRNA, shRNA, miRNA, and antisense DNA / RNA Inhibition (e.g., of MeCP2) can be achieved using post-transcriptional gene silencing (PTGS). Post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) is a conserved cellular defense mechanism for regulating the expression of exogenous genes. Random incorporation of elements such as transposons or viruses is thought to trigger the expression of dsRNAs that activate sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. The silencing effect is known as RNA interference (RNAi) (Ralph et al. (2005) Nat. Medicine 11:429-433). The RNAi mechanism involves processing long dsRNAs into double helixes of approximately 21-25 nucleotide (nt)RNAs. These products are called small interfering or silencing RNAs (siRNAs), which are sequence-specific mediators of mRNA degradation. In differentiated mammalian cells, dsRNAs exceeding 30 bp have been found to activate the interferon response, leading to the cessation of protein synthesis and nonspecific mRNA degradation (Stark et al. (1998) Ann. Rev. Biochem. 67:227-64). However, this response can be avoided by using 21 nt siRNA double helix (Elbashir et al. (2001) EMBO J. 20:6877-88, Hutvagner et al. (2001) Science 293:834-8), enabling the analysis of gene function in cultured mammalian cells.

[0244] shRNA consists of short, reverse-directed RNA repeats separated by small loop sequences. These are rapidly processed by cellular mechanisms into 19-22nt siRNAs, thereby repressing target gene expression.

[0245] MicroRNAs (miRNAs) are small (22-25 nucleotides long) non-coding RNAs that can effectively reduce the translation of target mRNA by binding to the 3' untranslated region (UTR) of the target mRNA. MicroRNAs are a very large group of small RNAs that are naturally produced in organisms, and at least some of them regulate the expression of target genes. The foundational members of the microRNA 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 insect development. The active RNA species is initially transcribed as a precursor of about 70 nt, which is processed after transcription to become the mature form of about 21 nt. Both let-7 and lin-4 are transcribed as hairpin RNA precursors, which are processed into their mature forms by the Dicer enzyme.

[0246] The concept of antisense involves selectively binding a short, possibly modified, DNA or RNA molecule to messenger RNA in a cell, thereby preventing the synthesis of the encoded protein.

[0247] Methods for designing siRNA, shRNA, miRNA, and antisense DNA / RNA to regulate the expression of target proteins are well known in the art.

[0248] vector A vector is a tool that enables or facilitates the transfer of an entity from one environment to another. According to the present invention, for example, some vectors used in recombinant nucleic acid techniques enable the transfer of entities such as nucleic acid segments (e.g., heterologous DNA segments, such as heterologous cDNA segments) into target cells. Vectors can serve the purposes of maintaining heterologous nucleic acids (DNA or RNA) within cells, promoting the replication of vectors containing nucleic acid segments, and / or promoting the expression of proteins encoded by nucleic acid segments.

[0249] The polynucleotide-containing vectors used in the present invention can be introduced into cells using various techniques known in the art, such as transfection, transduction, and transformation.

[0250] Transfection can refer to the general process of incorporating nucleic acids into cells, including the process of delivering polynucleotides to cells using non-viral vectors. Transduction can refer to the process of incorporating nucleic acids into cells using viral vectors.

[0251] Preferably, the vector used to transduce cells in the present invention is a viral vector. The vector of the present invention is preferably an adeno-associated virus (AAV) vector, but other viral vectors may be used. The vector may be, for example, an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or an adenovirus vector.

[0252] 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 configured to cross the blood-brain barrier.

[0253] In some embodiments, the viral vector particles configured to cross the blood-brain barrier for use in accordance with the present invention are retroviral, lentiviral, adeno-associated virus (AAV), or adenoviral vector particles. Preferably, the viral vector particles are lentiviral or AAV vector particles, more preferably AAV vector particles.

[0254] Although some embodiments of the present invention are described with respect to AAV vector particles, it will be understood that some embodiments may be applied mutatis mutandis to other viral vectors disclosed herein.

[0255] Transfection of cells with mRNA vectors can be achieved using, for example, nanoparticles such as liposomes.

[0256] In some embodiments, the vector is contained in a nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle, an inorganic nanoparticle, or a lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0257] Nanoparticles can be targeted to specific cell types using one or more ligands presented on the surface of the specific cell type.

[0258] In some embodiments, polynucleotide delivery is transposon-mediated.

[0259] In some embodiments, the polynucleotide is mRNA. The mRNA can be contained in a nanoparticle.

[0260] Adeno-associated virus (AAV) vector In one embodiment, the present invention provides an AAV vector particle comprising 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 shRNA having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the AAV vector particle is configured to cross the blood-brain barrier.

[0261] In one embodiment, the present invention provides an AAV vector particle comprising 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 shRNA having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the AAV vector particle is configured to cross the blood-brain barrier.

[0262] In one embodiment, the present invention provides AAV vector particles comprising 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 shRNA having at least 90% sequence identity with SEQ ID NO: 31. In some embodiments, the AAV vector particles are configured to cross the blood-brain barrier.

[0263] In one embodiment, the present invention provides AAV vector particles comprising 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 having at least 90% sequence identity with SEQ ID NO: 30, and / or (c) a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 31. In some embodiments, the AAV vector particles are configured to cross the blood-brain barrier.

[0264] Methods for preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0265] AAV vectors may contain the AAV genome or its fragments or derivatives.

[0266] The AAV genome is a polynucleotide sequence capable of encoding functions necessary for the production of AAV particles. These functions include those that operate in the AAV replication and packaging cycle in host cells, including capsid formation of the AAV genome onto AAV particles. Naturally occurring AAV is replication-deficient and relies on the provision of helper functions in trans to complete the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is typically replication-deficient.

[0267] The AAV genome may be in a single-stranded form, either positive-sense or negative-sense, or alternatively, a double-stranded form. The use of a double-stranded form allows for the avoidance of the DNA replication process in target cells, thereby potentially accelerating transgene expression.

[0268] The AAV genome can originate from any naturally occurring serotype, isolate, or clade of AAV. Therefore, the AAV genome can be the complete genome of a naturally occurring AAV. As is known to those skilled in the art, naturally occurring AAVs can be classified according to various biological systems.

[0269] Generally, AAV is referred to in terms of its serotypes. Serotypes correspond to variant subtypes of AAV, which have specific reactivity that can be used to distinguish them from other variant subtypes due to the expression profile of the capsid surface antigen. Typically, viruses with a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific to any other AAV serotype.

[0270] AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as recombinant serotypes such as Rec2 and Rec3, which have recently been identified from primate brains.

[0271] Several rAAV vectors have been reported to efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the central nervous system of neonatal mice (Zhang et al., Molecular Therapy 19:1440-1448).

[0272] In some embodiments, the AAV is the AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh39, or rh43 serotype. In some embodiments, the AAV vector particles contain the AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh39, or rh43 serotype capsid protein. In some embodiments, the AAV vector particles are the AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh39, or rh43 vector particles.

[0273] In some embodiments, the AAV is the AAV9, AAV9 PHP.B, AAV9 PHP.eB, or AAVrh10 serotype. In some embodiments, the AAV vector particle contains the AAV9, AAV9 PHP.B, AAV9 PHP.eB, or AAVrh10 serotype capsid protein.

[0274] In some embodiments, AAV is the AAV-DJ serotype. In some embodiments, the AAV vector particle contains an AAV-DJ capsid. AAV-DJ is described in Kondratov et al. (2021) Mol. Ther. 29:2806-2820.

[0275] The capsid protein may be an artificial or mutant capsid protein.

[0276] As used herein, the term “artificial capsid” means that the capsid particle contains an amino acid sequence that is not naturally occurring or that has been manipulated (e.g., modified) from a naturally occurring capsid amino acid sequence.

[0277] In other words, an artificial capsid protein, when its artificial capsid amino acid sequence is aligned with that of a parental capsid amino acid sequence, contains mutations or alterations in its amino acid sequence compared to the sequence of the parental capsid from which it is derived. Methods for sequence alignment are well known in the art and are referenced herein.

[0278] As used herein, the term “configured to cross the blood-brain barrier” means that the vector particles have the ability to cross the blood-brain barrier, and for example, the vector particles may include mutations or modifications from wild-type vector particles that improve the ability to cross the blood-brain barrier compared to unmodified or wild-type virus particles. The improvement in the ability to cross the blood-brain barrier may be measured, for example, by measuring the expression of a transgene possessed by the vector particles, such as GFP, where the expression of the transgene in the brain correlates with the ability of the virus particles to cross the blood-brain barrier.

[0279] In some embodiments, the AAV vector particles comprise an artificial capsid amino acid sequence that enables the viral particles to cross the blood-brain barrier.

[0280] In some embodiments, the AAV vector particles capable of crossing the blood-brain barrier comprise a VP1 capsid protein comprising an amino acid sequence comprising at least 4 consecutive amino acids derived from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

[0281] In some embodiments, the AAV vector particles capable of crossing the blood-brain barrier comprise a VP1 capsid protein comprising an amino acid sequence comprising at least 5 consecutive amino acids derived from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

[0282] In some embodiments, the AAV vector particles capable of crossing the blood-brain barrier comprise a VP1 capsid protein comprising an amino acid sequence comprising at least 6 consecutive amino acids derived from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

[0283] In some embodiments, the AAV vector particles capable of crossing the blood-brain barrier comprise a VP1 capsid protein comprising an amino acid sequence comprising the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19).

[0284] In some embodiments, the nucleic acid sequence encoding at least 4, at least 5, at least 6, or all 7 consecutive amino acids derived from the sequence TLAVPFK (SEQ ID NO: 18) or KFPVALT (SEQ ID NO: 19) is inserted at a position corresponding to the position between the sequences encoding amino acids 588 and 589 of AAV9 (SEQ ID NO: 20).

[0285] An example of the amino acid sequence of the (wild-type) AAV9 capsid is as follows: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (Sequence ID 20)

[0286] In some embodiments, the AAV vector particles contain an AAV9 PHP.B capsid, preferably an AAV-PHP.B VP1 capsid protein.

[0287] In some embodiments, the AAV vector particle that can cross the blood-brain barrier is AAV9 PHP.B.

[0288] In some embodiments, the amino acid sequence of the AAV-PHP.B capsid VP1 protein is as follows: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (Sequence ID 21)

[0289] In some embodiments, the AAV vector particle comprises a capsid having an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO: 21, more preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 21, wherein the AAV vector particle can cross the blood-brain barrier.

[0290] The AAV-PHP.B vector is described in Deverman et al. (2016) Nat Biotechnol 34:204-209 and International Publication No. 2015 / 038958, which are incorporated herein by reference.

[0291] In some embodiments, the AAV vector particles capable of crossing the blood-brain barrier include a VP1 capsid protein comprising an amino acid sequence containing the sequence DGTLAVPFKAQ (SEQ ID NO: 25).

[0292] In some embodiments, the AAV vector particle that can cross the blood-brain barrier is AAV9 PHP.eB.

[0293] In some embodiments, the amino acid sequence of the AAV-PHP.eB capsid VP1 protein is as follows: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (Sequence ID 22)

[0294] In some embodiments, the AAV vector particle comprises a capsid having an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity with SEQ ID NO: 22, more preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 22, wherein the AAV vector particle can cross the blood-brain barrier.

[0295] The AAV-PHP.eB vector is described in International Publication No. 2017 / 100671, which is incorporated herein by reference.

[0296] An overview of AAV serotypes can 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 elements of the AAV genome including ITR sequences, rep, or cap genes, for use in the present invention can be obtained from the following accession numbers for the whole AAV genome sequence: 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; Tuscan AAV ATCC VR-865 AY186198, AY629583, NC_004828; Tuscan AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

[0297] AAVs can also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally occurring AAVs, typically a phylogenetic group of AAVs that can be traced back to a common ancestor and include all of its descendants. Furthermore, AAVs can be referred to in terms of specific isolates, i.e., genetic isolates of a particular AAV found in nature. The term genetic isolate refers to a population of AAVs that has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a genetically distinct population.

[0298] A person skilled in the art can select a suitable serotype, clade, clone, or isolate of AAV for use in the present invention based on the common general knowledge of such persons.

[0299] AAV serotypes determine the tissue specificity (or tropism) of AAV virus infection.

[0300] Typically, the AAV genome of a naturally occurring serotype, isolate, or clade of AAV contains at least one terminal inversion sequence (ITR). The ITR sequence acts cis to provide a functional origin for replication, enabling the incorporation and excision of vectors from the cell's genome. In preferred embodiments, one or more ITR sequences are adjacent to a nucleotide sequence encoding a MeCP2 nucleotide sequence. The AAV genome may also contain packaging genes, such as rep and / or cap genes, which encode the packaging function of the AAV particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or their variants. The cap gene encodes one or more capsid proteins, such as VP1, VP2, and VP3 or their variants. These proteins constitute the capsid of the AAV particle.

[0301] Promoters are 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 commonly used to express rep genes, and the p40 promoter is commonly used to express cap genes.

[0302] Therefore, as discussed above, the AAV genome used in the AAV vector of the present invention may be a complete genome of naturally occurring AAV. For example, an AAV vector or vector particle can be prepared in vitro using a vector containing a complete AAV genome. However, although such vectors can, in principle, be administered to patients, this is rarely done in practice. Preferably, the AAV genome is derivatized for the purpose of administration to patients. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome and derivatives that can be produced by applying techniques known in the art. Derivatization of AAV genomes and AAV capsids is outlined in Coura and Nardi (2007) Virology Journal 4:99, as well as in Choi et al. and Wu et al. referenced above.

[0303] Derivatives of the AAV genome include any shortened or modified form of the AAV genome that enables in vivo expression of the transgene from the AAV vector of the present invention. Typically, it is possible to significantly cleave the AAV genome to include the smallest viral sequence while retaining the above-mentioned functions. This is preferred for safety reasons, as it reduces the risk of vector recombination with wild-type virus and also to avoid inducing a cellular immune response due to the presence of viral gene proteins in target cells.

[0304] Typically, derivatives contain at least one terminal inversion sequence (ITR), preferably more than one ITR, for example, two or more ITRs. One or more ITRs may originate from AAV genomes with different serotypes, or they may be chimeric ITRs or mutant ITRs. Preferred mutant ITRs have deletions of terminal resolution sites (trs). These deletions allow continuous replication of the genome to generate a single-stranded genome containing both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows for avoidance of DNA replication in target cells, thus enabling accelerated transgene expression.

[0305] In some embodiments, the AAV vector includes at least one, for example, two AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 ITRs. In some embodiments, the AAV vector includes at least one AAV9 ITR.

[0306] In some embodiments, the AAV vector includes two AAV9 ITRs.

[0307] One or more ITRs are preferably adjacent to any end of the nucleotide sequence encoding MeCP2. The inclusion of one or more ITRs is preferable, for example, to assist concatemer formation of the vector of the present invention in the nucleus of a host cell after conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase. The formation of such an episomal concatemer protects the vector construct for the lifetime of the host cell, thereby enabling the extension of transgene expression in vivo.

[0308] In preferred embodiments, the ITR element is the only sequence retained from the native AAV genome in the derivative. Therefore, the derivative preferably does not contain the rep and / or cap genes of the native genome, nor any other sequences of the native genome. This is preferred for the reasons mentioned above, and also to reduce the likelihood of vector integration into the host cell genome. Furthermore, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (e.g., regulatory elements) into the vector, in addition to the transgene.

[0309] Therefore, in the derivatives of the present invention, the following portions may be removed: one terminal inversion (ITR) sequence, a replication (rep) gene, and a capsid (cap) gene. However, in some embodiments, the derivatives may further include one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV is frequently incorporated into specific sites on human chromosome 19 and exhibits negligible frequency of random incorporation, and as a result, retention of incorporation ability in the vector may be acceptable in a therapeutic setting.

[0310] If the derivative includes 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 present invention encompasses providing capsid protein sequences derived from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., a pseudotype vector). Thus, in one embodiment, the AAV vector is in the form of a pseudotype AAV vector particle.

[0311] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide AAV vectors with one or more desired functionalities. Therefore, compared to AAV vectors containing naturally occurring AAV genomes such as the AAV2 genome, these derivatives may exhibit increased gene delivery efficiency, decreased immunogenicity (humoral or cellular), altered targeting range, and / or improved targeting of specific cell types. Increased gene delivery efficiency can be achieved through improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and nuclear transport, improved decoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Increased efficiency may also be associated with altered targeting range or targeting of specific cell populations, resulting in vector doses not being diluted by administration to tissues where it is not needed.

[0312] Chimeric capsid proteins include those produced by recombination between two or more capsid-coding sequences of naturally occurring AAV serotypes. This can be carried out, for example, by a marker-rescue approach in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and targeted selection is used to select the capsid sequence with the desired properties. The capsid sequences of the different serotypes may be modified by homologous recombination within the cell to produce novel chimeric capsid proteins.

[0313] Chimeric capsid proteins also include those produced by manipulating the capsid protein sequence 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.

[0314] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be constructed by randomly fragmenting the sequence of the relevant AAV gene, for example, the sequence encoding the capsid proteins of several different serotypes, and then reconstructing the fragments in a self-priming polymerase reaction, which can also cause cross-reactivity in regions of sequence homology. A library of hybrid AAV genes thus constructed by shuffling the capsid genes of several serotypes can be screened to identify viral clones with desired functionality. Similarly, error-prone PCR may be used to randomly mutate the AAV capsid gene to create a diverse library of variants, which can then be selected for desired properties.

[0315] The capsid gene sequence may also be genetically modified to introduce specific deletions, substitutions, or insertions from the natural wild-type sequence. In particular, the capsid gene may be modified by inserting sequences of unrelated proteins or peptides within the open reading frame of the capsid coding sequence, or at the N-terminus and / or C-terminus of the capsid coding sequence.

[0316] Unrelated proteins or peptides may, advantageously, act as ligands for specific cell types, thereby conferring improved binding to target cells or improving the specificity of vector targeting to specific cell populations (e.g., brain microvascular endothelial cells). Unrelated proteins may also assist in the purification of viral particles as part of the production process, i.e., they may be epitopes or affinity tags. Insertion sites are typically selected so as not to interfere with other functions of viral particles, such as their internalization and transport. Those skilled in the art can identify suitable insertion sites based on common general knowledge.

[0317] The present invention further encompasses providing sequences of the AAV genome in an order and configuration different from that of the natural AAV genome. The present invention also encompasses the substitution of one or more AAV sequences or genes with chimeric genes composed of sequences from another virus or more than one virus. Such chimeric genes may consist of sequences from two or more related viral proteins of different viral species.

[0318] The AAV vector of the present invention may take the form of a nucleotide sequence containing an AAV genome or a derivative thereof, and a sequence encoding a MeCP2 transgene or a derivative thereof.

[0319] The AAV particles of the present invention include a transcapsidized form in which an AAV genome or derivative having an ITR of one serotype is packaged on a capsid of a different serotype. The AAV particles of the present invention also include a mosaic form in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. The AAV particles also include a chemically modified form having ligands adsorbed to the capsid surface. For example, such ligands may include antibodies that target specific cell surface receptors.

[0320] Therefore, for example, the AAV particles of the present invention include those having the AAV2 genome and AAV9 capsid protein (AAV2 / 9), or the AAV9 PHP.B or PHP.eB capsid protein.

[0321] AAV vectors may contain multiple copies (e.g., two, three, etc.) of the nucleotide sequences referred to herein.

[0322] Retrovirus and lentiviral vectors Retroviral vectors may be derived from or derive from any suitable retrovirus. Numerous different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (avian Examples include erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin, J. Met. al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0323] Retroviruses can be broadly classified into two categories: "simple" and "complex." Retroviruses can also be further classified into seven groups. Five of these groups represent retroviruses with carcinogenic potential. The remaining two groups are lentiviruses and spumaviruses.

[0324] The basic structure of retroviral and lentiviral genomes shares many common features, such as the 5'LTR and 3'LTR. Between or within these are the packaging signals that enable the packaging of the genome, primer binding sites, integration sites that allow integration into the host cell genome, and the gag, pol, and env genes that encode packaging components—these are polypeptides necessary for the construction of the viral particle. Lentiviruses possess additional features, such as the rev and RRE sequences in HIV, which enable the efficient transport of the integrated proviral RNA transcript from the nucleus to the cytoplasm of the infected target cell.

[0325] In proviruses, these genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for provirus integration and transcription. LTRs also function as enhancer-promoter sequences, regulating the expression of viral genes.

[0326] The LTR itself is an identical sequence that can be classified into three elements: U3, R, and U5. U3 originates from a sequence specific to the 3' end of the RNA. R originates from a sequence repeated at both ends of the RNA. U5 originates from a sequence specific to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0327] In defective retroviral vector genomes, gag, pol, and env may be absent or non-functional.

[0328] In a typical retroviral vector, at least a portion of one or more protein-coding regions essential for replication may be removed from the virus. This results in a replication defect in the viral vector. A portion of the viral genome may also be replaced by a library encoding candidate modifiers operably linked to regulatory and reporter regions in the vector genome, in order to generate a vector containing candidate modifiers that can be transduced into target host cells and / or incorporate their genome into the host genome.

[0329] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin, J. Met. al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. Briefly, lentiviruses can be classified into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of 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 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).

[0330] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis, P et al. (1992) EMBO J.11:3053-8, Lewis, P et al. (1994) J. Virol.68:510-6). In contrast, other retroviruses such as MLV cannot infect non-dividing or slowly dividing cells, such as the cells that make up muscle, brain, lung, and liver tissue.

[0331] The lentiviral vectors used herein are vectors comprising at least one component moiety that can be derived from a lentivirus. Preferably, the component moiety is involved in the biological mechanism by which the vector infects cells, expresses genes, or replicates.

[0332] Lentiviral vectors can be "primate" vectors. Lentiviral vectors can be "non-primate" vectors (i.e., derived from viruses that do not primarily infect primates, especially humans). Examples of non-primate lentiviruses can be any member of the Lentiviridae family that does not naturally infect primates.

[0333] Examples of lentivirus-based vectors include those based on HIV-1 and HIV-2, which are listed below.

[0334] HIV-1 vectors contain cis-acting elements, also found in simple retroviruses. Sequences extending within the gag open reading frame have been shown to be crucial for HIV-1 packaging. Therefore, HIV-1 vectors often contain the relevant portion of the gag with a mutated translation start codon. Furthermore, most HIV-1 vectors also contain a portion of the env gene, including the RRE. Rev binds to the RRE, which enables the transport of full-length or single-spliced ​​mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, the need for Rev and RRE may be reduced by using constitutive transport elements derived from certain simple retroviruses, such as Mason-Pfizer salvir. The viral protein Tat is required for efficient transcription from the HIV-1 LTR promoter.

[0335] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Like HIV-1-based vectors, HIV-2 vectors also require RRE (Reactive Reproductive Energy) for efficient transport of full-length or single-spliced ​​viral RNA.

[0336] Preferably, the viral vector used in the present invention has a minimal viral genome.

[0337] It should be understood that the “minimal viral genome” is manipulated to remove non-essential elements and retain essential elements in order to provide the functionality necessary for infecting, transducing, and delivering the desired nucleotide sequence to target host cells. Further details of this strategy can be found in International Publication No. 1998 / 017815.

[0338] Preferably, the plasmid vector used to produce the viral genome within a host cell / packaging cell contains sufficient lentiviral genetic information to enable the packaging of the RNA genome into viral particles that can infect target cells in the presence of the packaging component but cannot independently replicate and produce infectious viral particles within the final target cell. Preferably, the vector lacks the functional gag-pol and / or env genes and / or other genes essential for replication.

[0339] However, plasmid vectors used to produce viral genomes within host / packaging cells also contain transcriptional regulatory sequences operably ligated to the lentiviral genome to guide genome transcription in the host / packaging cells. These regulatory sequences may be native sequences related to the transcribed viral sequence (i.e., the 5'U3 region), or they may be heterologous promoters, such as another viral promoter (e.g., the CMV promoter).

[0340] The vector may be a self-inactivating (SIN) vector lacking viral enhancer and promoter sequences. SIN vectors can be generated and transduced into non-dividing cells in vivo with efficacy similar to that of wild-type vectors. Transcriptional inactivation of the long-chain terminal repeat (LTR) in the SIN provirus should prevent recruitment by the reproducible virus. This should also allow for regulated gene expression from the internal promoter by eliminating any cis-effect of the LTR.

[0341] Vectors may have integration defects. Integration-defective lentiviral vectors (IDLVs) can be produced, for example, by packaging the vector with a catalytically inactive integrase (such as HIV integrase with 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. et al. (1996) J. Virol. 70:721-8), or by modifying or deleting the essential att sequence from the vector LTR (Nightingale, S. J. et al. (2006) Mol. Ther. 13:1121-32), or by a combination of the above.

[0342] Adenovirus vector Adenoviruses are double-stranded, linear DNA viruses that do not undergo RNA intermediates. There are over 50 different human serotypes of adenoviruses, classified into six subgroups based on genetic sequence homology. The natural targets of adenoviruses are the respiratory and gastrointestinal epithelium, generally causing only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are the most commonly used in adenovirus vector systems and are typically associated with upper respiratory tract infections in young people.

[0343] Adenoviruses are used as vectors for gene therapy and heterologous gene expression. Their large (36kb) genomes can accommodate up to 8kb of foreign insertion DNA and replicate efficiently in complementary cell lines, up to 10 12 Adenoviruses can produce extremely high titers. Therefore, adenoviruses are one of the best systems for studying gene expression in primary non-replicating cells.

[0344] The expression of viruses or exogenous genes from adenovirus genomes does not require replicating cells. Adenovirus vectors enter cells via receptor-mediated endocytosis. Once inside the cell, adenovirus vectors are hardly integrated into the host chromosome. Instead, they function episomal (independent of the host genome) as linear genomes in the host nucleus. Therefore, the use of recombinant adenoviruses mitigates the problems associated with random integration into the host genome.

[0345] Variants, derivatives, analogues, homologs, and fragments In addition to the specific proteins and nucleotides referred to herein, the present invention also encompasses their variants, derivatives, analogues, homologues, and fragments.

[0346] In the context of the present invention, a “variant” of any given sequence is a sequence in which a specific sequence of residues (either amino acid residues or nucleic acid residues) is modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, exchanging, and / or altering at least one residue present in the naturally occurring polypeptide or polynucleotide.

[0347] As used herein with respect to the protein or polypeptide of the present invention, the term “derivative” includes any substitution, alteration, modification, exchange, deletion, and / or addition of one (or more) amino acid residues to or from a sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0348] As used herein with respect to polypeptides or polynucleotides, the term “analog” includes any mimetic, i.e., a chemical compound having at least one of the endogenous functions of the polypeptide or polynucleotide it mimics.

[0349] Typically, amino acid substitutions may involve, for example, 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or capability. Amino acid substitutions may include the use of analogues that do not exist in nature.

[0350] The proteins used in this invention may also undergo silent changes, resulting in deletions, insertions, or substitutions of amino acid residues that produce functionally equivalent proteins. Intentional amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the endogenous function is preserved. 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 heads and similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0351] Conservative substitutions can be performed, for example, according to the following table. Amino acids in the same block in the second column, preferably in the same row in the third column, can be substituted for each other:

[0352] [Table 2]

[0353] As used herein, the term “homologous” means an entity having a specific homology to a wild-type amino acid sequence or wild-type nucleotide sequence. The term “homologous” can be considered equivalent to “identity.”

[0354] In the context of the present invention, homologous sequences are interpreted as containing amino acid sequences that are at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, preferably at least 95%, 96%, 97%, 98%, or 99% identical. Typically, homologous sequences contain the same active sites as the target amino acid sequence. Homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), but in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0355] In the context of the present invention, homologous sequences are interpreted as including nucleotide sequences that are at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, and preferably at least 95%, 96%, 97%, 98%, or 99% identical. While homology can also be considered in terms of similarity, in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0356] Preferably, a reference to a sequence having an identity percentage with any one of the sequence numbers detailed herein refers to a sequence having the described identity percentage with respect to the full length of the referenced sequence number.

[0357] Homology comparisons can be performed visually or, more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0358] Homology percentages can be calculated for consecutive sequences; that is, one sequence is aligned with the other, and each amino acid or nucleotide in one sequence is directly compared, one residue at a time, with the corresponding amino acid or nucleotide in the other sequence. This is called a "gapless" alignment. Typically, such gapless alignments are performed only over relatively short number of residues.

[0359] While this is a very simple and consistent method, it fails to consider, for example, that in otherwise identical sequence pairs, a single insertion or deletion in an amino acid or nucleotide sequence can deviate from the alignment of subsequent residues or codons, potentially leading to a significant reduction in the homology percentage when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without excessively penalizing the overall homology score. This is achieved by attempting to maximize local homology by inserting "gaps" into the sequence alignment.

[0360] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, resulting in sequence alignments with as few gaps as possible, reflecting a higher relevance between the two sequences being compared for the same number of identical amino acids or nucleotides, achieving a higher score than those with many gaps. A typical "affine gap cost" is used, which imposes a relatively high cost for the presence of gaps and a smaller penalty for each residue following a gap. This is the most commonly used gap scoring system. A high gap penalty naturally results in an optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0361] Therefore, calculating the maximum homology percentage first requires creating an optimal alignment, taking gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA, Develeux et al. (1984) Nucleic Acids Research 12:387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (Ausubel et al. (1999) ibid-Ch.18), FASTA (Atschul et al. (1990) J.Mol.Biol.403-410), and the GENEWORKS comparison tools suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferable 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).

[0362] While the final homology percentage can be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix (the default matrix for the BLAST program suite). The GCG Wisconsin program generally uses either publicly available default values ​​or custom symbol comparison tables, if provided (see the user manual for further details). For some applications, it is preferable to use the publicly available default values ​​for the GCG package, or, in the case of other software, a default matrix such as BLOSUM62.

[0363] Once the software creates the optimal alignment, it becomes possible to calculate the homology percentage, preferably the sequence identity percentage. The software typically does this as part of a sequence comparison and generates a numerical result.

[0364] A "fragment" is also a variant, and this term typically refers to a selected region of a polypeptide or polynucleotide that is functionally or, for example, in an assay, of interest. Therefore, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0365] Such variants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is performed, synthetic DNA encoding the insertion can be prepared along with 5' and 3' faciest regions corresponding to the naturally occurring sequence on either side of the insertion site. The faciest regions contain convenient restriction sites corresponding to locations in the naturally occurring sequence, and as a result, the sequence can be cleaved with a suitable enzyme, and the synthetic DNA can be ligated to the cleavage. The DNA is then expressed according to the present invention, producing the encoded protein. These methods are merely examples of numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0366] Codon optimization The polynucleotides used in this invention can be codon-optimized. Codon optimization has been previously described in International Publications 1999 / 41397 and 2001 / 79518. Different cells have different codon usage frequencies. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a given cell type. It is possible to increase expression by modifying the codons in the sequence to match the relative abundance of the corresponding tRNA. Similarly, it is possible to decrease expression by deliberately selecting codons for which the corresponding tRNA is known to be rare in a given cell type. Thus, an additional degree of translational control is available. Codon usage tables for mammalian cells and for various other organisms are known in the art.

[0367] Exemplary vector In some embodiments, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 23. Preferably, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having 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 with SEQ ID NO: 23.

[0368] In some embodiments, the vector of the present invention comprises or consists of the nucleotide sequence of SEQ ID NO: 23, or a fragment thereof.

[0369] In some embodiments, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 24. Preferably, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having 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 with SEQ ID NO: 24.

[0370] In some embodiments, the vector of the present invention comprises or consists of the nucleotide sequence of SEQ ID NO: 24, or a fragment thereof. (Sequence ID 23) Sequence ID 24)

[0371] Further exemplary vector sequences (AAV_CBA_hMECP2_mirT) are as follows:

[0372] [Table 3]

[0373] [Table 4] (Sequence ID 26) Bold (lowercase): 3' and 5' ITR Bold (uppercase): Human MECP2 CDS Uppercase: 3'UTR Underlined: Poly A Underlined: Chicken β-actin promoter

[0374] [Table 5]

[0375] In some embodiments, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 26. Preferably, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having 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 with SEQ ID NO: 26.

[0376] In some embodiments, the vector of the present invention comprises or consists of the nucleotide sequence of SEQ ID NO: 26, or a fragment thereof.

[0377] Further exemplary vector sequences (AAV_shRNA_CBA_hMECP2_mirT) are as follows:

[0378] [Table 6]

[0379] [Table 7]

[0380] [Table 8] (Sequence ID 27) Bold (lowercase): 3' and 5' ITR Upper case: shRNA_hMECP2 Underlined: H1 promoter Bold (uppercase): Human MECP2 CDS Uppercase: 3'UTR Underlined: Poly A Underlined: Chicken β-actin promoter

[0381] [Table 9]

[0382] In some embodiments, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having at least 70% sequence identity with SEQ ID NO: 27. Preferably, the vector of the present invention comprises or consists of a nucleotide sequence or fragment thereof having 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 with SEQ ID NO: 27.

[0383] In some embodiments, the vector of the present invention comprises or consists of the nucleotide sequence of SEQ ID NO: 27, or a fragment thereof.

[0384] Treatment method In some embodiments, the treatment method provides MeCP2 to the target central nervous system.

[0385] In some embodiments, the treatment method provides MeCP2 to the somatosensory cortex and / or striatum of the target.

[0386] In some embodiments, the treatment method provides MeCP2 to neuronal cells.

[0387] In some embodiments, the treatment method provides improvement in motor function in the subject. Methods for measuring motor function are known to those skilled in the art, such as beam balance tests.

[0388] In some embodiments, the treatment method provides improvement in learning and / or cognitive function in the 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 can be used. Alternative cognitive tests include, but are not limited to, the Mini Mental State Examination (MMSE), the Six-item Cognitive Impairment Test (6CIT), the Abbreviated Mental Test (AMT), and the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE).

[0389] Advantageously, the present invention provides a method for treatment by systemic administration of the vector particles of the present invention.

[0390] Pharmaceutical compositions and injectable solutions The agents used in this invention can be administered alone, but generally, especially for the treatment of humans, they are administered in mixture with a pharmaceutical carrier, excipient, or diluent.

[0391] The pharmaceuticals of the present invention, such as vector particles, can be formulated into pharmaceutical compositions. These compositions may include, in addition to the pharmaceuticals, pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers, 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 exact properties of the carrier or other materials can be determined by those skilled in the art according to the route of administration, such as intravenous or intra-arterial routes.

[0392] Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, magnesium chloride, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, surfactants such as 0.001% prulonic acid (PF68) may be used. In some cases, serum albumin may be used in the composition.

[0393] For injection, the active ingredient may be in the form of an aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the art can readily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer's injection, or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0394] For delayed release, the pharmaceutical may be contained in a pharmaceutical composition formulated for sustained release according to methods known in the art, for example, in microcapsules formed from biocompatible polymers or in liposome carrier systems.

[0395] The handling of cell therapy products is preferably carried out in accordance with the FACT-JACIE international standard for cell therapy.

[0396] Administration In some embodiments, polynucleotides, vectors, cells, or pharmaceutical compositions are administered systemically to a subject.

[0397] In some embodiments, polynucleotides, vectors, cells, or pharmaceutical compositions are administered topically to a subject.

[0398] In some embodiments, polynucleotides, vectors, cells, or pharmaceutical compositions are administered intracranially, intracerebrally, or intraparenchymically to a subject.

[0399] As used herein, the terms “systemic delivery” or “systemic administration” mean that the agent of the present invention is administered into the circulatory system, for example, to achieve broad distribution of the agent. In contrast, local or topical administration limits the delivery of the agent to a local area, and intracerebral administration, for example, involves direct injection into the brain.

[0400] In some embodiments, polynucleotides, vectors, cells, or pharmaceutical compositions are administered intravascularly, intravenously, or intra-arterially.

[0401] Preferably, in some embodiments, the polynucleotide, vector, cell, or pharmaceutical composition is administered into the internal carotid artery.

[0402] As used herein, the term “agent” may refer to the polynucleotides, vectors, cells, or pharmaceutical compositions of the present invention.

[0403] In some embodiments, polynucleotides, vectors, cells, or pharmaceutical compositions are administered simultaneously, sequentially, or separately in combination with immunosuppressants.

[0404] In some embodiments, the immunosuppressant is cyclosporine A (CsA).

[0405] As used herein, the terms “combination,” “in combination,” “used in combination,” or “combination preparation” may refer to the simultaneous, sequential, or separate combination administration of two or more drugs.

[0406] As used herein, the term “simultaneous” means that the drugs are administered concurrently, i.e., at the same time.

[0407] As used herein, the term “sequential” means that the drugs are administered one after another.

[0408] As used herein, the term “separate” means that the drugs are administered independently of each other, but within a time interval that allows the drugs to exert a combined, preferably synergistic, effect. Therefore, “separate” administration may allow one drug to be administered, for example, within 1 minute, 5 minutes, or 10 minutes of the other.

[0409] Dosage Those skilled in the art can easily determine the appropriate dose of the drug of the present invention for administration to a subject. Typically, a physician determines the most suitable actual dose for an individual patient, which depends on various factors including the activity of the particular compound used, its metabolic stability and duration of action, age, weight, overall health, sex, diet, mode and timing of administration, excretion rate, drug combination, severity of the particular condition, and the individual receiving treatment. Naturally, there may be individual cases where a higher or lower dose range is advantageous, and such cases are within the scope of the present invention.

[0410] subject As used herein, the term “subject” refers to either a human or a non-human animal.

[0411] Examples of non-human animals include vertebrates, such as non-human primates (especially higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs, and mammals such as cats. Non-human animals can also be companion animals.

[0412] Preferably, the subject is a human being.

[0413] In one embodiment, the subject is a mouse model of Rett disease.

[0414] Those skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the invention as disclosed.

[0415] Preferred features and embodiments of the present invention are described herein as non-limiting examples.

[0416] The implementation of this invention will utilize conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the scope of the skills of those skilled in the art, unless otherwise specified. Such techniques are described in the literature. For example, Sambrook, J., Fritsch, EFand Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Ausubel, FM et al. Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley&Sons, Polak, JMand McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press, Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, and Lilley, DMand. Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part See A:Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. [Examples]

[0417] Example 1 result Considering the highly differentiated expression of Mecp2 between neuronal and glial cells in the brain, the inventors attempted to realize a system that differentiates the expression of therapeutic genes and is compatible with the strict packaging size (less than 4.8 kb) of AAV vectors. To this end, the inventors devised a method to place the virally transmitted therapeutic Mecp2 gene under the control of cell-type specific microRNAs. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules that may contain 21-23 nucleotides involved in RNA silencing. miRNAs form base pairs with complementary sequences in mRNA molecules, leading to gene silencing through mRNA cleavage, destabilization, and translation arrest. The inventors hypothesized that by incorporating target sequences of miRNAs selectively expressed in glial cells into a viral vector, transgene expression in these cells would be regulated, but not in neurons. Furthermore, the inventors hypothesized that increasing the number of targets for a specific miRNA would increase its silencing efficiency, and therefore, selecting the appropriate number of miRNA target sequences in the vector would provide a method for finely regulating the expression level of the transgene. To test this hypothesis, the inventors prepared cM2 AAV virus vectors containing a strong CBA promoter upstream of the V5-Mecp2 isoform-1 gene, with two copies, four copies, or no copies inserted of the well-known miR-124 target sequence (miR-124TS) that is specifically expressed in neurons (Figure 1). Next, the inventors prepared AAV9 particles and transduced them into primary mouse neuron cultures in a dish. Seven days after transduction, protein lysates were prepared from the neuron cultures and Western blotting was performed to test the levels of V5, a surrogate for the Mecp2 protein with a tag fused at the 5' end. Interestingly, V5-Mecp2 protein levels were strongly reduced in vectors incorporating miR-124TS, with the silencing level being highest when four copies of miR-124TS were included (Figure 1).These results suggest that including miR target sites in AAV vectors can suppress viral gene expression, and that the level of silencing is proportional to the number of miR target site copies incorporated into the vector. To utilize this system, we generated novel cM2 vectors in which two or four copies of miR-31 and miR-338 target sites, respectively, were cloned to be specifically expressed in astrocytes or oligodendrocytes (Figures 2 and 3). cM2 vectors containing miR-31TS were produced in AAV9 particles in transduced primary mouse astrocyte cultures. Seven days after transduction, astrocytes were harvested and protein lysates were analyzed by Western blotting. Interestingly, V5-Mecp2 protein levels were significantly reduced when miR-31TS was incorporated into the vector, and the most potent silencing was obtained with four miR-31TS copies (Figure 2). A very similar trend to V5-Mecp2 protein silencing was observed in primary mouse oligodendrocyte cultures transduced with a cM2 vector incorporating an increased number of miR-338 target sites (Figure 3).

[0418] Next, the inventors decided to construct multiple miR target sequences together in a specific viral vector. Considering the very low levels of MeCP2 in glial cells, the inventors decided to incorporate four copies of miR-31TS and miR-338TS (Figure 4). Although Mecp2 is highly expressed in neurons, viral Mecp2 expression is likely to be higher given the use of multiple copies of the AAV vector and a potent CBA promoter entering neurons. Therefore, the inventors decided to add one single miR-124TS copy to partially reduce the total gene level in transduced neuronal cells (Figure 4A). The resulting viral vector, named cM2-mR, was produced in AAV9 particles in either transduced mouse primary neuron cultures, astroglial cultures, or oligodendrocyte cultures in a dish. The transduced cell cultures were lysed and Western blotting was performed for protein quantification. V5-Mecp2 levels were high in neuronal cell lysates but significantly lower in astrocytic and oligodendrocyte cultures (Figure 4B). Notably, this protein profile closely matches that of endogenous Mecp2 protein levels. Therefore, cM2-mR viral vectors with a predetermined number of target sequences incorporated for cell type-specific miRNAs can differentiate viral Mecp2 expression levels in different cell types to achieve a pattern equivalent to that of the endogenous gene expression profile. Next, cM2-mR AAV9 particles were inoculated into the striatum of adult wild-type mice by stereotactic guided injection (Figure 5A). Three weeks after transduction, 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). Notably, the V5-Mecp2 immunostaining was assessed by NeuN, as assessed by unbiased intensity signal quantification. + GFAP compared to neurons +The expression level was significantly lower in astrocytes (Figure 5B). These findings support the efficiency of the miR-TS cassette in differentially reducing the expression of the viral Mecp2 transgene in brain cells in vivo.

[0419] Another overlooked challenge in establishing safe gene therapy for RTT is the somatic brain mosaic, where both wild-type and mutant MECP2 cells coexist. Therefore, transduction of a standard Mecp2-expressing viral vector into wild-type cells boosts its total expression to hyperphysiological levels that can cause undesirable side effects. To address this, we devised adding an shRNA cassette to the vector that can silence endogenous Mecp2 expression but not viral Mecp2 expression. By thus silencing only endogenous Mecp2, the vector ensures a uniform level of viral Mecp2 in both wild-type and mutant brain cells. Furthermore, this silencing approach may also be useful in suppressing mutant Mecp2 morphologies that may compete with functional Mecp2 through gain-of-function pathological mechanisms. Therefore, the inventors designed two shRNAs (shRNA-U1 (SEQ ID NO: 16) and shRNA-U2 (SEQ ID NO: 15)) on the 3'-UTR sequence of mouse Mecp2 and cloned them downstream of the PolIII promoter H1 either in a viral vector alone or within cM2 (Figure 6A). Next, these vectors were produced in AAV9 particles and transduced into primary mouse neuronal cultures. Seven days after transduction, qPCR and Western blotting were performed using neuronal cell lysates for mRNA and protein analysis, respectively. Both shRNAs were able to potently downregulate endogenous Mecp2 mRNA levels (Figure 6B). Furthermore, shRNA-U2 (shU2) was able to reliably downregulate endogenous Mecp2 protein levels, as assessed by Western blotting (Figure 6C). However, in neurons transduced with CM2-shU2, the Mecp2 protein corresponding to the V5-Mecp2 morphology expressed by the viral vector was readily detectable by Western blotting. These results confirm that shRNA-U2 effectively silences endogenous mouse Mecp2 but does not silence viral Mecp2 morphology.

[0420] At this point, the inventors incorporated the H1-shRNA-U2 cassette into cM2-mR to generate a final vector called CM2-ELO (Endogenous Level Optimization). CM2-ELO contains both a miR-TS cassette for Mecp2 regulatory expression in brain cells and a Mecp2-shRNA sequence for silencing the endogenous Mecp2 gene while expressing a functional copy of the gene under a strong CBA promoter with an engineered Mecp2 3'-UTR sequence. The final composition of the CM2-ELO vector is shown in Figure 7.

[0421] Materials and methods Genesis of gene transfer vectors Mecp2 CDS containing the 3'-UTR (223 bp) was PCR amplified to add a V5 tag to the 5' end of the coding sequence and inserted into the CBA-CreNLS vector described in Morabito (2017) Mol Ther. 25:2727-2742 to generate the cM2 vector. Target sequences of selected microRNAs were designed using 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 TargetScan software (https: / / www.targetscan.org / vert_80 / ).

[0422] AAV vector creation Non-replicating recombinant AAV virus particles were generated in 293T cells and cultured in Dulbecco's 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 passaged every 3-4 days using 0.25% trypsin (Sigma-Aldrich). Non-replicating recombinant virus particles were generated in 293T cells by simultaneous transfection with polyethyleneimine (polyethylenimine, PEI, Polyscience) of three different plasmids: a transgene-containing plasmid, a packaging plasmid for the rep and cap genes, and pHelper (Agilent) for three adenovirus helper genes. Cells and supernatant were harvested after 120 hours. Cells were lysed in a hypertonic buffer (40 mM Tris, 500 mM NaCl, 2 mM MgCl2, pH=8) containing 100 U / ml Salt Active Nuclease (SAN, Arcticzymes) at 37°C for 1 hour. Virus particles present in the supernatant were concentrated by precipitation using 8% PEG8000 (polyethylene glycol 8000, Sigma-Aldrich) and then added to the supernatant for further incubation at 37°C for 30 minutes. To clarify the lysate, cell debris was separated by centrifugation (4000 g, 30 min). The viral phase was isolated in the 40% fraction using an iodixanol step gradient (15%, 25%, 40%, 60% Optiprep, Sigma-Aldrich) and concentrated in PBS (phosphate-buffered saline) using a 100K cutoff concentrator (Amicon Ultra15, MERCK-Millipore). The viral titer was determined using the AAVpro(c) titration kit Ver2 (TaKaRa).

[0423] cultures of primary mouse neurons Primary neuron cultures were prepared from male mouse embryos at embryonic day 18.5 (E18.5). The cortex was dissected individually, and HBSS (Ca 2+ and Mg 2+ Cells were sequentially incubated in trypsin (0.005%, 15 minutes at 37°C, Sigma-Aldrich) and DNAse (0.1 mg / mL, 3 minutes at room temperature, Sigma-Aldrich) in a Hanks buffer solution (Euroclone) that did not contain 0.6% glucose (Sigma-Aldrich), 0.2% penicillin / streptomycin (Sigma-Aldrich), 0.25% L-glutamine (Sigma-Aldrich), and 1% B27 (ThermoFisher Scientific) in Neurobasal medium (ThermoFisher Scientific), which was then plated on a poly-L-lysine (Sigma-Aldrich) coated dish (2.0 × 10⁻¹⁴). 5 cells / cm 2 ).

[0424] Primary mouse astrocytic culture Astrocyte cultures were established from P1 mouse offspring. After dissection, the hippocampus and cortex were dissociated by treatment with trypsin (0.25%, Gibco, Thermo Fisher) and DNase-I (Sigma-Aldrich) at 37°C for 15 minutes, followed by fragmentation with a pipette. The dissociated cells were plated onto 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, microglia cells were harvested from 10-14 day old cultures by orbital shaking at 200 rpm for 30 minutes.

[0425] culture of first-generation mouse oligoglia P3 mouse brains were harvested for cell dissociation and O4-positive cell enrichment using magnetic-activated cell sorting (MACS, Miltenyi Biotec). Pooled brains (n = 4) were dissociated using the Papain Neural Tissue Dissociation Kit. Brain homogenate cells were first incubated with anti-O4 microbead antibody to isolate O4 + cells. O4 + cells (approximately 60,000 OPCs per pup) were cultured 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) on poly-d-ornithine (Sigma Aldrich)-coated 24-well plates (30,000 cells / well). 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.

[0426] Western blot[[ID=⑨]] Protein extracts were prepared in RIPA buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 0.5% Triton, and a complete 1% protease and phosphatase inhibitor mixture, Roche Diagnostics). Primary neuron, brain, and liver lysate samples (50 μg of protein lysate) were separated using an 8% polyacrylamide gel and then transferred to a PVDF membrane. The membranes were incubated overnight at 4°C with the following primary antibodies in 1×PBST containing 5% w / v defatted dry: mouse anti-V5 (1:1000, ThermoFisher Scientific), rabbit anti-calnexin (1:50000, Sigma), and mouse anti-β-actin (1:50000, Sigma). Subsequently, the membranes were incubated with the corresponding horseradish peroxidase (HRP) conjugated secondary antibody (1:10000, Dako). Next, the signal was identified using a chemiluminescent solution (ECL reagent, RPN2232, GE Healthcare) and detected using a ChemiDoc imaging system (Bio-Rad).

[0427] Immunofluorescence Cells (neurons, astrocytes, and oligodendrocytes) were fixed with ice-cold 4% paraformaldehyde (PFA) at 4°C for 30 minutes, washed with PBS (3 times), and incubated with 10% donkey serum to saturate nonspecific binding sites and 0.1% Triton X-100 at room temperature for 1 hour to create pores on the cell membrane and promote antibody binding to intracellular antigens. Subsequently, the cells were incubated overnight at 4°C with primary antibodies. After washing with PBS (3 times), the cells were incubated at room temperature for 1 hour in a blocking solution containing DAPI (1:1000, Sigma-Aldrich) and Alexa Fluor-488 and Alexa Fluor-594 anti-rabbit or anti-mouse secondary antibodies (1:1000, ThermoFisher Scientific). After washing with PBS (3 times), the cells were mounted with fluorescent mounting medium (Dako). Images were acquired using a Nikon Eclipse600 fluorescence microscope.

[0428] Brain tissue was sectioned using a cryostat after embedding in an optimal cutting temperature compound (OCT) in dry ice. Floating 50 μm thick coronal sections were rinsed in PBS and incubated with 10% donkey serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) at room temperature for 1 hour, followed by incubation overnight at 4°C with primary antibody (diluted in blocking solution). After washing with PBS three times, the sections were incubated at room temperature for 1 hour in a blocking solution containing DAPI (1:1000, Sigma-Aldrich) and Alexa Fluor-488 and Alexa Fluor-594 anti-rabbit or anti-mouse secondary antibodies (1:1000, ThermoFisher Scientific). After washing with PBS three times, the sections were mounted with fluorescent mounting medium (Dako). Confocal images were acquired at 125x magnification using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems Ltd). Cells and tissues were stained with the following primary antibodies: rabbit anti-MeCP2 (1:500, Cell Signaling Technology) and anti-chicken MAP2 (1:500, Abcam).

[0429] Surgical procedures for animals After anesthetizing the animals with isoflurane and positioning them on a stereotactic support, two holes were drilled in the skull to allow the nerve needle containing the virus to be injected into the mouse striatum according to the following coordinates: anterior-posterior (AP) -2.0 mm, medial-lateral (ML) +1.5 mm, dorsal-ventral (DV) -2.0 mm. Throughout the surgical procedure, oxygen and isoflurane levels were adjusted according to the health status of the mice. The animals were then sacrificed two weeks after viral transduction, and the brains were subsequently harvested.

[0430] statistics Numerical values ​​are expressed as mean ± standard deviation, as shown. All statistical analyses were performed using GraphPad Prism 8.0 with one-way ANOVA, two-way ANOVA, and independent t-tests. P-values ​​less than 0.05 were considered statistically significant. In multi-group comparisons, multiple test correction for paired tests between groups was applied using Tukey's post-hoc analysis.

[0431] Example 2 result Downregulation of the endogenous human MECP2 gene To generate a transgene cassette applicable to human cells where MECP2 expression is finely regulated, as in the mouse model, we began by selecting an shRNA to downregulate the endogenous MECP2 gene. As described in the mouse model, this shRNA was designed to target the MECP2 3'UTR region present at the endogenous locus, although it is not included in the transgene cassette (Figure 8A). We then evaluated the efficiency in human HeLa cells by transfecting plasmids expressing the selected shRNA (or scrambled control) with GFP as a marker for transfected cells using qRT-PCR and immunofluorescence (Figure 8A). Two days after transfection, we performed qRT-PCR and confirmed approximately 70% downregulation of the MECP2 gene compared to the control (Figure 8B). Finally, the inventors demonstrated the actual reduction of MeCP2 protein by immunofluorescence and evaluated the intensity of the MeCP2 signal in treated transfected cells (GFP+) compared to scrambled controls (Figure 8C).

[0432] Verification of MECP2 cell type-specific expression in Ret iPSC-derived neurons and astrocytes. After validating shRNA, the inventors confirmed the efficiency of their miRNA target sequence cassette (mirT: 1 copy of mir-124, 4 copies of mir-31, 4 copies of mir-338) in directly regulating MeCP2 expression in human cell conditions. To do this, the inventors generated neurons from human neuron precursors (NPCs) derived from hIPSCs. After 4 weeks of differentiation, human neurons were transduced with AAVs expressing MECP2 CDS with or without the mirT cassette (Figure 9A). Ten days after infection, the cells were fixed, and exogenous MeCP2 expression was evaluated using V5 tags (Figure 9B). In this way, the inventors demonstrated that the presence of one copy of the mir-124 target sequence can mitigate the expression of transgene MeCP2 in neurons while maintaining high levels of expression (Figure 9B).

[0433] Next, the inventors repeated the same experiment in human astrocytes derived from neuronal precursors (Figure 10A). In this case as well, the inventors used the V5 tag and immunofluorescence to analyze MeCP2 expression in astrocytes with and without mirT cassettes (Figure 10B).

[0434] Analysis of V5 signal intensity revealed a significant reduction in expression in GFAP+ cells in the presence of mirT, confirming the cassette's ability to differentially regulate MeCP2 expression in human cells as well (Figure 10B).

[0435] Verification of the AAV9_V5-Mecp2-mirT+shRNA vector in mutant Mecp2-KO animals.

[0436] The inventors validated a cassette (mirT+shRNA) containing mouse Mecp2 CDS in vivo in a Mecp2-KO mouse model. Mice were treated by intracerebroventricular injection during the neonatal period and injected with a Mecp2 cassette modified using AAV9 as a vehicle to target the brain parenchyma, resulting in 2 × 10⁶ mice per mouse. 10The animals were injected with vg (Figure 11A). After four weeks, the animals were sacrificed, their brains were fixed, and transduction and expression were studied by immunofluorescence using V5 tags (Figure 11B). In this way, we demonstrated that the presence of mirT can distinguish exogenous Mecp2 expression between neurons and astrocytes by mimicking the endogenous profile. Indeed, V5 fluorescence intensity was found to be significantly higher in NeuN+ neurons than in Sox9+ astrocytes (Figure 11B).

[0437] Materials and methods HeLa cell culture HeLa cells were maintained in Dulbecco's 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 passaged every 3-4 days using 0.25% trypsin. Cells were plated (2 × 10⁶) for shRNA validation. 5 Cells ( / cm²) were transfected in vitro on day 1 (DIV1) with a plasmid vector expressing shRNA (or scrambled sequence as a control) and GFP under a constitutive promoter. Cells were fixed in DIV3 for immunofluorescence staining (4% PFA, 4°C, 30') or lysates were fixed for total RNA extraction.

[0438] RT-qPCR Total RNA was isolated from HeLa cells using TRI reagent (sigma). MECP2 mRNA levels were then evaluated by qRT-PCR using the following primers: 5'-GATCAATCCCCAGGGAAAAGC-3' and 5'-TCTCCCAGTTACCGTGAAGTC-3'. RNA levels were normalized to 18S using the following primers: 5'-GTAACCCGTTGAACCCCATT-3' and 5'-CCATCCAATCGGTAGTAGCG-3'. The results were compared to the fold change in MECP2 expression in shRNA-treated HeLa cells compared to scrambled controls (2 -ΔΔCt ) was reported as such.

[0439] Immunofluorescence Cells and tissues were fixed in 4% PFA at 4°C for 10 minutes. Brain was dissected using a cryostat to obtain 50 μm slices. Cells and slices were then washed with PBS (3 times) and incubated with 10% donkey serum and Triton X-100 (Sigma) at RT for 1 hour to saturate nonspecific binding sites, followed by incubation overnight at 4°C with primary antibody. After washing with PBS (3 times), cells were incubated at RT for 1 hour in a blocking solution containing DAPI and Alexa Fluor-488 and Alexa Fluor-594 anti-rabbit or anti-mouse secondary antibodies (1:1000, ThermoFisher Scientific). After washing with PBS (3 times), cells and slices were mounted with fluorescent mounting medium (Dako). Images were acquired using a Nikon Eclipse 600 fluorescence microscope or a Leica TCS SP5 laser scanning confocal microscope. Cells and slices were stained with the following primary antibodies: 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, Abcam), and chicken-GFAP (1:500, Abcam). Signal fluorescence intensity was quantified using Imaged software (NIH, US).

[0440] Differentiation of iPSC-derived neurons iPSCs were first differentiated into neural progenitor cells (NPCs). The NPCs were then dissociated with Accutase and plated in NPC medium onto a Matrigel-coated 6-well plate (3 × 10⁶ cells per well). 5(100 cells). After 2 days, the medium was replaced with differentiation medium containing Neurobasal, 1% Pen / Strep, 1% glutamine, 1:50 B27, 10 μM SU5402, and 8 μM PD0325901, and 10 μM DAPT was added and maintained for 3 days. After 3 days, the cells were dissociated with Accutase and placed in neuronal maturation medium supplemented with ROCK inhibitor Y27632 (10 μM) in 12-well plates coated with poly-L-lysine / laminin / fibronectin (100 μg / ml, 2 μg / ml, 2 μg / ml) (2 × 10⁶ cells per well). 5 (individual cells) and 24-well plates (1 x 10⁶ cells per well) 5 The cells were plated over the first 24 hours. Neuronal maturation medium consisted of Neurobasal, 1% Pen / Strep, 1% glutamine, 1:50 B27, 20 ng / ml human BDNF, 200 μM ascorbic acid, 250 μM dibutyryl cAMP, 10 μM DAPT, and 1 μg / μl laminin. The following day, the medium was changed to remove the ROCK inhibitor, and at this stage, half of the medium was changed every 2-3 days. After 6 weeks of differentiation, the viral particles were raised to a final concentration of 5 × 10⁶. 10 The substance was directly added to cultured neurons at vg / ml. All analyses were performed one week after infection, at the time of specimen fixation (4% PFA, 4°C, 30').

[0441] Differentiation of NPC-derived astrocytes Neural progenitor cells (NPCs) were differentiated into astrocytes. Briefly, on day 1 of differentiation, 90-95% confluent NPC cultures were washed with PBS and incubated with accutase at 37°C for 5 minutes. The accutase reaction was then stopped by resuspending the NPCs in NPC medium, followed by centrifugation at 300×g for 5 minutes. The supernatant was removed, and the cells were resuspended by gentle pipetting to obtain a single-cell suspension. The NPCs were then seeded in low-adhesion 35 mm dishes and incubated at 37°C for 24 hours with shaking at 90 rpm to obtain NPC spheres. The following day, the NPC medium was replaced with DMEM-F12 supplemented with 1:100 B-27, 1:200 N-2, and 5 μM ROCK inhibitor. After 48 hours, the culture medium was replaced with astrocyte growth medium (AGM Bullet Kit, Lonza, #CC-3186) for 15 days, shaken at 37°C and 90 rpm, and the medium was changed every 3 days. After 2 weeks, the spheres thus obtained were plated in astrocyte growth medium on dishes coated with polyornithine and laminin. When the cells reached approximately 95% confluence, the spheres were aspirated with a tip and the adherent cells were passaged into a new dish. The culture was considered to be pure astrocytes after the third passage. The viral particles were reduced to a final concentration of 5 × 10⁶. 10 The solution was directly added to cultured astrocytes at vg / ml. All analyses were performed one week after infection, at the time of specimen fixation (4% PFA, 4°C, 30').

[0442] animal Mice were maintained under sterile conditions in microisolates at the San Raffaele Scientific Institute Institutional Mouse Facility (Milan, Italy), and supplied with autoclaved food and water. Mecp2-KO mice (The Jackson Laboratory stock #003890) were maintained in a C57BL / 6J background. All procedures were carried out according to protocols approved by the Internal IACUC and reported to the Italian Ministry of Health in accordance with European Council Directive 2010 / 63 / EU.

[0443] AAV vector injection and tissue harvesting The mouse genotype was determined in P0 using primers provided by Jackson Laboratory (https: / / www.jax.org / Protocol?stockNumber=003890&protocolID=2082). In P1, Mecp2-KO mice were given 4 × 10⁶ 12 5 µl of AAV9 containing CBA-V5-Mecp2-mirT+shRNA was injected intracerebroventricularly at a starting concentration of vg / ml. After injection, all mice were weighed twice weekly and sacrificed at 1 month of age to isolate the brain. The brains were then fixed in 4% PFA for 2 days and then immersed in cryoprotection solution (30% sucrose in PBS) for immunofluorescence analysis.

[0444] statistics Numerical values ​​are expressed as mean ± standard deviation, as shown. All statistical analyses were performed using independent t-tests in Prism 8.0 (GraphPad). P-values ​​less than 0.05 were considered statistically significant.

[0445] Various preferred features and embodiments of the present invention will be described with reference to the following numbered paragraphs. 1. The nucleotide sequence encoding methyl-CpG binding protein 2 (MeCP2), (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 polynucleotide comprising a nucleotide sequence encoding a MeCP2 expression inhibitor, and optionally a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NO: 15. 2. The polynucleotide described in paragraph 1, comprising one miR-124 target sequence, four miR-31 target sequences, and four miR-338-3p target sequences. 3. (a) The miR-124 target sequence contains or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 5 (b) The miR-31 target sequence contains or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 6, and / or (c) The polynucleotide according to paragraph 1 or 2, wherein the miR-338-3p target sequence includes, or consists of, a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7. 4. A polynucleotide described in any of the preceding paragraphs, comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8. 5. The nucleotide sequence encoding MeCP2 is, (a) A nucleotide sequence encoding an amino acid sequence having at least 70% identity with SEQ ID NO: 1 or 2, (b) A nucleotide sequence having at least 70% identity with SEQ ID NO: 3 or 4, and (c) A polynucleotide as described in either of the preceding paragraphs, comprising a sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3 or 4. 6. A polynucleotide as described in any of the preceding paragraphs, wherein the nucleotide sequence encoding MeCP2 is operably ligated to a strong promoter and / or 3'-UTR, the 3'-UTR being approximately 1000 bp or less in length. 7. A polynucleotide as described in any of the preceding paragraphs, further comprising a nucleotide sequence encoding an inhibitor of MeCP2 expression. 8. A vector containing the polynucleotide described in any of the preceding paragraphs. 9. The vector described in paragraph 8, comprising a nucleotide sequence having at least 70% sequence identity with sequence number 23 or 24. 10. A vector described in paragraph 8 or 9, which is an AAV, retrovirus, lentivirus, or adenovirus vector. 11. A vector described in any one of paragraphs 8-10, which is a viral vector particle. 12. The vector according to paragraph 11, wherein the AAV vector particles contain a capsid selected from the group consisting of AAV9, AAV9 PHP.B, AAV9 PHP.eB, and AAVrh10 capsids. 13. Cells containing the polynucleotide or vector described in any of the preceding paragraphs. 14. A pharmaceutical composition comprising a polynucleotide, vector, or cell as described in any of the preceding paragraphs, and a pharmaceutically acceptable carrier, diluent, or excipient. 15. A polynucleotide, vector, cell, or pharmaceutical composition described in any of the preceding paragraphs, for use in therapy. 16. A polynucleotide, vector, cell, or pharmaceutical composition described in any one of paragraphs 1 to 14, for use in the treatment or prevention of Rett syndrome.

[0446] All publications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the disclosed polynucleotides, vectors, cells, compositions, uses, and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention is disclosed in relation to certain preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. Indeed, various modifications of the disclosed forms for carrying out the invention, which will be apparent to those skilled in the art, are intended to be within the scope of the following claims.

Claims

1. The nucleotide sequence encoding methyl-CpG binding protein 2 (MeCP2), (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 polynucleotide comprising a nucleotide sequence encoding a MeCP2 expression inhibitor, and optionally a nucleotide sequence encoding an shRNA having at least 90% sequence identity with SEQ ID NOs: 15, 30, or 31.

2. The polynucleotide according to claim 1, comprising one miR-124 target sequence, four miR-31 target sequences, and four miR-338-3p target sequences.

3. (a) The miR-124 target sequence contains or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 5 (b) The miR-31 target sequence contains or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 6, and / or (c) The polynucleotide according to claim 1 or 2, wherein the miR-338-3p target sequence includes or consists of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

7.

4. A polynucleotide according to any one of the prior claims, comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

8.

5. The nucleotide sequence encoding MeCP2 is, (a) A nucleotide sequence encoding an amino acid sequence having at least 70% identity with SEQ ID NO: 1, 2, or 28, (b) A nucleotide sequence having at least 70% identity with SEQ ID NOs: 3, 4, or 29, and (c) The polynucleotide according to any one of claims 1 to 4, comprising a sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 3, 4, or 29.

6. The polynucleotide according to any one of claims 1 to 5, wherein the nucleotide sequence encoding MeCP2 is operably linked to a strong promoter and / or a 3'-UTR, and the 3'-UTR is about 1000 bp in length or less.

7. The polynucleotide according to any one of claims 1 to 6, further comprising a nucleotide sequence encoding an inhibitor of MeCP2 expression.

8. A vector comprising a polynucleotide according to any one of claims 1 to 7.

9. The vector according to claim 8, comprising a nucleotide sequence having at least 70% sequence identity with sequence numbers 23, 24, 26, or 27.

10. The vector according to claim 8 or 9, which is an AAV, retrovirus, lentivirus, or adenovirus vector.

11. A vector according to any one of claims 8 to 10, which is in the form of a viral vector particle.

12. The vector according to claim 11, wherein the AAV vector particles include a capsid selected from the group consisting of AAV9, AAV9 PHP. B, AAV9 PHP. eB, AAVrh10, and AAV-DJ capsid.

13. A cell comprising a polynucleotide or vector according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising a polynucleotide, vector, or cell according to any one of claims 1 to 13, and a pharmaceutically acceptable carrier, diluent, or excipient.

15. A polynucleotide, vector, cell, or pharmaceutical composition according to any one of claims 1 to 14, for use in therapeutic purposes.

16. A polynucleotide, vector, cell, or pharmaceutical composition according to any one of claims 1 to 14, for use in the treatment or prevention of Rett syndrome.

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