Expression vectors for cholesterol 24-hydrolase in the treatment of Rett syndrome

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder that affects almost exclusively girls (approximately 1 in 10,000 females). Rett syndrome is characterized by normal early growth and development, along with developmental delays, loss of purposeful hand use, distinctive hand movements, delayed brain and head growth, gait problems, epileptic seizures, and intellectual disability. Herein, the inventors demonstrated that intravenous delivery of a vector expressing the cholesterol 24-hydroxylase (CYP46A1) gene in a mouse model of RTT can prevent / correct the development of motor deficits in mild and severe models of the disease in both male and female mice. Additionally, in males, the inventors demonstrated prevention of Purkinje cell loss and amelioration of astrogliosis and microgliosis in a mild MECP2 KO model. Thus, the present invention relates to a vector for use in treating Rett syndrome or a related autism spectrum disorder, comprising a nucleic acid encoding cholesterol 24-hydroxylase.

Description

[Technical field]

[0001] The present invention relates to the treatment of Rett Syndrome.

[0002] Rett syndrome (RTT) is a neurodevelopmental disorder that affects almost exclusively girls (approximately 1 in 10,000 females). It is characterized by normal early growth and development combined with developmental delays, loss of purposeful hand use, peculiar hand movements, delayed brain and head growth, gait problems, epileptic seizures, and intellectual disability (Hagberg et al. 1985).

[0003] The course of Rett syndrome, including the age of onset and severity of symptoms, varies from child to child. However, before symptoms appear, children generally appear to grow and develop normally, although subtle abnormalities are often present in infancy, such as low muscle tone (hypotonia), feeding difficulties, and clumsy limb movements. Then, gradually, physical and mental symptoms appear. As the syndrome progresses, children lose the purposeful use of their hands and the ability to speak. Other early symptoms may include problems with crawling or walking and reduced eye contact. Loss of functional use of the hands is followed by compulsive hand movements, such as wringing and hand washing. The onset of this regression phase can be sudden (Armstrong 1997).

[0004] Children with Rett syndrome often exhibit autistic-like behaviors at an early stage. Other symptoms may include toe walking, sleep disorders, drunken gait, teeth grinding and chewing difficulties, growth retardation, epileptic seizures, cognitive impairment, and breathing difficulties during awakening, such as hyperventilation, apnea (breath holding), and air swallowing. Rett syndrome is caused by mutations in the X-linked gene methyl-CpG binding protein 2 (MECP2), a ubiquitously expressed transcriptional regulator (Amir et al. 1999; Duncan Armstrong 2005).

[0005] Mouse models recapitulate many of the symptoms of RTT, and their study has provided insight into the physiological basis of the disease. Female Mecp2 / + mice show partial phenotypic variance due to random X-chromosome inactivation, whereas male Mecp2 / Y mice have a fully penetrant phenotype. Null males are normal at birth and weaning, but develop limb clasping, tremors, lethargy, and respiratory abnormalities. These symptoms progressively worsen until death at 6-16 weeks of age (Guy et al. 2001).

[0006] In animal models of RTT disease, several studies have already demonstrated that Mecp2 deficiency disrupts cholesterol homeostasis. Indeed, genes in the mevalonate pathway were slightly upregulated in the brain but not in the liver (mild effect) in 1-month-old Mecp2 / Y mice. At 2 months of age, total cholesterol concentrations were elevated in the brains of symptomatic mutant mice (Nagy and Ackerman 2013). It has also been reported that Cyp46a1 expression is increased by 38% over wild-type levels in MECP2-deficient males, indicating an increased need for cholesterol turnover in neurons (Buchovecky et al. 2013;Lutjohann et al. 2018a).

[0007] A therapeutic approach was also attempted by administering statins, which inhibit the first rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR). Statins increased the median survival time and improved motor coordination and activity in mutant male mice, but did not improve respiratory irregularities or abnormal acoustic startle response. To more closely model the treatment of female patients with RTT, Mecp2 / + female mice were also treated with statins, which improved symptoms (Buchovecky et al. 2013).

[0008] So far, a cure for Rett syndrome is unknown. The only molecules currently in use are aimed at reducing the secondary effects associated with Rett syndrome. Medications may be required for respiratory irregularities and movement disorders, and anticonvulsants may be used to control epileptic seizures. Regular monitoring should be performed for possible scoliosis and cardiac abnormalities. However, several approaches are actually being evaluated in mouse models of the disease. They mainly aim to restore MECP2 expression using gene therapy delivery (Garg et al. 2013) (KK Gadalla et al. 2013;KKE Gadalla et al. 2017). This increases survival but does not necessarily improve the phenotype, especially the coordination performance. In addition, hepatotoxicity is highlighted when delivered in high doses. MECP2 is a transcription factor whose expression is strongly regulated to maintain synaptic regulation and neuronal homeostasis. Thus, elevated MECP2 levels (up to twice the endogenous levels) result in a Rett-like disorder with the opposite phenotype of MECP2 loss of function (Na et al. 2013). Thus, there is an urgent need to develop novel strategies for the treatment of Rett patients.

[0009] Summary of the Invention Here, the inventors propose to combat Rett Syndrome by modulating the cholesterol metabolic pathway, more specifically by a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase, which causes the expression of cholesterol 24-hydroxylase in target cells. It is therefore an object of the present invention to provide a vector for use in the treatment of Rett Syndrome, comprising a nucleic acid encoding cholesterol 24-hydroxylase.

[0010] In an embodiment, the vector comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2. Alternatively, the vector comprises the nucleic acid sequence of SEQ ID NO: 1.

[0011] In an embodiment, the vector is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors, preferably AAV vectors, more preferably AAV9, AAV10 (AAVrh.10) or AAVPHP.eB vectors, even more preferably AAVPHP.eB.

[0012] In an embodiment, the vector is administered directly to the patient's brain, preferably to all areas of interest in the brain (hippocampus, cortex, striatum, pons and cerebellum).In an embodiment, the vector is administered by intravascular, intravenous, intranasal, intraventricular, intrathecal or retro-orbital injection.In a particular embodiment, the vector is administered intravenously.In a particular embodiment, the vector is administered to the cerebrospinal fluid.

[0013] Another object of the present invention is to provide a pharmaceutical composition for use in the treatment of Rett Syndrome, including a vector, comprising a nucleic acid encoding cholesterol 24-hydroxylase. [Brief description of the drawings]

[0014] [Figure 1] - Basal levels of CYP46A1 in brain regions (cerebellum, pons, striatum, hippocampus and cortex) of the exacerbated MECP2 KO male mouse model at 45 days compared to wild-type (control) littermates (WT). [Diagram 2] - Exacerbated lipid abnormalities in the brains of MECP2 KO males at 45 days compared to wild-type (control) littermates (WT). [Figure 3A] - Behavioral assessment of mild MECP2 KO male mice (KO MECP2 AAV) 3 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 3B]- Behavioral assessment of mild MECP2 KO male mice (KO MECP2 AAV) 3 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 3C] - Behavioral assessment of mild MECP2 KO male mice (KO MECP2 AAV) 3 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 4A] -Biodistribution of AAVPHP.eB-CYP46A1 in the central nervous system (A) and peripheral organs (B) of mild male KO MECP2 mice. [Figure 4B] -Biodistribution of AAVPHP.eB-CYP46A1 in the central nervous system (A) and peripheral organs (B) of mild male KO MECP2 mice. [Diagram 5] - Assessment of target engagement by quantification of 24OH cholesterol levels in mild KO MECP2 males at 16 weeks of age. Brain 24S-hydroxycholesterol content in mild KO MECP2 male mice assessed by UPLC-HRMS. Results are expressed as mean ± SEM. [Figure 6] - Assessment and histological analysis of Purkinje cells at 16 weeks following intravenous delivery of AAVPHP.eB-CYP46A1 into mild KO MECP2 males (quantification of Purkinje cell numbers in the cerebellum. Results are expressed as mean ± SEM). [Figure 7] - Assessment and histological analysis of astrogliosis at 16 weeks following intravenous delivery of AAVPHP.eB-CYP46A1 as treatment in mild KO MECP2 males. Quantification of astrocyte numbers in different regions of the brain. Results are expressed as mean ± SEM. [Figure 8]Assessment and histological analysis of microgliosis at 16 weeks following intravenous delivery of AAVPHP.eB-CYP46A1 as treatment in -KO MECP2 mild male mice. Quantification of microglial cell numbers in different brain regions. Results are expressed as mean ± SEM. [Figure 9A] - Behavioral assessment of exacerbated MECP2 KO male mice (KO MECP2 AAV) 3 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 9B] - Behavioral assessment of exacerbated MECP2 KO male mice (KO MECP2 AAV) 3 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 9C] - Behavioral assessment of exacerbated MECP2 KO male mice (KO MECP2 AAV) 3 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 10A] Biodistribution of AAVPHP.eB-CYP46A1 in the central nervous system (A) and peripheral organs (B) of exacerbated KO MECP2 males at day -40. [Figure 10B] Biodistribution of AAVPHP.eB-CYP46A1 in the central nervous system (A) and peripheral organs (B) of exacerbated KO MECP2 males at day -40. [Figure 11A]-mRNA levels of genes involved in cholesterol metabolism in the lumbar brain of exacerbated KO MECP2 males at 6 weeks. mRNA was extracted from whole brain of 6 week old mice and mouse and human (A) CYP46A1 levels and (B) other genes involved in the cholesterol pathway were quantified. mRNA levels were normalized to the actin housekeeping gene. Data are presented as mean ± SEM (n=6 per group). Data are presented as mean ± SEM (n=7-9 per group). Statistical analysis: Student's t-test. *p<0.05, **p<0.01. [Figure 11B] -mRNA levels of genes involved in cholesterol metabolism in the lumbar brain of exacerbated KO MECP2 males at 6 weeks. mRNA was extracted from whole brain of 6 week old mice and mouse and human (A) CYP46A1 levels and (B) other genes involved in the cholesterol pathway were quantified. mRNA levels were normalized to the actin housekeeping gene. Data are presented as mean ± SEM (n=6 per group). Data are presented as mean ± SEM (n=7-9 per group). Statistical analysis: Student's t-test. *p<0.05, **p<0.01. [Figure 12] 6-week assessment and histological analysis of Purkinje cells following intravenous delivery of AAVPHP.eB-CYP46A1 as treatment in -exacerbated KO MECP2 males. Quantification of Purkinje cell numbers in the cerebellum. Results are presented as mean ± SEM. [Figure 13] Evaluation and histological analysis of astrogliosis at 6 weeks following intravenous delivery of AAVPHP.eB-CYP46A1 as treatment in -exacerbated KO MECP2 males. Quantification of astrocyte numbers in different regions of the brain. Results are expressed as mean ± SEM. [Figure 14] -Assessment and histological analysis of microgliosis at 16 weeks following intravenous delivery of AAVPHP.eB-CYP46A1 as treatment in exacerbated KO MECP2 male mice. Quantification of microglial cell numbers in different regions of the brain. Results are expressed as mean ± SEM. [Figure 15A]- Behavioral assessment of exacerbated MECP2 KO female mice (KO MECP2 AAV) 12 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 15B] - Behavioral assessment of exacerbated MECP2 KO female mice (KO MECP2 AAV) 12 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM. [Figure 15C] - Behavioral assessment of exacerbated MECP2 KO female mice (KO MECP2 AAV) 12 weeks after intravenous administration of AAVPHP.eB-CYP46A1 compared to untreated male MECP2 KO mice (KO MECP2) and controls (WT). (A) Grasping test; (B) Rotarod; (C) Body weight tracking. Results are presented as mean ± SEM.

[0015] Detailed Description of the Invention We have demonstrated that intravenous delivery of a vector expressing the CYP46A1 gene in a mouse model of RTT can prevent / correct the development of motor impairment in mild and exacerbated models of the disease in both male and female mice. In addition, in males, we have demonstrated prevention of Purkinje cell loss and amelioration of astrogliosis and microgliosis in a mild KO MECP2 model.

[0016] Based on this, the present inventors provide a viral vector for the treatment of Rett syndrome, which expresses CYP46A1 in cells of the central nervous system, particularly neurons in the cortex, hippocampus, striatum and cerebellum. This strategy can be useful for treating intellectual disability associated with Rett syndrome or other pathologies associated with autism spectrum disorder.

[0017] Rett Syndrome The present invention particularly relates to the treatment of Rett syndrome.Preferably, the present invention relates to the treatment of Rett syndrome caused by mutation in the X-linked gene methyl-CpG binding protein 2 (MECP2), a ubiquitously expressed transcriptional regulator.In an embodiment, the present invention relates to the treatment of Rett syndrome associated with intellectual disability.

[0018] In the context of the present invention, the terms "treatment," "treat," or "treating" are used herein to characterize a therapeutic approach or process that aims to: (1) slow or halt the progression, worsening, or exacerbation of the symptoms of the disease state or condition to which such term applies; (2) relieve or bring about the amelioration of the symptoms of the disease state or condition to which such term applies; and / or (3) reverse or cure the disease state or condition to which such term applies.

[0019] As used herein, the term "subject" or "patient" refers to animals, preferably mammals, and even more preferably humans, including adults and children, although the term "subject" can also refer to non-human animals, mammals, such as mice and non-human primates.

[0020] CYP46A1 sequence A first object of the present invention relates to a vector for use in the treatment of Rett Syndrome, comprising the complete sequence of a nucleic acid encoding cholesterol 24-hydroxylase.

[0021] As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed or translated.

[0022] As used herein, the terms "coding sequence" or "sequence encoding a particular protein" refer to a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.

[0023] The CYP46A1 gene encodes cholesterol 24-hydroxylase. This enzyme is a member of the cytochrome P450 superfamily of enzymes. This enzyme converts cholesterol to 24S-hydroxycholesterol (24S-OH-Chol), which can dynamically cross the BBB. This allows peripheral circulation to be excreted from the body (Bjorkhem, I. et al.1998), thus maintaining cholesterol homeostasis. The cDNA sequence for CYP46A1 is disclosed in Genbank Access Number AF094480 (SEQ ID NO:1). Its amino acid sequence is shown in SEQ ID NO:2.

[0024] The present invention uses a nucleic acid construct comprising the sequence of SEQ ID NO:1 or a variant thereof for the treatment of Rett Syndrome and optionally related autism spectrum related disorders.

[0025] Variants include natural variants due to, for example, allelic variations (e.g., polymorphisms) between individuals, alternative splicing forms, etc. The term variant also includes CYP46A1 gene sequences from other sources or organisms. Variants are preferably substantially homologous to SEQ ID NO:1, i.e. typically exhibit at least about 75%, preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95% nucleotide sequence identity with SEQ ID NO:1. Variants of the CYP46A1 gene also include nucleic acid sequences that hybridize to the above-defined sequences (or their complements) under stringent hybridization conditions. Exemplary stringent hybridization conditions include temperatures above 30° C., preferably above 35° C., more preferably above 42° C., and / or salts below about 500 mM, preferably below 200 mM. Hybridization conditions can be adjusted by one of skill in the art by modifying temperature, salt and / or concentrations of other reagents such as SDS, SSC, and the like.

[0026] Non-viral vectors In an embodiment, the vector used in the present invention is a non-viral vector.Typically, the non-viral vector can be a plasmid encoding CYP46A1.This plasmid can be administered directly or via liposome, exosome or nanoparticle.

[0027] Viral Vectors Gene delivery viral vectors useful in practicing the present invention can be constructed using methods well known in the art of molecular biology. Typically, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements required for the production of viral proteins that mediate cell transduction.

[0028] The terms "gene transfer" or "gene delivery" refer to methods or systems for reliably inserting foreign DNA into a host cell. Such methods can result in transient expression of unintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of the transferred genetic material into the genomic DNA of the host cell.

[0029] Examples of viral vectors include adenoviral, lentiviral, retroviral, herpes viral and adeno-associated viral (AAV) vectors.

[0030] Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, 96 / 22378, US 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO 94 / 19478.

[0031] In a preferred embodiment, an adeno-associated virus (AAV) vector is utilized.

[0032] "AAV vector" refers to a vector derived from an adeno-associated virus serotype (including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, e.g., AAVrh.10, AAVPHP.eB, etc.). An AAV vector can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the rep and / or cap genes, but retaining functional flanking ITR sequences. Functional ITR sequences are required for rescue, replication and packaging of AAV virions. Thus, an AAV vector is defined herein to include at least those sequences (e.g., functional ITRs) that are required in cis for viral replication and packaging. The ITRs need not be wild-type nucleotide sequences and can be modified, for example, by insertion, deletion or substitution of nucleotides, so long as the sequences provide functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to provide control elements including at least the transcriptionally operably linked components, a transcription initiation region, a DNA of interest (i.e., the CYP46A1 gene), and a transcription termination region. As a self-complementary construct 14, two copies of the DNA of interest can be included.

[0033] In a more preferred embodiment, the AAV vector is an AAV9, AAV10 (AAVrh.10), AAVPHP.B or AAVPHP.eB vector or a vector derived from one of these serotypes. In a most preferred embodiment, the AAV vector is an AAVPHP.eB vector. The AAVPHP.eB vector is an evolved AAV-PHP.B variant that efficiently transduces CNS neurons (Ken Y Chan et al. 2017; WO 2017100671). Other vectors can also be used, such as those described in WO 2015038958 and WO 2015191508.

[0034] The control elements are selected to be functional in mammalian cells. The resulting construct containing the operably linked components is bounded (5' and 3') by functional AAV ITR sequences. "Adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to the art-recognized regions found at each end of the AAV genome. The AAV ITRs function in cis as origins of DNA replication and packaging signals for the virus. The AAV ITRs, together with the AAV rep coding region, provide for efficient excision and rescue of the nucleotide sequence interposed between the two adjacent ITRs from the mammalian cell genome and integration of the nucleotide sequence interposed between the two adjacent ITRs into the mammalian cell genome. Nucleotide sequences of AAV ITR regions are known for AAV-2 sequences (see, e.g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (BN Fields and DM Knipe, eds.)). As used herein, "AAV ITR" need not necessarily comprise a wild-type nucleotide sequence, but may be modified, for example, by nucleotide insertion, deletion, or substitution. Furthermore, AAV ITRs can be derived from any of several AAV serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV vector are not required to function as intended, i.e., to allow for excision and rescue of a sequence of interest from a host cell genome or vector and to provide a nucleotide sequence that is consistent with the AAV serotype. If the Rep gene products are present in the cell, they need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they allow integration of the heterologous sequence into the recipient cell genome.

[0035] Particularly preferred are vectors derived from AAV serotypes that have tropism for and high transduction efficiency in cells of the mammalian central nervous system (CNS), especially neurons. A review and comparison of the transduction efficiency of various serotypes is provided in Davidson et al, 2000. In one preferred example, AAV2-based vectors have been shown to direct long-term expression of transgenes in the CNS, preferably in transduced neurons. In another non-limiting example, preferred vectors include vectors derived from AAV4 and AAV5 serotypes. This vector has also been shown to transduce cells of the CNS (Davidson et al, supra). In particular, this vector can be an AAV vector that includes a genome derived from AAV5 (particularly, the ITRs are AAV5 ITRs) and a capsid derived from AAV5.

[0036] In a particular embodiment of the present invention, the vector is a pseudotyped AAV vector. Specifically, the pseudotyped AAV vector comprises an AAV genome derived from a first AAV serotype and a capsid derived from a second AAV serotype. Preferably, the genome of the AAV vector is derived from AAV2. Furthermore, the capsid is preferably derived from AAV5. Specific non-limiting examples of pseudotyped AAV vectors include an AAV vector comprising a genome derived from AAV2 in a capsid derived from AAV5, an AAV vector comprising a genome derived from AAV2 in a capsid derived from AAVrh.10, etc.

[0037] The selected nucleotide sequence is operably linked to a control element that directs its transcription or expression in a subject in vivo. Such control elements can include the control sequences normally associated with the selected gene. In particular, such control elements can include the promoter of the CYP46A1 gene, in particular the promoter of the human CYP46A1 gene (Ohyama Y et al., 2006).

[0038] Alternatively, heterologous control sequences can be utilized. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PGK) promoter, the SV40 early promoter, the mouse mammary tumor virus LTR promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, e.g., the mouse metallothionein gene, will also find use herein. Such promoter sequences are commercially available, e.g., from Stratagene (San Diego, Calif.). For purposes of the present invention, both heterologous promoters and other control elements, e.g., CNS-specific and inducible promoters, enhancers, and the like, will be of particular use.

[0039] Examples of heterologous promoters include the CMV promoter. Examples of CNS-specific promoters include those isolated from genes derived from myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), synapsin (e.g., the human synapsin 1 gene promoter), and neuron-specific enolase (NSE).

[0040] Examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and aufin.

[0041] AAV expression vectors having a DNA molecule of interest linked to AAV ITRs can be constructed by directly inserting a selected sequence into the AAV genome. This genome has the main AAV open reading frame ("ORF") excised from it. Other parts of the AAV genome can also be deleted, so long as sufficient parts of the ITRs remain to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, US Pat. Nos. 5,173,414 and 5,139,941; WO 92 / 01070 (published Jan. 23, 1992) and WO 93 / 03769 (published Mar. 4, 1993); Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992; Muzyczka, 1992; Kotin, 1994; Shelling and Smith, 1994, and Zhou et al., 1994. Alternatively, AAV ITRs can be excised from the viral genome or the AAV vector containing them, and fused to the 5' and 3' ends of a selected nucleic acid construct present in another vector using standard ligation techniques. AAV vectors containing ITRs are described, for example, in US Pat. No. 5,139,941. In particular, several AAV vectors are described therein and are available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225 and 53226. Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences located 5' and 3' of one or more selected nucleic acid sequences. Codons preferred for expression of the chimeric gene sequence in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods (see, e.g., Edge, 1981; Nambair et al., 1984; Jay et al., 1984). To produce rAAV virions, the AAV expression vector is introduced into a suitable host cell, for example, by transfection, using known techniques.Many transfection techniques are generally known in the art (see, e.g., Graham et al., 1973; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981). Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome-mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Felgner et al., 1987), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., 1987).

[0042] For example, a preferred viral vector, e.g., AAVPHP.eB, in addition to a nucleic acid sequence encoding cholesterol 24-hydroxylase, comprises an AAV vector backbone with ITRs derived from AAV-2, a promoter, e.g., the cytomegalovirus / β-actin hybrid promoter (CAG), consisting of an enhancer, promoter, splice donor and intron from the chicken β-actin gene, from the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus immediate early gene, a splice acceptor from rabbit β-globin, or any neuronal promoter, e.g., the dopamine-1 receptor or dopamine-2 receptor promoter, with or without a wild-type or mutant Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).

[0043] Vector Delivery A method for treating Rett syndrome is disclosed. The method comprises administering to a patient in need thereof a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase. The vector can be delivered to the subject's brain and / or spinal cord directly or by intravascular, intravenous, retroorbital, intranasal, intraventricular or intrathecal injection.

[0044] In certain embodiments, a method for treating Rett Syndrome (RTT) in a subject, comprising: (a) providing a vector as defined above comprising a nucleic acid encoding cholesterol 24-hydroxylase; (b) delivering the vector to the subject's brain and / or spinal cord, thereby transducing cells in the brain and / or spinal cord with the vector, whereby cholesterol 24-hydroxylase is expressed at therapeutically effective levels by the transduced cells.

[0045] Advantageously, the vector is a viral vector, more advantageously an AAV vector, even more advantageously an AAV vector selected from the group consisting of AAV9, AAV10 or AAVPHP.eB, preferably AAVPHP.eB.

[0046] In certain embodiments, the vector is delivered to the brain, particularly the motor cortex and / or spinal cord, hi certain embodiments, the vector is delivered only to the spinal cord.

[0047] Methods of delivery or administration of viral vectors to neurons and / or astrocytes and / or oligodendrocytes and / or microglia generally include any method suitable for delivering the vector to cells, either directly or via hematopoietic cell transduction, such that at least a portion of the cells of a selected synaptically connected cell population are transduced. The vector can be delivered to any cell of the central nervous system, cells of the peripheral nervous system, or both. Preferably, the vector is delivered to cells of the brain and / or spinal cord. Generally, the vector is delivered to cells of the brain, including, for example, cells of the motor cortex, spinal cord, or a combination thereof, or any suitable subpopulation thereof.

[0048] To specifically deliver vectors to specific regions and populations of cells in the brain or spinal cord, the vectors can be administered by stereotactic microinjection. For example, the patient has a stereotactic frame base fixed in place (screwed into the skull). The brain with the stereotactic frame base (MRI fitted with fiducial markings) is imaged using high resolution MRI. The MRI images are then transferred to a computer running stereotactic software. A series of coronal, sagittal and axial images are used to determine the target (vector injection site) and the projection path. The software directly converts the projection path into three-dimensional coordinates suitable for the stereotactic frame. A cranial burr hole is positioned and drilled with a needle embedded at a predetermined depth above the entry site and the stereotactic device. The vector is then injected into the target site and finally mixed with a contrast agent. Because the vector is integrated into the target cells rather than producing viral particles, the subsequent spread of the vector is low and is primarily a function of passive diffusion from the injection site prior to integration and of course the desired trans-synaptic transport. The degree of diffusion can be controlled by adjusting the ratio of vector to fluid carrier.

[0049] Further routes of administration can also include local application of the vector under direct visualization, for example, superficial cortical application, intranasal application or other non-stereotaxic application.

[0050] The target cells of the vectors of the invention are brain cells (neurons), preferably neural cells, in the hippocampus, cortex, striatum and cerebellum of subjects affected by RTT. Preferably, the subject is a human, generally a child or infant, in particular a female infant, but may be an adult.

[0051] On the other hand, the present invention encompasses the delivery of vectors to biological models of disease, where the biological model can be any mammal at any stage of development at the time of delivery, e.g., embryonic, fetal, infant, juvenile or adult, preferably adult. Furthermore, the target cells can essentially come from any source, in particular non-human primates and mammals of the orders Rodent (mouse, rat, rabbit, hamster), Carnivora (cat, dog) and Artiodactyla (cow, pig, sheep, goat, horse) as well as any other non-human system (e.g., zebrafish model system).

[0052] Preferably, the method of the present invention includes intracerebral administration by stereotactic injection. However, other known delivery methods can also be adapted according to the present invention. For example, for a broader distribution of the vector through the brain, it can be injected into the cerebrospinal fluid, for example, by lumbar puncture, cisterna magna or ventricular puncture. To target the vector to the brain, it can be injected into the spinal cord or peripheral ganglia or into the flesh (subcutaneous or intramuscular) of the target body part. In certain situations, the vector, for example, the vector containing AAVPHP.eB here, can be administered via an intravascular approach. For example, the vector can be administered intra-arterially (carotid artery) in situations where the blood-brain barrier is disrupted. Furthermore, for more global delivery, the vector can be administered during the "opening" of the blood-brain barrier, which is achieved by injection of a hypertonic solution containing mannitol or by ultrasound local delivery.

[0053] The vectors used herein can be formulated in any suitable medium for delivery. For example, the vectors can be placed in a pharma- ceutically acceptable suspension, solution, or emulsion. Suitable vehicles include saline and liposomal preparations. More specifically, pharma-ceutically acceptable carriers can include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline, buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like.

[0054] Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0055] Colloidal dispersion systems can also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or exosomes.

[0056] The preferred dose and schedule can be determined by a physician and will depend on the age, sex, weight and stage of the disease of the subject. By way of example, for delivery of cholesterol 24-hydroxylase using a viral expression vector, each unit dose of cholesterol 24-hydroxylase expression vector can comprise 2.5 to 1 ml of a composition comprising the viral expression vector in a pharma- ceutically acceptable fluid, providing 1010 to 1015 cholesterol 24-hydroxylase-expressing viral particles per ml of composition.

[0057] Pharmaceutical Compositions A further object of the present invention relates to a pharmaceutical composition for use in the treatment of Rett Syndrome, comprising a therapeutically effective amount of the vector of the present invention.

[0058] By "therapeutically effective amount" is meant a sufficient amount of a vector of the invention to treat Rett Syndrome at a reasonable benefit / risk ratio applicable to any medical treatment.

[0059] It will be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used; the age, weight, general health, sex and diet of the patient; the timing of administration, route of administration and excretion rate of the specific compound being used; the duration of treatment; drugs used in combination or concomitantly with the specific polypeptide being used, as well as factors well known in the medical field. For example, it is well within the skill of the art to start administering the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can vary over a wide range per adult per day. The therapeutically effective amount of the vector of the present invention to be administered and the dose for the treatment of a pathological condition with a certain number of viral or non-viral particles and / or pharmaceutical compositions of the present invention will depend on a number of factors, including the age and condition of the patient, the severity of the disturbance or disorder, the method and frequency of administration, and the specific peptide being used.

[0060] The pharmaceutical composition containing the vector of the present invention can be provided in any form suitable for intramuscular, intracerebral, intranasal, intrathecal, intraventricular or intravenous administration. In the pharmaceutical composition of the present invention for intramuscular, intranasal, retroorbital, intravenous, intracerebral, intrathecal or intraventricular administration, the active ingredient can be administered alone or in combination with another active ingredient in a unit dosage form, in a mixture with a conventional pharmaceutical carrier, to animals and humans.

[0061] Preferably, the pharmaceutical composition contains a pharma- ceutically acceptable medium for injectable preparations.These can be, in particular, isotonic and sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts) or dry, in particular lyophilized compositions.Lyophilized compositions can be composed of sterile injectable solutions, optionally based on the addition of sterile water or saline.

[0062] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, for example, certain injectable solutions, although drug release capsules and the like are also available.

[0063] Multiple doses may also be administered.

[0064] It is also possible to link therapeutic approaches with small molecule compounds that can activate CYP46A1, such as efavirenz (Mast, N. et al. 2017; Mast, N. et al. 2014) or antifungals (Mast, N. et al. 2013; Fourgeux, C. et al. 2014), or conversely, small molecule compounds that can inhibit CYP46A1, and thus be a way to discontinue gene therapy approaches if necessary.

[0065] The present invention will be further illustrated by the following examples, which, however, and the accompanying figures should not be construed in any way as limiting the scope of the invention.

[0066] Working Example In this experiment, we used a severe mouse model of Rett syndrome and demonstrated a striking rescue of the phenotype.

[0067] Materials and Methods animal Two strains of mice were used in this study.

[0068] 3 female B6.129P2(C)-Mecp2 tm1Bird / J homozygotes were obtained from Jackson Laboratories (stock 007177) and bred with C57BL / 6 mice to generate a colony subsequently designated mild MECP2 KO.

[0069] 10 female B6.129P2(C)-Mecp2 tm.11Bird / J heterozygotes were obtained from Jackson Laboratories (stock 003890) and bred with C57BL / 6 mice to generate a colony that was subsequently designated exacerbated MECP2 KO.

[0070] Mice were housed in a temperature-controlled room and maintained on a 12-h light / dark cycle. Food and water were available ad libitum. Experiments were performed in accordance with the European Communities Council Directive (2010 / 63 / EU) on the care and use of laboratory animals.

[0071] AAV plasmid design and vector production AAV vectors were produced and purified by Atlantic Gene therapies (INSERM U1089, Nantes, France). Vector production has been described elsewhere (Hudry et al., 2010). The viral construct for AAVPHP.eB-CYP46A1-HA contained an expression cassette consisting of the human Cyp46a1 gene driven by the CMV early enhancer / chicken β-actin (CAG) synthetic promoter (CAG) surrounded by AAV2 inverted terminal repeat (ITR) sequences.

[0072] MECP2 genotyping Mice were genotyped according to the Jackson lab procedures (https: / / www2.jax.org / protocolsdb / f?p=116:5:0::NO:5:P5_MASTER_PROTOCOL_ID,P5_JRS_CODE:28595,007177) and (https: / / www.jax.org / Protocol?stockNumber=003890&protocolID=24870) depending on the colony.

[0073] Intravenous infusion 3-week-old MECP2 - / y Male (mild and aggravated) mice and 12-week-old MECP2+ / - aggravated females were anesthetized with 4% isoflurane and then maintained at 2% with 80% air and 20% oxygen. - / y Animals (n=14) were injected intravenously via retro-orbital injection with a total volume of 5.10e11 vg (volume 100ul). WT and non-injected exacerbated MECP2 - / y Animals (n = 10-13 per group) were injected with 100 μl of saline. - / y Animals (n=11) were injected intravenously via retro-orbital injection with a total volume of 5.10e11 vg (volume 100 μl). - / y Animals (n=17-19 per group) were injected with 100 μl of saline.

[0074] Deteriorated MECP2 + / - Female animals (n=8) were intravenously injected with a total volume of 5.10e11 vg (volume 100 μL) via retro-orbital injection. + / - Animals (n=9-10 per group) were injected with 100 μl of saline.

[0075] Behavioral testing Grasping test The grasping test evaluates consistency and is classical to the Rett assessment. Animals were scored before injection and weekly thereafter from 3 weeks up to 32 weeks (for mild KO males) and up to 6 weeks for deteriorated KO males. For deteriorated females, they were scored 1 week after injection and every 3 weeks thereafter (up to 25 weeks, although this is currently ongoing). The animal was held by the tail and if the mouse twitched, a score of "1" was given, and then for each hind limb grasp, a point was added to the score. Results were expressed as mean ± SEM for each group for each test and subjected to a two-way analysis of variance.

[0076] Rotarod General motor performance was tested using an accelerating rotarod LE8200 (Bioseb, France) and measured as previously described (https: / / www.mousephenotype.org / ). Animals were scored weekly from 3 weeks of age until the longest survival period (6 weeks for deteriorated KO males, 28 weeks for mild KO males, and 19 weeks so far for deteriorated KO females).

[0077] Tissue collection Mice were anesthetized with pentobarbital (180 mg / kg Euthasol) solution and perfused transcardially with phosphate-buffered saline (PBS). Brains, spinal cords, and sciatic nerves as well as peripheral organs (liver, heart, lungs, kidneys, spleen, diaphragm, gastrocnemius) were collected and post-fixed in 4% PFA, then either paraffin-embedded (cut at 6-10 μm with a microtome) for histology or immediately frozen in liquid nitrogen for biomolecular analysis.

[0078] The different tissues were ground / disintegrated in liquid nitrogen and dissociated to analyze RNA, DNA expression or for lipid analysis.

[0079] Primary antibody The antibodies used for immunohistochemistry (IHC) analysis are listed in Table 1 below.

[0080] [Table 1]

[0081] immunostaining Paraffin Sections Immunohistochemical labeling was performed using the ABC method. Briefly, tissue sections were treated with peroxide for 30 min to inhibit endogenous peroxidase. After washing in PBS, sections were treated with 10 mM Tris / 1 mM EDTA / 0.1% Tween (pH 8.75) at 95°C for 45 min (for anti-HA only) or 10 mM citrate (pH 6) at 95°C for 45 min (for anti-CB only). After washing in PBS, sections were incubated with blocking solution (10% goat serum in PBS / 0.3% TritonX-100) for 1 h. Primary antibodies were diluted in blocking solution and incubated on tissue sections at 4°C overnight. After washing in PBS, sections were sequentially incubated with goat anti-rabbit or goat anti-mouse antibodies conjugated to biotin (Vector Laboratories) for 30 min at room temperature followed by ABC complex (Vector Laboratories). After washing in PBS, peroxidase activity was detected using diaminobenzidine (Dako, Carpinteria, CA) as a chromogen. In some cases, slides were counterstained with hematoxylin. Slides were mounted with Depex (VWR International).

[0082] Nissl staining was performed on brain sections according to standard protocols.

[0083] Image acquisition For all IHC and staining, sections were acquired using a Hamamatsu slide scanner.

[0084] DNA extraction DNA was extracted from the brain, spinal cord and peripheral organs using a chloroform / phenol protocol.

[0085] RNA extraction Total RNA was extracted from brain portions using Trizol or TriReagent (Sigma). One microgram total RNA was transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions.

[0086] q-PCR cDNA was amplified with SyberGreen (Roche). Primers for RT-qPCR were as in the table above. The amplification protocol for all primers was hot start (95°C for 5 min), 45 amplification cycles (95°C for 15 s, 60°C for 1 min) and melting curve analysis. Data was analyzed using Lightcycler 480 software with efficiency factors for each gene and normalized to actin.

[0087] [Table 2]

[0088] Vector genome copy numbers were measured by qPCR on genomic DNA extracted from spinal cord, brain, sciatic nerve and peripheral organs using Light Cycler 480 SYBR Green I Master (Roche, France). Results (vector genome copy numbers per cell) were expressed as n-fold difference in transgene sequence copy numbers relative to Adck3 gene copies as internal standard (viral genome copy numbers for 2N genomes).

[0089] Cholesterol and oxysterol measurements Cholesterol and oxysterol analysis followed the "gold standard" method (Dzeletovic et al., 1995) to minimize the formation of autoxidation artifacts. Briefly, mouse brain tissue samples were weighed and homogenized in a TissueLyser II instrument (Qiagen) in 500 μl of a solution containing butylated hydroxytoluene (BHT, 50 μg / ml) and EDTA (0.5 M). At this point, a mixture of internal standards [epicoprostanol, 2H7-7-latosterol, 2H6-desmosterol, 2H6-lanosterol and 2H7-24(R / S)-hydroxycholesterol] (Avanti Polar Lipids) was added. Alkaline hydrolysis was performed using 0.35 M ethanolic KOH under Ar for 2 h at room temperature. After the solution was neutralized with phosphoric acid, sterols were extracted with chloroform. The lower phase was collected and dried under a stream of nitrogen, and the residue was dissolved in toluene. Oxysterols were then separated from cholesterol and its precursors on a 100 mg Isolute silica cartridge (Biotage). Cholesterol was eluted with 0.5% propan-2-ol in hexane, followed by oxysterols with 30% propan-2-ol in hexane. Sterol and oxysterol fractions were independently silylated with Regisil® + 10% TMCS [bis(trimethylsilyl)trifluoro-acetamide + 10% trimethylchlorosilane] (Regis technologies) as previously described (Chevy et al, 2005). The trimethylsilyl ether derivatives of sterols and oxysterols were separated by gas chromatography (Hewlett-Packard 6890 series) on a medium polarity capillary column RTX-65 (65% diphenyl 35% dimethylpolysiloxane, 30 m length, 0.32 mm diameter, 0.25 μm film thickness; Restesk). A mass spectrometer (Agilent 5975 inert XL) in series with the gas chromatograph was set up for detection of positive ions. Ions were generated in electron impact mode at 70 eV.The ions were identified by fragmentograms in scanning mode and quantified by selective monitoring of specific ions after normalization and calibration using appropriate internal and external standards [epicoprostanol m / z 370, 2H7-7-latosterol m / z 465, 2H6-desmosterol m / z 358, 2H6-lanosterol m / z 504, 2H7-24(R / S)-hydroxycholesterol m / z 553, cholesterol m / z 329, 7-latosterol m / z 325, 8-dehydrocholesterol m / z 325, desmosterol m / z 343, lanosterol m / z 393 and 24(R / S)-hydroxycholesterol m / z 413].

[0090] Western blot analysis Total protein was extracted from several regions of 45-day-old MECP2- / y males. Total protein concentration was determined using a BCA kit (Pierce). Equal amounts of total protein extracts (30 μg) were electrophoretically separated using SDS-PAGE on 4-12% Bis-tris gels (NuPAGE® Novex Bis-tris midi gel 15 or 26 well, Life Technologies, Carlsbad, USA) and transferred to nitrocellulose membranes. Blocked membranes (5% nonfat dry milk in TBS-0.1% Tween-20) were incubated with primary antibodies overnight at 4°C and washed three times for 10 min with TBS-0.1% Tween-20 (T-BST). The membranes were then labeled with secondary IgG-HRP antibodies raised against each corresponding primary antibody. After three washes with T-BST, the membrane was incubated with ECL chemiluminescence reagent (Clarity Western ECL Substrate; GE Healthcare, Little Chalfont, UK) according to the supplier's instructions. Peroxidase activity was detected with a camera system Fusion TX7 (Fisher Scientific). Normalization was performed by densitometric analysis using Quantity One 1D image analysis software (version 4.4; Biorad, Hercules, CA, USA). Optical density was normalized to a "standard protein" (GAPDH). Partition ratios were calculated and normalized to the sample with the highest value defined as 1.

[0091] statistical analysis Statistical analysis was performed using unpaired Student's t-test and two-way ANOVA. Results are expressed as mean ± SEM. Significance thresholds were set at P<0.05, P<0.01, and P<0.001, as defined in the text. All analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, USA).

[0092] result C57BL6 / Mecp2 for Rett syndrome tm1.1BirdReduced CYP46A1 protein levels in several brain regions in a mouse model CYP46A1 levels were measured in 45-day-old mice with MECP2 - / y CYP46A1 protein levels were assessed in frozen biopsies from affected animals. In particular, CYP46A1 protein levels were analyzed in the cerebellum, pons, striatum, hippocampus, and cortex of affected animals and compared to WT littermates (Figure 1). Western blot analysis demonstrated a significant decrease in CYP46A1 protein levels in the cerebellum, striatum, and a trend was observed in the pons and hippocampus, but no differences were observed in the cortex, compared to control littermates (Figure 1).

[0093] Overall, these data support the relevance of CYP46A1, a key enzyme in the cholesterol metabolism pathway, as a therapeutic target in human Rett syndrome.

[0094] C57BL6 / Mecp2 for Rett syndrome tm1.1Bird Deregulation of cholesterol metabolic pathways in model brains Previously, alterations in cholesterol and global lipid changes in Rett syndrome have been reported (Lutjohann et al., 2018b). To determine the impact of CYP46A1 deficiency in Rett syndrome, cholesterol metabolism was assessed using MECP2 deficiency assays in four 45-day-old rats. - / yQuantitative measurements of both sterols and oxysterols in whole brain extracts from animals and age-matched littermates were analyzed by gas chromatography-mass spectrometry (GC / MS). Thereby, lanosterol, the first sterol in the cholesterol metabolic pathway, was measured along with cholesterol intermediates from the Kandutsch-Russell (lasosterol) and Bloch (desmosterol) pathways. First, cholesterol and 24-hydroxycholesterol (24S-OHC) content were compared, and a statistically significant decrease was observed in 24S-OHC levels in MECP2 KO mice compared to wild-type littermates (Figure 2). A significant increase was observed for cholesterol. Significant decreases in lanosterol and 7-lasosterol were also measured in the brains of KO MECP2 compared to WT littermates. Finally, non-significant decreases were measured for desmosterol and 8DHC (Figure 2). No statistically significant differences were found for the oxysterols 25S-OHC and 27S-OHC and the remaining sterol (7-DHC) (Figure 2).

[0095] Collectively, these results suggest alterations in the cholesterol metabolic pathway in Rett syndrome affected regions.

[0096] Overexpression of CYP46A1 in a mild mouse model of Rett syndrome reduced Rett syndrome-associated motor deficits and improved the histological and molecular phenotypes associated with Rett syndrome We investigated whether upregulation of the cholesterol metabolic pathway, via increasing levels of CYP46A1, could ameliorate motor alterations in an in vivo model of Rett syndrome: a mild KO MECP2 model, particularly in the more severely affected male KO. - / yOverexpression of CYP46A1 by intravenous administration of AAVPHP.eB-CYP46A1-HA in mice clearly attenuates motor alterations in the mouse model, as seen in significant corrections of motor impairment measures by the grasp test (Figure 3A) and rotarod (Figure 3B) over a 29-week period after injection (the longest period studied).

[0097] Overexpression of CYP46A1 also significantly resulted in better growth in mice and restored normal body weight compared to untreated animals, whose growth was comparable to that of WT littermates (FIG. 3C).

[0098] A group of mice was euthanized at 16 weeks of age to assess histological and molecular rescue, and the remaining cohort was maintained until 32 weeks of age.

[0099] Biodistribution studies demonstrated that there were about 8 vector genome copies (VGCs) in the brain (Figure 4A), about 4 VGCs in the spinal cord, 1 VGC in the sciatic nerve, and the transduction of peripheral tissues was really low, with a maximum of 0.4 VGCs in the liver and less than 0.2 VGCs in other peripheral organs (Figure 4B).

[0100] This biodistribution correlated perfectly with the abundant expression of CYP46A1-HA in all brain structures, particularly in the cortex, striatum, hippocampus and cerebellum (data not shown), also confirming the results previously obtained in WT animals (data not shown).

[0101] Binding to the target has been validated for quantification of 24-hydroxycholesterol in the lumbar spinal cord of animals, revealing a 1.9-fold increase in treated animals compared to untreated animals (Figure 5). In addition, we reported a global increase in the cholesterol pathway with increased levels of 7-lathosterol, lanosterol, 25-OH and 27-OH cholesterol compared to untreated mild KO MECP2 males (Figure 5).

[0102] A partial preservation of Purkinje cells (PCs) was demonstrated in the cerebellum (effect of lobule and treatment by ANOVA test) with a general improvement (p-value < 0.0001) that could also be observed in the various lobules (Figure 6).

[0103] We then investigated the effect of treatment on neuroinflammation. We demonstrated an improvement in astrogliosis, especially in the hippocampus, in treated animals compared to untreated animals (Figure 7). In addition, we also improved microgliosis, especially in the striatum, hippocampus and cerebellum, in treated animals compared to untreated animals (Figure 8). Overall, a key finding is also the fact that AAVPHP.eB delivery did not cause any long-term inflammation in the brain (Figures 7 and 8).

[0104] Overexpression of CYP46A1 attenuates behavioral changes and ameliorates Rett syndrome-associated phenotypes in an exacerbated male model of Rett syndrome Based on treatments in mildly affected animals, we conducted an investigation to determine whether upregulation of cholesterol metabolic pathways, via elevated CYP46A1 levels, could ameliorate locomotor alterations in a more severe in vivo model of Rett syndrome: exacerbated KO MECP2.

[0105] Overexpression of CYP46A1 by intravenous administration of AAVPHP.eB-CYP46A1-HA in 3-week-old mice clearly attenuates motor alterations in an exacerbated mouse model.

[0106] AAVPHP.eB-CYP46A1 treatment clearly reduces motor impairment measured by the grasp test (Figure 9A) and attenuates changes measured by rotarod (Figure 9B) assessment by 4 weeks without attributable to COVID19 restraint (experiment ongoing). KO mice exhibited growth impairment, which was not ameliorated by 3 weeks of treatment (Figure 9C).

[0107] Mice were euthanized at 6 weeks of age (40 days) and histological and molecular analyses were performed. Biodistribution studies showed approximately 12 vector genome copies (VGCs) in the brain (Figure 10A) and approximately 7 VGCs in the spinal cord (Figure 10A) only 20 days after injection, with low transduction in peripheral tissues, with up to 2 VGCs in the liver, 1 VGC in the heart, and less than 0.5 VGCs in other peripheral organs (Figure 10B). This biodistribution correlated with the expression of a large amount of hCYP46A1 mRNA (Figure 11A), which did not affect endogenous mouse levels (Figure 11A). Regarding other genes involved in the cholesterol pathway, we reported a trend towards normalization of ApoE levels in treated animals compared to untreated animals (Figure 11B). None of the other genes were affected at 40 days of age, but survival needs to be evaluated later in cohorts of mice.

[0108] Preservation of Purkinje cells (PCs) was demonstrated in the cerebellum (effect of lobule and treatment by ANOVA test) with a general improvement (p-value < 0.0001) that could also be observed in the different lobules (Figure 12).

[0109] We then studied the effect of treatment on neuroinflammation. We did not demonstrate any major effect on astrogliosis, except for an increase in the cerebral cortex (Figure 14) and hippocampus. For both astrogliosis and microgliosis, no major differences were observed at this age in the deteriorated KO MECP2 males. Further investigations need to be performed at a later stage. Another cohort of treated animals is in progress to evaluate the effect on long-term improvement and survival.

[0110] Overexpression of CYP46A1 ameliorates behavioral changes in an exacerbated female model of Rett syndrome Because Rett Syndrome affects human females, we wished to investigate the effects of overexpression of CYP46A1 in affected KO MECP2 females.

[0111] Administration of AAVPHP.eB-CYP46A1 clearly and significantly reduces motor impairment measures measured by the grasp test (Figure 15A) and improves changes measured by rotarod (Figure 15B) assessment by 19 weeks without attributable to COVID19 constraints (experiment ongoing).

[0112] KO mice had growth retardation accompanied by a tendency to obesity, and a clear amelioration of this phenotype was observed in treated animals (FIG. 15C).

[0113] Collectively, these data suggest that overexpression of CYP46A1 attenuates behavioral changes in both males and females in mild and exacerbated models of Rett syndrome and represents a therapeutic target.

[0114] References Throughout this application various references are included to describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. [Table 3] TIFF0007624410000004.tif226165

Claims

1. A composition for treating Rett Syndrome (RTT), comprising: comprising a nucleic acid encoding cholesterol 24-hydroxylase, composition.

2. 2. The composition of claim 1, wherein the RTT is associated with at least one autism spectrum disorder.

3. The composition of claim 2, wherein the RTT is associated with intellectual disability.

4. The composition according to any one of claims 1 to 3, wherein the nucleic acid encodes the amino acid sequence of SEQ ID NO:

2.

5. The composition of any one of claims 1 to 4, wherein the nucleic acid sequence comprises codons preferred for expression in a mammalian CNS cell.

6. The composition according to any one of claims 1 to 5, wherein the nucleic acid has the sequence of SEQ ID NO:

1.

7. The composition according to any one of claims 1 to 6, which is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors.

8. The composition of any one of claims 1 to 7, which is an AAV vector.

9. 9. The composition of claim 8, which is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 vector.

10. 10. The composition of claim 9, wherein the AAV10 vector is AAVrh.10 or AAVPHP.eB.

11. The composition according to any one of claims 1 to 10, which is administered intravenously.

12. The composition of any one of claims 1 to 11, which is administered directly to the brain or cerebrospinal fluid of a patient.

13. The composition of claim 12 , which is administered to a neuron.

14. 14. The composition of claim 13, wherein the neuron is a cortical neuron, a hippocampal neuron, a striatal neuron, or a cerebellar neuron.

15. The composition of any one of claims 1 to 13, which is administered by intravascular, intravenous, intranasal, intraventricular, retroorbital, intrathecal injection, stereotactic microinjection, or superficial cortical application.

16. A pharmaceutical composition for use in treating Rett Syndrome, comprising a therapeutically effective amount of a composition according to any one of claims 1 to 13.

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