Expression vector for cholesterol 24-hydroxylase for use in amyotrophic lateral sclerosis therapy

Abstract

The present invention relates to a vector for the treatment of amyotrophic lateral sclerosis, which may or may not be associated with frontotemporal dementia and related motor neuron disorders, comprising a nucleic acid encoding cholesterol 24-hydroxylase.

Description

Technical Field

[0001] The present invention relates to the treatment of amyotrophic lateral sclerosis. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a rare neurological disease belonging to the group of motor neuron disorders, which are mainly related to neurons responsible for controlling autonomous muscle movements (such as chewing, walking and speaking) (Zarei S et al., 2015). ALS is characterized by the gradual degeneration and death of motor neurons, extending from the brain to the spinal cord, and extending to the muscles of the whole body. ALS involves both upper motor neurons (information from motor neurons in the brain is transmitted to the spinal cord) and lower motor neurons (motor nuclei in the brain), which degenerate and therefore stop sending information to muscles, which gradually weaken, start to twitch and become atrophic (Rowland LP et al., 2001).

[0003] Most people with ALS die within 3 to 5 years after the first symptoms appear, usually due to respiratory failure. Only 10% of people with ALS survive more than 10 years after the onset of symptoms.

[0004] In ALS, several potential risk factors have been associated: age, with symptoms typically appearing between the ages of 55 and 75; sex, with ALS being slightly more common in men; and race and ethnicity, with Caucasians and non-Hispanics being most likely to develop ALS.

[0005] However, the vast majority of ALS cases (90%) are sporadic. In contrast, about 10% of ALS cases are familial (FALS). For these familial cases, several genes have been implicated: C9ORF72, SOD1, FUS, TARDBP, and more recently, SMCR8 (Greenway et al. 2006; Kabashi et al., 2008; Kaur et al., 2016; Turner MR et al., 2017; Ticozzi et al., 2011; Valdmanis PN et al., 2008).

[0006] There are several aspects under consideration regarding the pathophysiology of ALS, one of the leading theories is related to RNA processing, particularly based on the finding that TDP-43 and genes such as TARDBP and FUS are implicated as genetic causes of ALS in the majority of ALS cases. Both genes are involved in pre-mRNA splicing, RNA transport and translation. Pre-mRNAs containing C9orf72 repeats can sequester nuclear RNA binding proteins, preventing them from properly splicing other mRNAs. Another well-recognized aspect of ALS is protein aggregation, including SOD1, TDP43 and FUS, which can be observed in both ALS patients and animal models. It has been proposed that aggregates interfere with normal protein homeostasis and induce cellular stress. In addition, protein aggregates can sequester other proteins or RNAs that are essential for normal cell function.

[0007] So far, there is no known cure for ALS. Only two drugs are FDA-approved for ALS: riluzole and edaravone. Riluzole is believed to work by reducing glutamate levels to reduce damage to motor neurons. Riluzole extended survival by several months, but did not reverse damage already present in neurons (Zoccolella et al., 2007). Regarding edaravone, it has only been shown to reduce clinical assessments of daily impairment (Brooks et al., 2018). Therefore, there is an urgent need to develop new strategies for ALS therapy. Summary of the invention

[0008] The present inventors now propose to combat ALS by modulating the cholesterol metabolic pathway, more specifically by means of a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase, which vector expresses cholesterol 24-hydroxylase in target cells.

[0009] Therefore, an object of the present invention is to provide a vector for treating amyotrophic lateral sclerosis, the vector comprising a nucleic acid encoding cholesterol 24-hydroxylase.

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

[0011] In an embodiment, the vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes virus vector and an adeno-associated virus (AAV) vector, preferably an AAV vector, more preferably an AAV9, AAV10 (AAVrh.10) or AAVPHP.eB vector, and even more preferably AAVPHP.eB.

[0012] In an embodiment, the vector is administered directly into the brain and / or spinal cord of a patient, preferably into the spinal cord and / or motor cortex.

[0013] Another object of the present invention is to provide a pharmaceutical composition for treating amyotrophic lateral sclerosis, comprising a vector containing a nucleic acid encoding cholesterol 24-hydroxylase. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 -WT and SOD1 G93A mRNA levels of genes involved in cholesterol metabolism in the lumbar spinal cord of animals at 8 weeks and those of WT and SOD1 G93A 24-OH cholesterol levels in the spinal cord of animals at week 15. (A) 24-OH cholesterol levels in the spinal cord of WT and SOD1 mice at 8 weeks of age. G93A mRNA was extracted from the lumbar spinal cord of mice. mRNA levels were normalized to the actin housekeeping gene. Data are presented as mean ± SEM (n = 4-5 per group). (B) SOD1 evaluated by UPLC-HRMS G93A 24S-hydroxycholesterol content in lumbar spinal cord of mice. Data are expressed as mean ± SEM (n = 7-9 per group). Statistical analysis: Student's t test. *p < 0.05, **p < 0.01.

[0015] Figure 2 - Expression of CYP46A1-HA was evaluated after 8 weeks of intravenous delivery of AAVPHP.eB-CYP46A1-HA in WT animals and analysis was performed 3 weeks after low, medium or high dose injection. HA staining on cervical, thoracic and lumbar sections of the spinal cord.

[0016] Figure 3 - Evaluation of inflammation after 8 weeks of intravenous delivery of AAVPHP.eB-CYP46A1-HA in WT animals and analysis performed 3 weeks after low, medium or high dose injection. GFAP staining on lumbar sections of spinal cord. NI: Not injected

[0017] Figure 4 -Behavioral evaluation of SOD1 mice after intravenous administration of AAVPHP.eB-CYP46A1 during the preventive phase (3 weeks). (A) Weight tracking, (B) grasping test, and (C) inversion test. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Black * corresponds to p-values ​​relative to WT animals, gray to those relative to SOD1 animals.

[0018] Figure 5- Biodistribution of AAVPHP.eB-CYP46A1 after prophylactic treatment in SOD1 animals and target engagement assessment by quantification of 24OH cholesterol levels at 15 weeks of age after prophylactic treatment. (A) Biodistribution in the central nervous system and (B) peripheral organs, and (C) lumbar spinal cord 24S-hydroxycholesterol content in SOD1G93A mice assessed by UPLC-HRMS. Results are expressed as mean ± SEM.

[0019] Figure 6 - Evaluation of CYP46A1-HA expression and histological analysis of the spinal cord at 15 weeks of age after intravenous delivery of AAVPHP.eB-CYP46A1 as a preventive treatment. (A) HA staining on cervical, thoracic and lumbar sections of the spinal cord of SOD1 animals treated with AAV; (B) Co-staining for HA and VachT on cervical, thoracic and lumbar sections of the spinal cord of WT, SOD1 and SOD1 AAV animals; (C) Quantification of the number of motor neurons; (D) Luxol staining of lumbar sections of the spinal cord, and (E) evaluation of the percentage of myelin. The results are expressed as mean ± SEM. *p<0.05, **p<0.01. Black * corresponds to the p value relative to WT animals, and gray is relative to SOD1 animals.

[0020] Figure 7 – Muscle phenotype of AAV-treated SOD1 animals is improved after prophylactic treatment. (A) Oxidized hematoxylin and eosin staining to analyze fiber size in muscle. Mean fiber area of ​​tibialis (B), gastrocnemius (D), and quadriceps (F) muscles is represented, and the percentage of fibers is further separated according to their cross-sectional size in tibialis (C), gastrocnemius (E), and quadriceps (G). Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01.

[0021] Figure 8 - Preservation of neuromuscular junctions in AAV-treated SOD1 animals after prophylactic treatment. (A) Co-staining of neurofilaments (NF) and bungarus toxin (BTX) in 15-week-old WT, SOD1, and SOD1-treated animals after intravenous delivery of AAVPHP.eB-CYP46A1. (BC) Scoring of NMJs according to their innervation status and integrity at 15 weeks of age. (D) Evaluation of NMJ formation in WT and SOD1 animals at 3 weeks using triple staining of pan NF, bungarus toxin, and VachT. (E) Quantification of NMJs according to their maturation status from M1 to M4 at 3 weeks of age.

[0022] Fig. 9- Molecular analysis of NMJs during preventive treatment at 15 weeks. Analysis of Musk expression in tibialis (A) and gastrocnemius (B) muscles and quantification of nAchR expression in tibialis (C) and gastrocnemius (D) muscles.

[0023] Fig.10 -Behavioral evaluation of SOD1 mice after intravenous administration of AAVPHP.eB-CYP46A1 during the therapeutic phase (8 weeks). (A) Weight tracking, (B) grasping test, (C) inversion test, and (D) survival. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Black * corresponds to p-values ​​relative to WT animals, gray to relative to SOD1 animals.

[0024] Fig.11 - Biodistribution of AAVPHP.eB-CYP46A1 in SOD1 animals after preventive treatment and target engagement evaluation by quantification of 24OH cholesterol levels at 15 weeks of age after therapeutic treatment. (A) Biodistribution in the central nervous system and (B) peripheral organs, and (C) lumbar spinal cord 24S-hydroxycholesterol content in SOD1G93A mice assessed by UPLC-HRMS. Results are expressed as mean ± SEM.

[0025] Fig.12 - Evaluation of CYP46A1-HA expression at 15 weeks of age and histological analysis of the spinal cord after intravenous delivery of AAVPHP.eB-CYP46A1 as therapeutic treatment. (A) Co-staining for HA and VachT on lumbar sections of the spinal cord of WT, SOD1 and SOD1 AAV animals. (B) Quantification of the number of motor neurons on lumbar spinal cord sections, and (C) Luxol staining on lumbar sections of the spinal cord. Results are expressed as mean ± SEM. *p<0.05, **p<0.01. Black * corresponds to p-values ​​relative to WT animals, gray to SOD1 animals.

[0026] Fig.13 - Partial correction of muscle phenotype in AAV-treated SOD1 animals after therapeutic treatment. Mean fiber area of ​​tibialis (A), gastrocnemius (C) and quadriceps (E) muscles, as well as the percentage of re-segmented fibers according to the cross-sectional size of fibers in tibialis (B), gastrocnemius (D) and quadriceps (F) muscles are represented. Results are expressed as mean ± SEM. *p<0.05, **p<0.01.

[0027] Fig.14- Preservation of neuromuscular junctions in AAV-treated SOD1 animals after therapeutic treatment. (A) Co-staining of WT, SOD1 and SOD1-treated animals at 15 weeks of age for neurofilament (NF) and bungarotoxin (BTX) after therapeutic intravenous delivery of AAVPHP.eB-CYP46A1. (BC) Scoring of NMJs according to their innervation status and integrity at 15 weeks of age.

[0028] Fig.15 - Molecular analysis of NMJ at 15 weeks of therapeutic treatment. Analysis of Musk expression in tibialis (A) and gastrocnemius (B) muscles and quantification of nAchR expression in tibialis (C) and gastrocnemius (D) muscles.

[0029] Fig.16 - Behavioral evaluation of SOD1 mice after intravenous administration of AAVPHP.eB-CYP46A1 at high doses during the therapeutic phase (8 weeks). Clasping test. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Black * corresponds to p-values ​​relative to WT animals, gray to those relative to SOD1 animals.

[0030] Fig.17 -Behavioral evaluation of C9ORF72 mice after high-dose intravenous administration of AAVPHP.eB-CYP46A1 during the preventive phase (4 weeks). (A) Weight tracking and (B) notched bar performance. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Black * corresponds to p-values ​​relative to WT animals, gray relative to C9ORF72 animals.

[0031] Fig.18 – (A) Evaluation of p62 aggregates in cells after overexpression of SMCR8 WT or mutants and overexpression of CYP46A1. (B) Quantification of the total number of positive transduced cells (HA) reflecting neuronal survival under different conditions. DETAILED DESCRIPTION

[0032] The inventors demonstrated that intravenous delivery of a vector expressing the CYP46A1 gene in a mouse model of ALS was able to prevent / correct the development of motor impairment. More specifically, the inventors demonstrated that delivery of a plasmid expressing the CYP46A1 gene into primary striatal neurons mimicking the pathology of amyotrophic lateral sclerosis caused a significant reduction in p62 aggregates (which is a hallmark of SMCR8 dysfunction and is involved in ALS through impairment of autophagy) and improved neuronal survival.

[0033] On this basis, the inventors provide a viral vector for treating ALS, wherein the vector expresses CYP46A1 in cells of the central nervous system, particularly in motor neurons and neurons within the motor cortex. This strategy is useful in treating any motor neuron disorder that is a continuum with some ALS that are primarily formed through common mutant genes. In particular, the inventors provide a viral vector for treating frontotemporal dementia (FTD) associated with ALS.

[0034] Amyotrophic lateral sclerosis

[0035] The present invention specifically relates to the treatment of amyotrophic lateral sclerosis. Preferably, the present invention relates to the treatment of ALS caused by sporadic factors and / or when disease-related proteins (i.e., SOD1, FUS, C9ORF72, TDP43...) contain gain-of-function mutations. In an embodiment, the present invention relates to the treatment of ALS associated with at least one motor neuron-related disorder. Such related motor neuron disorders are preferably selected from lower motor neuron disorders or upper motor neuron disorders, including spastic paraplegia. In a specific embodiment, the present invention relates to the treatment of ALS associated with frontotemporal dementia (FTD). FTD is a fatal neurodegenerative disease characterized by neuronal degeneration in the frontal and temporal lobes, leading to a progressive deterioration of language, personality and behavior. At least 15-20% of ALS patients receive a companion diagnosis of FTD (Prudencio et al., 2015). In another specific embodiment, the present invention relates to the treatment of ALS without frontotemporal dementia.

[0036] In the context of the present invention, the terms "treatment", "treat" and "treating" are used herein to characterize a therapeutic method or process intended to: (1) slow or arrest the progression, aggravation or worsening of the symptoms of the disorder or disease state to which such terms apply; (2) alleviate the symptoms of or bring about improvement in the symptoms of the disorder or disease state to which such terms apply; and / or (3) reverse or cure the disorder or disease state to which such terms apply.

[0037] The term "subject" or "patient" as used herein refers to an animal, preferably a mammal, and even more preferably a human, including adults and children. However, the term "subject" may also refer to non-human animals, mammals (such as mice) and non-human primates.

[0038] CYP46A1 sequence

[0039] The first object of the present invention relates to a vector for treating amyotrophic lateral sclerosis, said vector comprising the complete sequence of a nucleic acid encoding cholesterol 24-hydroxylase.

[0040] The term "gene" as used herein refers to a polynucleotide containing at least one open reading frame, which can encode a specific polypeptide or protein after being transcribed or translated.

[0041] The term "coding sequence" or "sequence encoding a specific protein" as used herein refers to a nucleic acid sequence that is transcribed (for DNA) and translated (for 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' (carboxyl) terminus. Coding sequences may include, but are not limited to, cDNA from prokaryotic mRNA or eukaryotic mRNA, genomic DNA sequences from prokaryotic DNA or eukaryotic DNA, and even synthetic DNA sequences.

[0042] The CYP46A1 gene encodes cholesterol 24-hydroxylase. This enzyme is a member of the cytochrome P450 enzyme superfamily. The enzyme converts cholesterol into 24S-hydroxycholesterol (24S-OH-Chol), which can dynamically cross the BBB and complete the peripheral circulation to be excreted from the body ( et al., 1998), thereby maintaining cholesterol homeostasis. The cDNA sequence of CYP46A1 is disclosed in Genbank Accession No. AF094480 (SEQ ID NO: 1). The amino acid sequence is shown in SEQ ID NO: 2.

[0043] The present invention utilizes a nucleic acid construct comprising the sequence of SEQ ID NO: 1 or a variant thereof for treating amyotrophic lateral sclerosis and optionally related motor neuron disorders (eg, FTD).

[0044] Variants include naturally occurring variants due to, for example, allelic variation between individuals (e.g., polymorphisms), alternative splicing forms, and the like. 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., exhibiting generally at least about 75%, preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95% nucleotide sequence identity to SEQ ID NO: 1. Variants of the CYP46A1 gene also include nucleic acid sequences that hybridize to the sequences defined above (or their complementary strands) under stringent hybridization conditions. Typical stringent hybridization conditions include temperatures above 30° C., preferably above 35° C., more preferably above 42° C., and / or a salinity of less than about 500 mM, preferably less than 200 mM. The skilled artisan can adjust the hybridization conditions by varying the temperature, salinity, and / or the concentration of other reagents (e.g., SDS, SSC, etc.).

[0045] Non-viral vectors

[0046] In an embodiment, the vector used in the present invention is a non-viral vector. Generally, the non-viral vector can be a plasmid encoding CYP46A1. Such a plasmid can be administered directly or via liposomes, exosomes or nanoparticles.

[0047] Viral vectors

[0048] Gene delivery viral vectors useful in the practice of the present invention can be constructed using well-known methodologies in the field of molecular biology. Generally, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, appropriate regulatory elements, and elements necessary to produce viral proteins that mediate cell transduction.

[0049] The term "gene transfer" or "gene delivery" refers to a method or system for reliably inserting exogenous DNA into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, expression and extrachromosomal replication of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of the host cell.

[0050] Examples of viral vectors include adenoviral vectors, lentiviral vectors, retroviral vectors, herpes virus vectors, and adeno-associated virus (AAV) vectors.

[0051] Such recombinant viruses can be produced by techniques known in the art, such as 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 in, for example, WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056, and WO94 / 19478.

[0052] In a preferred embodiment, an adeno-associated virus (AAV) vector is used.

[0053] "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. The AAV vector may have one or more AAV wild-type genes (preferably rep genes and / or cap genes) that are deleted in whole or in part, but retains functional flanking ITR sequences. Functional ITR sequences are essential for the rescue, replication, and packaging of AAV virus particles. Therefore, the AAV vector is defined herein as at least including those sequences (e.g., functional ITRs) required for cis viral replication and packaging. ITRs do not have to be wild-type nucleotide sequences and can be changed (e.g., by insertion, deletion, or substitution of nucleotides) as long as the sequence provides functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to provide at least components that are operably linked in the direction of transcription, control elements comprising a transcription initiation region, DNA of interest (i.e., the CYP46A1 gene), and a transcription termination region. Two copies of the DNA of interest may be included as a self-complementary construct.

[0054] In a more preferred embodiment, the AAV vector is an AAV9, AAV10 (preferably 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 effectively transduces CNS neurons (Chan KY et al., 2017; WO2017100671). Other vectors, such as those described in WO2015038958 and WO2015191508, may also be used.

[0055] The control elements are selected to be functional in mammalian cells. The resulting construct comprising operably linked components is combined with a functional AAV ITR sequence (5' and 3'). "Adeno-associated virus inverted terminal repeats" or "AAV ITRs" refer to regions recognized in the art that are present at each end of the AAV genome, which act together in cis as the starting point of DNA replication and as a packaging signal for the virus. AAV ITRs together with the AAV rep coding region provide the following: effectively excised and rescued therefrom, and the nucleotide sequence inserted between the two flanking ITRs is integrated into the mammalian cell genome. The nucleotide sequence in the AAV ITR region is known. For AAV-2 sequences (see, for example, Kotin, 1994; Berns, KI, "Parvoviridae and their Replication", Fundamental Virology, Second Edition (BN Fields and DMKnipe, ed.)). "AAV ITRs" used herein do not necessarily include wild-type nucleotide sequences, but may be altered (e.g., by insertion, deletion or substitution of nucleotides). Furthermore, the AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. Furthermore, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector need not necessarily be identical to or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing excision and rescue of the sequence of interest from the host cell genome or vector, and allowing integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0056] 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), particularly neurons. A review and comparison of the transduction efficiencies of different serotypes is provided in Davidson et al., 2000. In a preferred embodiment, AAV2-based vectors have been demonstrated to direct long-term expression of transgenes in the CNS, preferably transducing neurons. In other non-limiting examples, preferred vectors include vectors derived from AAV4 and AAV5 serotypes, which have also been demonstrated to transduce cells of the CNS (Davidson et al., supra). In particular, the vector may be an AAV vector comprising a genome derived from AAV5 (particularly the ITRs are AAV5 ITRs) and a capsid derived from AAV5.

[0057] In a specific 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. In addition, 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, and the like.

[0058] The selected nucleotide sequence is operably linked to a control element that directs its transcription or expression in a subject. Such control elements may comprise control sequences that are generally associated with the selected gene. In particular, such control elements may include a promoter of a CYP46A1 gene, in particular a promoter of a human CYP46A1 gene (Ohyama Y et al., 2006).

[0059] Alternatively, heterologous control sequences may be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, phosphoglycerate kinase (PGK) promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter (such as CMV immediate early promoter region (CMVIE)), Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes (such as murine metallothionein genes) will also be found useful herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA). For purposes of the present invention, heterologous promoters and other control elements (such as CNS-specific and inducible promoters, enhancers, and the like) will be particularly useful.

[0060] Examples of heterologous promoters include the CMV promoter.Examples of CNS-specific promoters include promoters isolated from genes from: myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), synapsins (eg, human synapsin 1 gene promoter), and neuron-specific enolase (NSE).

[0061] Examples of inducible promoters include the DNA response elements for ecdysone, tetracycline, hypoxia and aufin.

[0062] AAV expression vectors with a DNA molecule of interest bound by AAV ITRs can be constructed by inserting the selected sequence directly into the AAV genome from which the major AAV open reading frame ("ORF") has been excised. Other portions of the AAV genome may also be deleted as long as sufficient portions of the ITRs are retained to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 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, the AAV ITR can be excised from the viral genome or from an AAV vector comprising the AAV ITR, and the AAV ITR can be fused to the 5' and 3' of the selected nucleic acid construct present in another vector using standard ligation techniques. AAV vectors comprising ITRs have been described in, for example, U.S. Patent No. 5,139,941. In particular, several AAV vectors that can be obtained from the American Type Culture Collection ("ATCC") with accession numbers 53222, 53223, 53224, 53225 and 53226 are described. In addition, chimeric genes can be synthesized to include 5' and 3' AAV ITR sequences arranged in one or more selected nucleic acid sequences. Codons preferred for expression of chimeric gene sequences in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. (See, for example, Edge, 1981; Nambaier et al., 1984; Jay et al., 1984). To produce rAAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques (e.g., by transfection). 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-speed microprojectiles (Klein et al., 1987).

[0063] For example, a preferred viral vector (e.g., AAVPHP.eB) comprises, in addition to a nucleic acid sequence encoding cholesterol 24-hydroxylase, a backbone of an AAV vector having ITRs derived from AAV-2, a promoter, or any neuronal promoter, such as the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus / β-actin hybrid promoter (CAG), wherein the CAG is composed of an enhancer from the cytomegalovirus immediate gene, a promoter, a splice donor and an intron from the chicken β-actin gene, and a splice acceptor from rabbit β-globin; the neuronal promoter is, for example, a promoter of a dopamine 1 receptor or a dopamine 2 receptor, wherein the promoter of the dopamine 1 receptor or the dopamine 2 receptor has or does not have a wild-type or mutant form of a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[0064] Vector delivery

[0065] Disclosed is a method for treating amyotrophic lateral sclerosis, comprising administering to a patient in need thereof a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase. The vector can be delivered directly to the brain and / or spinal cord of the subject, or delivered by intravascular, intravenous, intranasal, intracerebroventricular, or intrathecal injection. In a specific embodiment, the vector is AAVrh10 or AAVPHP.eB and is delivered by intravenous injection.

[0066] In a specific embodiment, a method is provided for treating amyotrophic lateral sclerosis (ALS) in a subject, the method comprising:

[0067] (a) providing a vector as defined above, said vector comprising a nucleic acid encoding cholesterol 24-hydroxylase; and

[0068] (b) delivering the vector to the brain and / or spinal cord of a subject, whereby the vector transduces cells in the brain and / or spinal cord, and whereby the transduced cells express cholesterol 24-hydroxylase at therapeutically effective levels.

[0069] Advantageously, the vector is a viral vector, more advantageously an AAV vector, and even advantageously an AAV vector selected from the group consisting of AAV9, AAV10 or AAVPHP.eB.

[0070] In a specific embodiment, the vector is delivered to the brain (particularly the motor cortex) and / or the spinal cord. In a specific embodiment, the vector is delivered only to the spinal cord.

[0071] In another specific embodiment, the vector is administered by intravenous injection.

[0072] The method of delivering or administering a viral vector to neurons and / or astrocytes and / or oligodendrocytes and / or microglia generally includes any method suitable for (directly or by hematopoietic cell transduction) delivering the vector to the cells so that at least part of the cells in the selected synaptic connected cell population are transduced. The vector can be delivered to any cell of the central nervous system, the cell of the peripheral nervous system, or both. Preferably, the vector is delivered to cells of the brain and / or spinal cord. Typically, the vector is delivered to cells of the brain (including, for example, cells of the motor cortex), the spinal cord, or a combination thereof, or any suitable subpopulation thereof.

[0073] In order to deliver the vector specifically to specific areas and specific cell groups of the brain or spinal cord, the vector can be given by stereotaxic microinjection. For example, the patient has a stereotactic frame base fixed in place (screwed into the skull). The brain with a stereotactic frame base (MRI compatible with fiducial markers) is imaged using high-resolution MRI. Then, the MRI image is transferred to a computer running stereotactic software. A series of coronal, sagittal and axial images are used to determine the target (site of vector injection) and trajectory. The software converts the trajectory directly into 3-dimensional coordinates suitable for the stereotactic frame. A borehole is drilled above the entry site, and the stereotactic instrument is positioned with an implanted needle at a given depth. Then, the vector is injected into the target site and eventually mixed with a contrast agent. Since the vector is integrated into the target cell, rather than producing viral particles, the subsequent diffusion of the vector is very small, and is mainly passive diffusion from the injection site before integration, and of course the desired transsynaptic transport effect. The degree of diffusion can be controlled by adjusting the ratio of the vector to the fluid carrier.

[0074] Additional routes of administration may also include topical application of the vector under direct visualization, for example, superficial dermal application, intranasal application, or other non-stereotaxic application.

[0075] The target cells of the vector of the present invention are cells of the spinal cord (motor neurons) and cells of the brain (motor cortex), preferably nerve cells, of a subject suffering from ALS.

[0076] Preferably, the subject is a human being, typically an adult, but may be a child or infant. However, the present invention encompasses the delivery of the vector to a biological model of the disease. In this case, the biological model may be any mammal at any stage of development when delivered, such as an embryonic stage, a fetal stage, an infant stage, a juvenile stage, or an adult stage, preferably an adult. In addition, the target cell may be substantially from any source, especially non-human primates and rodents (mice, rats, rabbits, hamsters), carnivores (cats, dogs) and artiodactyls (cattle, pigs, sheep, goats, horses) mammals, and any other non-human system (e.g., zebrafish model system).

[0077] Preferably, the method of the present invention includes intracerebral administration by stereotactic injection. However, according to the present invention, other known delivery methods may also be used. For example, in order to distribute the vector more widely in the brain, it can be injected into the cerebrospinal fluid, for example, by lumbar puncture, cisterna magna or ventricular puncture. In order to guide the vector to the brain, it can be injected into the spinal cord or into the flesh (subcutaneous or intramuscular) of the peripheral ganglia or the body part of interest. In some cases, such as here for AAVPHP.eB, the vector can be administered by an intravascular method. For example, in the case where the blood-brain barrier is disturbed, the vector can be administered intra-arterially (carotid artery). In addition, in order to deliver more comprehensively, the vector can be administered during the "opening" of the blood-brain barrier achieved by infusion of a hypertonic solution including mannitol or local delivery by ultrasound.

[0078] The carrier used herein can be formulated in any suitable vehicle for delivery. For example, they can be placed in a pharmaceutically acceptable suspension, solution or emulsion. Suitable media include saline and liposome preparations. More specifically, pharmaceutically 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 (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (e.g., supplements based on Ringer's dextrose), etc.

[0079] Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.

[0080] Colloidal dispersion systems can also be used for targeted gene delivery. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes or exosomes.

[0081] The preferred dosage and regimen can be determined by a physician and depends on the age, sex, weight, and stage of the disease of the subject. As an example, for the delivery of cholesterol 24-hydroxylase using a viral expression vector, each unit dose of a vector expressing cholesterol 24-hydroxylase may contain 2.5 μL to 100 μL of a composition containing a viral expression vector in a pharmaceutically acceptable fluid, and it provides from 10 μL to 10 μL of the composition per mL. 10 Up to 10 15 The virus particles expressed cholesterol 24-hydroxylase.

[0082] The vector can be used in therapeutic treatment and / or prophylactic treatment.

[0083] Therefore, an object of the present invention is to provide a vector for preventive treatment of ALS, wherein the vector comprises a nucleic acid encoding cholesterol 24-hydroxylase.

[0084] Another object of the present invention is to provide a vector for therapeutic treatment of ALS, wherein the vector comprises a nucleic acid encoding cholesterol 24-hydroxylase.

[0085] Pharmaceutical composition

[0086] A further object of the present invention relates to a pharmaceutical composition for treating ALS, said pharmaceutical composition comprising a therapeutically effective amount of a vector according to the present invention.

[0087] "Therapeutically effective amount" refers to an amount of the vector of the invention sufficient to treat ALS at a reasonable benefit / risk ratio applicable to any medical treatment.

[0088] It should be understood that the total daily dosage 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 dosage 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 patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific compound being used; the duration of treatment; drugs used in combination or concurrently with the specific polypeptide being used; and factors well known in the medical field, etc. For example, it is well known to those skilled in the art to start the dosage of the compound at a level lower than the dosage required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product per adult per day may vary within a wide range. The therapeutically effective amount of the vector according to the present invention that should be administered, as well as the dosage of a certain amount of the virus or non-viral particle and / or pharmaceutical composition of the present invention for treating a pathological condition will depend on a variety 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 to be used.

[0089] The presentation of the pharmaceutical composition comprising the carrier according to the present invention can be any form suitable for intramuscular, intracerebral, intranasal, intrathecal, intracerebroventricular or intravenous administration. In the pharmaceutical composition of the present invention for intramuscular, intranasal, intravenous, intracerebral, intrathecal or intracerebroventricular administration, the active ingredient can be administered to animals and humans alone or in combination with another active ingredient in a unit administration form (such as a mixture with a conventional pharmaceutical support).

[0090] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for a formulation that can be injected. These vehicles may be in particular isotonic sterile saline solutions (sodium dihydrogen phosphate or sodium dihydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or dry (especially lyophilized) compositions that allow the formation of sterile injections when sterile water or physiological saline is added, as appropriate.

[0091] After formulation, the solution will be administered in a manner compatible with the dosage form and in an amount that is therapeutically effective. The preparation can be easily administered in a variety of dosage forms, such as the type of injectable solution, but drug release capsules and the like can also be used.

[0092] Multiple doses may also be given.

[0093] It is also possible to link the therapeutic approach to small molecules that can activate CYP46A1, such as efavirenz (Mast N et al., 2013; Mast N et al., 2017; Mast N et al., 2014; Mast N et al., 2017) or antifungal drugs (Mast N et al., 2013; Fourgeux et al., 2014), which may in turn inhibit CYP46A1 and therefore serve as a method to terminate gene therapy if necessary.

[0094] The present invention will be further illustrated by the following examples. However, the examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0095] Example

[0096] In this experiment, an in vitro model of ALS was established based on the overexpression of SMCR8 D928V (a mutant of SMCR8), which forms a complex with C9ORF72 and WDR1 and acts as a GDP / GTP exchange factor for RAB8 and RAB39b, thereby controlling autophagic flux (Sellier et al., 2016). WT SMCR8 or mutant SMCR8 (which alters autophagy with siRNA expression of SMCR8) were overexpressed.

[0097] Materials and methods

[0098] animal

[0099] Two male B6SJL-Tg(SOD1*G93A)1Gur / J were obtained from Jackson Laboratory (Cat. No. 002726) and mated with C57BL / 6 mice to generate colonies.

[0100] Three male FVB / NJ-Tg(C9orf72)500Lpwr / J were obtained from Jackson Laboratory (Cat. No. 029099) and mated with FVB / NJ mice to generate colonies.

[0101] 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 on the care and use of experimental animals (2010 / 63 / EU).

[0102] AAV plasmid design and vector production

[0103] AAV vectors were produced and purified by Atlantic Gene therapies (INSERM U1089, Nantes, France). The production of the vector has been described elsewhere (Hudry E et al., 2010). The viral construct of AAVPHP.eB-CYP46A1-HA contains an expression cassette consisting of the human Cyp46a1 gene, driven by the CMV early enhancer / chicken β-actin (CAG) synthetic promoter (CAG) and surrounded by the inverted terminal repeats (ITRs) of AAV2. The final titer of the batch was between 1.5 and 2.10 13 vg / mL.

[0104] Genotyping

[0105] Mice were typed according to Jackson Laboratory protocols: for SOD1G93A mouse line (https: / / www2.jax.org / protocolsdb / f?p=116:5:0::NO:5:P5_MASTER_PROTOCOL_ID,P5_JRS_CODE:24173,002726); for C9ORF72 500r line, https: / / www2.jax.org / protocolsdb / f?p=116:5:0::NO:5:P5_MASTER_PROTO COL_ID,P5_JRS_CODE:30889,029099.

[0106] Intravenous injection

[0107] To determine the dose of AAVPHP.eB-CYP46A1-HA to be injected in the ALS mouse model, C57BL6 animals were injected with 3 doses totaling 2.5.10 11 vg(low), total 5.10 11 vg(medium) and total 1.10 12 vg (high dose) (n=3 per group).

[0108] For preventive treatment, 4% isoflurane was used to treat SOD1 G93A Anesthesia was induced in C9ORF72 500r mice aged 4 weeks and then maintained with 2% isoflurane in 80% air and 20% oxygen. SOD1 animals received a total of 2.5.10 11 vg (low dose) or total 5.10 11 vg (medium dose) (100 μL volume) was injected intravenously. WT and uninjected SOD1 animals received an injection of 100 μL of saline solution.

[0109] Details of the preventive group:

[0110] - SOD1 study: n=10 WT; n=12 SOD1 and n=14 SOD1 AAV, with a total of 2.5.10 11 vg

[0111] - C9 study: n = 16 WT; n = 16 C9 and n = 17 C9 AAV, with a total of 5.10 11 vg

[0112] For therapeutic treatment, anesthesia was induced in 8-week-old SOD1G93A or 16-week-old C9ORF72 500r mice with 4% isoflurane and then maintained with 2% isoflurane in 80% air and 20% oxygen. SOD1 animals received a total of 2.5.10 11 vg (low dose) or total 5.10 11 vg (medium dose) (100 μL volume) was injected intravenously. WT and uninjected SOD1 animals received an injection of 100 μL of saline solution.

[0113] Details of the therapeutic groups:

[0114] -SOD1 Research:

[0115] n=24WT; n=27SOD1 and n=22SOD1 AAV, with a total of 2.5.10 11 vg

[0116] n = 5WT; n = 5SOD1 and n = 7SOD1 AAV, for a total of 5.10 11 vg

[0117] - C9 study: n = 14 WT; n = 13 C9 and n = 12 C9 AAV, with a total of 5.10 11 vg

[0118] Behavioral testing

[0119] Weight Tracking

[0120] All animals were weighed prior to injection, weekly for SOD1 animals, and biweekly for C9ORF72 animals.

[0121] Clasping test

[0122] The clasping test evaluates coordination and is classical for ALS evaluation. Animals were scored prior to injection and then every 2 weeks from 6 or 8 weeks until 15 weeks. Animals were held from the tail and if the mouse twitched, the score was 1, and then the score was increased for each hindlimb clasping. The results of each test are expressed as the mean ± SEM for each group and analyzed with a t-test.

[0123] Inversion test

[0124] The inversion test evaluates strength and is classic for ALS evaluation. Animals were scored before injection and then every 2 weeks from 6 or 8 weeks until 15 weeks. The animals were placed on a grid and the grid was inverted, and the remaining feet attached to the grid were scored immediately (temporary inversion). The results of each test were expressed as mean ± SEM for each group and analyzed by t-test.

[0125] For the C9 model, mice were challenged even more with the inversion test by three 5-minute trials with 15-minute recovery between each trial, resulting in more robust results. For each trial, the time they maintained attachment was scored and the average was calculated for each animal.

[0126] Notched Rods

[0127] Coordination was assessed using the notched bar test (number of scores for upper or lower limb drops) as previously described (Piguet F et al., 2018).

[0128] Survival evaluation

[0129] Mice were observed daily and immediately euthanized if they reached endpoint criteria (hind limb dragging or body weight loss > 20%).

[0130] Tissue collection

[0131] Mice were anesthetized with pentobarbital (Euthasol 180 mg / kg) solution and perfused transcardially with phosphate buffered saline (PBS). Brain, spinal cord and sciatic nerve and peripheral organs (liver, heart, lung, kidney, spleen) were collected and post-fixed in 4% PFA before paraffin embedding for histology (cut into 6-10 μm with a microtome) or immediately frozen in liquid nitrogen for biomolecular analysis.

[0132] Limb muscles were dissected and postfixed overnight in 4% PFA / PBS at 4°C. Samples were rinsed 3 times in PBS and cryoprotected in 20% sucrose / PBS for 48 hours. Tissues were embedded in cryomatrix (Thermoscientific) and cut (20 μm) using a cryostat (Leica CM3050S). Cryosections were dried at room temperature and stored at -20°C.

[0133] Gastrocnemius, tibialis, quadriceps, and portions of the spinal cord were dissected and frozen in liquid nitrogen.

[0134] The different tissues were ground / crushed in liquid nitrogen and then isolated for analysis of RNA or DNA expression.

[0135] Primary striatal cell culture and transfection

[0136] Primary striatal neurons were dissected from day 14 embryos from pregnant Swiss mice (Janvier) as previously described (Charvin D et al., 2005; Deyts et al., 2009). After 7 days in vitro, transient transfection of striatal cells was performed using LipofectamineTM2000 (Invitrogen). At this stage, very few glial cells were observed (55%; data not shown). Cells were transfected under 5 conditions (CYP46A1-HA; SMWR8 WT-HA; SMCR8 D928V-HA; SMWR8 WT-HA+CYP46A1-HA; SMCR8 D928V-HA+CYP46A1HA), and untransfected cells were used as controls. N=6 replicates were performed for each condition. 100 ng DNA of each plasmid was transfected in 8-well labtek chambers for 16 h before cell fixation using Lipofectamine.

[0137] Primary Antibody

[0138] Table 1 below lists the antibodies used for immunohistochemistry (IHC) analysis.

[0139] Table 1: Antibodies used for immunofluorescence and immunohistochemistry (IHC) analysis

[0140] Primary Antibody source IF Mouse anti-HA Biolegend(901514) 1:1000 Mouse anti-p62 Abcam(ab56416) 1:250 Rabbit anti-HA Cell signaling(#3724) 1:500 Rabbit anti-Iba1 Wako (019-19741) 1:500 Mouse anti-GFAP Sigma(G-3893) 1:500 Mouse anti-Pan-NF Biolegend(837904) 1:1000 Alexa 488-αBTX Lifesciences(B13422) 1:2000 Rabbit anti-VachT Sigma(SABA2000559) 1:1000

[0141] Immunostaining

[0142] On the cell

[0143] The immunofluorescence procedure was started by fixing the cells in PFA for 15 minutes. After washing 3 times, the cells were permeabilized in PBS / 0.3% Triton X-100 and then blocked at RT for 1 h in PBS / 0.1% TritonX-100 containing 5% normal goat serum (NGS, Gibco). The cells were then incubated with the corresponding primary antibodies at 4 ° C overnight. After washing 3 times, the cells were incubated with the corresponding secondary antibodies (1:1000; Vector Laboratories Inc., CA, USA) diluted in PBS / 0.1% Triton X-100 at RT for 1 h. After washing three times, the cells were dried and sealed in mounting medium with DAPI.

[0144] On paraffin sections

[0145] Muscle: Before processing, muscle frozen sections were treated with 0.1M glycine in PBS for 30 min. After washing in PBS, the sections were permeabilized with PBS / 0.3% TritonX-100 for 10 min and saturated with PBS / 0.3% Triton / 10% BSA for 45 min. The primary antibody was diluted in a saturated solution and incubated O / N at 4°C. After washing in PBS / 0.1% Triton, the secondary antibody and α-bungarotoxin were diluted in PBS / 0.1% Triton / 10% BSA and added for 1 hour at room temperature. After washing in PBS / 0.1% Triton, the slides were mounted with fluorescent aqueous mounting medium (F4680, Sigma).

[0146] The primary antibodies for immunofluorescence were mouse anti-Pan-Neurofilament (1:1000; Biolegend, 837904) and rabbit anti-vesicular acetylcholine transporter (VAChT; 1:1000; Sigma, 2000559). The secondary antibodies were diluted 1:1000 and were donkey anti-rabbit / AlexaFluor 594 and anti-mouse / AlexaFluor Cy3 (Life Technologies, Carlsbad, CA, USA).

[0147] α-Bungarotoxin-Alexa594 (1:2000; Life Technologies, B13422) was incubated with the secondary antibody. Photos were taken with a confocal SP8 Leica DLS inverted microscope (Carl Zeiss, Zaventem, Belgium). For all images, brightness and contrast were adjusted using ImageJ software after acquisition to match the observation results.

[0148] Spinal cord: For immunofluorescence, the spinal cord was treated with 10mM Tris / 1mM EDTA / 0.1% Tween pH 8.75 at 95°C for 45min. After washing in PBS, the sections were incubated with permeabilization solution (PBS / 0.3% TritonX-100) for 15min and then incubated with saturation solution (PBS / 0.1% TritonX-100 / 10% horse serum) for 1 hour. The primary antibody was diluted in 10% horse serum / PBS / 0.1% Triton X-100 and incubated overnight at 4°C on tissue sections. After washing in PBS / 0.1% TritonX-100, the sections were incubated with secondary antibodies (donkey anti-rabbit Alexa 594 and donkey anti-mouse alexa488, 1:1000, Life Technologies).

[0149] Immunohistochemical labeling was performed using the ABC method. Briefly, tissue sections were treated with peroxide for 30 minutes to inhibit endogenous peroxidase. After washing in PBS, the sections were treated with 10mM Tris / 1mM EDTA / 0.1% Tween pH 8.75 at 95°C for 45min (for anti-HA only). After washing in PBS, the sections were incubated with blocking solution (goat serum or 10% goat serum in PBS / 0.3% Triton X-100) for 1 hour. The primary antibody was diluted in blocking solution and incubated overnight at 4°C on tissue sections. After washing in PBS, the sections were sequentially incubated with goat anti-rabbit or goat anti-mouse antibodies conjugated to biotin (Vector Laboratories), followed by ABC complex (Vector Laboratories) at room temperature for 30 minutes. After washing in PBS, peroxidase activity was detected using diaminobenzidine as a chromogen (Dako, Carpinteria, CA). In some cases, the slides were counterstained with hematoxylin. Slides were mounted with Depex (VWR International).

[0150] Luxol staining

[0151] Myelin staining / visualization on spinal cord sections using classic luxol staining.

[0152] Image acquisition

[0153] Images of immunofluorescence slides were collected at room temperature using a Leica DM 5000B microscope equipped with a Leica DFC310FX digital camera using macroscope (Leica) and LAS V3.8 (Leica) software. The pictures used for comparison were taken under the same image acquisition conditions, and all adjustments of brightness and contrast were uniformly applied to all images used for cell culture analysis.

[0154] Muscle and VachT images were acquired using axioscan, Zeiss and ZEN 2.6 software or nanozoomer. For all images, brightness and contrast were adjusted using ImageJ software after acquisition to match the observations.

[0155] For all IHC and staining, sections were acquired using a Hamamatsu slide scanner.

[0156] Muscle fiber analysis

[0157] Tibialis anterior (TA), gastrocnemius (gastro) and quadriceps (quadri) were dissected at 15 weeks. They were fixed with formalin, embedded in paraffin, cut (10 mm) and stained with hematoxylin / eosin. Images were collected using axioscan, Zeiss and ZEN 2.6 software. The muscle fiber cross-sectional area (CSA) of TA, Gastro, Quadri was measured on a minimum of 150 fibers in each animal (n=4-13) using ImageJ software.

[0158] Neuromuscular junction analysis

[0159] NMJ morphology was quantified on at least 100 TA junctions from three different animals. Endplate distribution could be classified into six categories: (1) normal endplates, (2) modified endplates, (3) fragmented endplates, (4) degenerated endplates, (5) nonporous endplates, and (6) ectopic endplates. At least 100 junctions were analyzed for each animal (n = 3-4). Motor endplate maturation was assessed in P21 mice according to previously described criteria (Audouard et al.).

[0160] Motor neuron quantification

[0161] The number of motor neurons was quantified in cervical, thoracic and lumbar spinal cord sections after immunohistochemistry for VAChT. The number of transduced motor neurons was quantified after immunohistochemistry for HA. For each mouse, motor neurons were counted in the left and right ventral sides of three spinal cord sections (n=3).

[0162] Luxol Quantification

[0163] The total area of ​​myelin has been measured on the lumbar spinal cord using Fiji software and normalized by the total spinal cord area. The percentage of myelin in WT is then reported as 100% and the results are reported in comparison to WT animals.

[0164] DNA extraction

[0165] DNA was extracted from brain, spinal cord, and peripheral organs using a chloroform / phenol protocol.

[0166] RNA extraction

[0167] Total RNA was extracted from gastrocnemius, tibialis, quadriceps and parts of lumbar spinal cord in mice and lumbar spinal cord of patients using Trizol or TriReagent (Sigma). One microgram of total RNA was transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions.

[0168] q-PCR

[0169] cDNA was amplified with SyberGreen (Roche). Primers used for RT-qPCR are 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 were analyzed using Lightcycler 480 software with efficiency factors for each gene and normalized to actin.

[0170]

[0171] The vector genome copy number was measured by qPCR of genomic DNA extracted from DRG, spinal cord (cervical, thoracic and lumbar levels), brain, cerebellum and peripheral organs using Light Cycler 480SYBR Green I Master (Roche, France). The results (vector genome copy number per cell) were expressed as n-fold differences in transgenic sequence copy number relative to Adck3 gene copies as internal standard (viral genome copy number of 2N genome).

[0172] Measurement of cholesterol and oxysterols

[0173] Analysis of cholesterol and oxysterols follows "gold standard" methods 25, so that the formation of self-oxidation products is minimized. In short, mouse striatal tissue samples are weighed and homogenized in 500 μL solutions containing butylated hydroxytoluene (BHT, 50 μg / mL) and EDTA (0.5M) using Tissue Lyser II instruments (Qiagen). At this point, a mixture of internal standards [epicoprostanol, 2H7-7-enecholesterol, 2H6-desmosterol, 2H6-lanosterol and 2H7-24 (R / S)-hydroxycholesterol] (Avanti Polar Lipids) is added. Alkaline hydrolysis is carried out at room temperature using 0.35M ethanol KOH under Ar for 2h. After the solution is neutralized with phosphoric acid, sterols are extracted in chloroform. The lower phase is collected, dried under a stream of nitrogen, and the residue is dissolved in toluene. Oxysterols were then separated from cholesterol and its precursors on a 100 mg Isolute silica column (Biotage); cholesterol was eluted in 0.5% propan-2-ol in hexane, and oxysterols were then eluted in 30% propan-2-ol in hexane. [Bis(trimethylsilyl)trifluoroacetamide + 10% trimethylsilyl chloride] (Regis Technologies) was used to silylate the sterol and oxysterol fractions, respectively. 26 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, length 30 m, diameter 0.32 mm, film thickness 0.25 μm; Restesk). A mass spectrometer (Agilent 5975 inert XL) connected in series with the gas chromatograph was set up for detection of positive ions. Ions were generated in electron impact mode at 70 eV. They were identified by fragmentation spectra in scanning mode and quantified by selective monitoring of specific ions after normalization and calibration with appropriate internal and external standards [epicostanol m / z 370, 2H7-7-enocholanol m / z 465, 2H6-alkanosterol m / z 358, 2H6-lanosterol m / z 504, 2H7-24(R / S)-hydroxycholesterol m / z 553, cholesterol m / z 329, 7-enocholanol m / z, 7-dehydrocholesterol m / z 325, 8-dehydrocholesterol m / z 325, alkanosterol m / z 343, lanosterol m / z 393 and 24(R / S)-hydroxycholesterol m / z 413].

[0174] Quantification of neuronal survival by immunofluorescence

[0175] Neuronal survival was assessed by direct quantification of HA positive cells under par condition in 5 wells per condition at 10x magnification on a Leica microscope.

[0176] Statistical analysis

[0177] Statistical analysis was performed using an unpaired Student's t-test. Results are expressed as mean ± SEM. Significant 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).

[0178] result

[0179] Basic evaluation of cholesterol pathways in SOD1 animals

[0180] At 8 weeks of age, SOD1 G93A The expression levels of several genes involved in the cholesterol pathway have been quantified in the lumbar spinal cord of WT and WT animals ( Figure 1 Significant decreases in CYP46A1 and ApoE have been observed ( Figure 1 A), and the trends of SREBP1, SREBP2, and HMGCR ( Figure 1 A) In addition, at 15 weeks of age, SOD1 G93A 24-Hydroxycholesterol was measured in the lumbar spinal cord of WT and WT animals, indicating that SOD1 G93A Its content is greatly reduced (about 60% reduction) ( Figure 1 B) These results confirm the hypothesis that modulation of CYP46A1 in ALS models also rescues the ALS phenotype.

[0181] Validation of AAVPHP.eB as a good vector for ALS

[0182] The inventors first investigated whether AAVPHP.eB is a good vector for ALS. For this purpose, 8-week-old WT animals were injected with a low dose (2.5.10 11 vg), medium dose (5.10 11 vg) or high dose (1.10 12 vg) encoding CYP46A1-HA. The good targeting of motor neurons by these 3 doses has been highlighted ( Figure 2 ), there was no significant difference between low and high doses, with transduction rates ranging from 50% to 60% ( Figure 2 ).

[0183] As shown by GFAP ( Figure 3 ) and Iba1 (data not shown) staining (did not induce any microgliosis or astrogliosis), and even in the high-dose group, this extensive transduction was not associated with any immune response in the spinal cord ( Figure 3 ).

[0184] These results prompted continuation with a lower dose of the vector.

[0185] Prevention of ALS phenotype in the SOD1G93A mouse model of ALS

[0186] CYP46A1 overexpression prevents behavioral changes in a prophylactically treated ALS mouse model.

[0187] The present inventors investigated whether upregulation of the cholesterol metabolism pathway, through increased CYP46A1 levels, could improve motor changes in an in vivo model of ALS (SOD1G93A model). CYP46A1 overexpression by prophylactic intravenous administration of AAVPHP.eB-CYP46A1-HA in 3-week-old mice significantly prevented motor changes in the mouse model. First, AAV-treated SOD1G93A mice had an improved growth curve ( Figure 4 A). Prophylactic AAVPHP.eB-CYP46A1-HA delivery significantly corrected motor impairment measured by the grasp test ( Figure 4 B) and completely prevented the changes measured by the inversion test ( Figure 4 C).

[0188] Biodistribution studies showed approximately 1 vector genome copy (VGC) in all levels of the spinal cord and between 2 and 4 copies ( Figure 5 A), and very low transduction in peripheral tissues, with a maximum of 0.4 VGC in the heart and less than 0.1 VGC in other peripheral organs ( Figure 5 B). Engagement of the target has been verified with quantification of 24-hydroxycholesterol in the lumbar spinal cord of the animals, showing a 3-fold increase in treated animals compared to WT animals ( Figure 5 C).

[0189] This increase in 24OH cholesterol was completely associated with the abundant expression of CYP46A1-HA at all levels of the spinal cord, especially in motor neurons (MNs) ( Figure 6 A), which also confirms the results obtained previously in WT animals ( Figure 2 ).

[0190] Partial preservation of MNs (approximately 60% compared to WT) was confirmed at the lumbar level, as assessed by quantification of co-staining for VachT and HA ( Figure 6 B and Figure 6 C), the number of MNs in the thoracic segment was similar compared to WT, and there was no difference at the cervical level ( Figure 6 C). This 60% preservation is sufficient to avoid demyelination in the lumbar spinal cord, as demonstrated by Luxol staining and quantification of myelin percentage ( Figure 6 D and Figure 6 E). AAV-treated SOD1 animals had an equal percentage of myelin as WT animals and significantly higher than non-SOD1-treated animals.

[0191] Another aspect that the inventors wanted to study was the muscle phenotype, indeed, muscles are severely affected in ALS, mainly due to the loss of innervation and neuromuscular junctions (NMJ) subsequent to the loss of MNs.

[0192] They first demonstrated significant preservation of muscle architecture, assessed by measuring mean fiber area ( Figure 7 A). Tibialis muscles of SOD1-treated animals showed a significant increase in mean fiber area compared to untreated animals ( Figure 7 B) and repartitioning according to fiber cross-sectional area, clearly demonstrating the retention of large cross-sectional fibers ( Figure 7 B). For the gastrocnemius muscle ( Figure 7 D and Figure 7 E) and quadriceps ( Figure 7 F and Figure 7 G), similar results were observed.

[0193] They then focused on NMJ phenotypes, demonstrating that the number of innervated junctions was increased in SOD1 treated animals compared to untreated animals ( Figure 8 A and Figure 8 B); and structural improvements in NMJs, notably an increase in the number of NMJs with normal or thicker endplates and a decrease in the number of ectopic and fragmented endplates ( Figure 8 C).

[0194] This result is particularly important and has significant therapeutic implications because when the inventors evaluated the status of NMJs in WT and untreated SOD1G93A animals at 3 weeks, NMJs already showed pathology at 3 weeks, with an increased number of immature junctions in SOD1 animals compared to WT ( Figure 8 D and Figure 8 E). This means that even if AAV-CYP46A1 is injected when NMJs are not fully formed, it is still able to significantly improve the phenotype of treated animals.

[0195] To complete the study, the inventors also quantified the levels of Musk (a receptor involved in agrin binding) and nAchR (a receptor for acetylcholine) in the tibialis muscle at 15 weeks of age, both of which are involved in synaptic stability and maintenance of NMJs. In both the tibialis and gastrocnemius muscles, the expression levels of both MuSK and nAchR were improved ( Fig. 9 ).

[0196] CYP46A1 overexpression alleviates behavioral changes in an ALS mouse model after therapeutic treatment

[0197] Based on the preventive treatment of animals, a study was conducted to determine whether upregulation of the cholesterol metabolism pathway by increasing CYP46A1 levels could improve motor alterations in a post-symptomatic ALS in vivo model (SOD1G93A model). Overexpression of CYP46A1 by intravenous administration of AAVPHP.eB-CYP46A1-HA in 8-week-old mice significantly alleviated motor alterations in the mouse model. The mice had improved growth ( Fig.10 A).

[0198] Administration of therapeutic AAVPHP.eB-CYP46A1 significantly reduced motor impairment as measured by the cling grip test ( Fig.10 B), and mitigates the variation measured by the inversion test ( Fig.10 C). In addition, the treatment resulted in an increase in the survival of the treated mice compared to untreated animals, with the life expectancy increased by an average of 14 days ( Fig.10 D).

[0199] Mice were euthanized at 15 weeks of age for histological and molecular analyses.

[0200] Biodistribution studies showed approximately 3-4 vector genome copies (VGC) in all levels of the spinal cord and between 10 and 20 copies in the brain ( Fig.11 A), and very low transduction in peripheral tissues, with a maximum of 0.5 VGC in the liver and less than 0.2 VGC in other peripheral organs ( Fig.11 B). Engagement of the target has been verified with quantification of 24-hydroxycholesterol in the lumbar spinal cord of the animals, showing a 1.3-fold increase in treated animals compared to WT animals ( Fig.11 C).

[0201] Partial preservation of MNs (approximately 60% compared to WT) was demonstrated at the lumbar level, as assessed by quantification of co-staining of VachT and HA ( Fig.12 A and Fig.12 B), similar to the results of preventive treatment ( Figure 6 C).

[0202] Here again this 60% preservation was sufficient to avoid demyelination in the lumbar spinal cord, as demonstrated by Luxol staining and quantification of myelin percentage ( Fig.12 C). AAV-treated SOD1 animals had an equal percentage of myelin as WT animals and significantly higher than untreated SOD1 animals.

[0203] By measuring the average fiber area ( Fig.13 ) showed a significant improvement in the phenotype in the treated animals compared to the untreated animals. The quadriceps muscles of the SOD1 treated animals showed a significant increase in mean fiber area compared to the untreated animals ( Fig.13 E and Fig.13 F). For the tibialis muscle ( Fig.13 A and Fig.13 B) Gastrocnemius Fig.13 C and Fig.13 D), similar results were observed.

[0204] They then focused on NMJ phenotypes, demonstrating that the number of innervated junctions was increased in SOD1 treated animals compared to untreated animals ( Fig.14 A and Fig.14 B); and improved NMJ structure, notably a higher number of NMJs with normal endplates and a decrease in the number of fragmented and immature endplates ( Fig.14 C).

[0205] Finally, the effects of treatment on the expression levels of both MuSK and nAchR were milder than those of preventive treatment ( Fig.15 ), but improved as in the gastrocnemius ( Fig.15 D).

[0206] Taken together, these data strongly support CYP46A1 as a relevant target in ALS. The inventors then decided to evaluate whether increasing doses of AAV-CYP46A1 could still improve the beneficial effects and used therapeutic treatment with 5.10 11 vg was injected into cohorts of SOD1G93A animals. Preliminary behavioral results showed a strong effect, with significant improvements in behavior as measured by clasping scores ( Fig.16 ). Mice are at 12 weeks and will be evaluated for survival.

[0207] Finally, the inventors claim that CYP46A1 may be a relevant target for both familial and sporadic forms of ALS, thus targeting not only patients with SOD1 mutations, but also patients with C9ORF72 mutations in TARDP patients. For this purpose, the inventors decided to test their therapeutic strategy on other models of ALS, the first being a C9ORF72 mouse model with 500 repeats of GGGGCC.

[0208] In the C9ORF72 model, the inventors administered 5.10 11 vg AAV-CYP46A1-HA was used as a preventive treatment and has been followed up to 16 weeks. No differences in growth curves were observed ( Fig.17 A). Based on the notched bar test, significant improvements in athletic performance have been demonstrated ( Fig.17 B) Tracking of mice is ongoing and survival will be assessed.

[0209] Overall, these data support that overexpression of CYP46A1 has therapeutic properties, promoting improvements in neuronal physiology, particularly autophagy, in in vitro models of ALS ( Fig.18 ), and data on post-symptomatic delivery of treatments after symptoms have already appeared are encouraging for ALS treatment.

[0210] References

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Sequence Listing <110> INSERM <120> Expression vector for cholesterol 24-hydroxylase for use in amyotrophic lateral sclerosis therapy <130> B2868 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 1503 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(1503) <400> 1 atg agc ccc ggg ctg ctg ctg ctc ggt agc gcc gtc ctg ctc gcc ttc 48 ggc ctc tgc tgc acc ttc gtg cac cgc gct cgc agc cgc tac gag cac 96 atc ccc ggg ccg ccg cgg ccc agt ttc ctt cta gga cac ctc ccc tgc 144 ttt tgg aaa aag gat gag gtt ggt ggc cgt gtg ctc caa gat gtg ttt 192 ttg gat tgg gct aag aag tat gga cct gtt gtg cgg gtc aac gtc ttc 240 cac aaa acc tca gtc atc gtc acg agt cct gag tcg gtt aag aag ttc 288 ctg atg tca acc aag tac aac aag gac tcc aag atg tac cgt gcg ctc 336 cag act gtg ttt ggt gag aga ctc ttc ggc caa ggc ttg gtg tcc gaa 384 tgc aac tat gag cgc tgg cac aag cag cgg aga gtc ata gac ctg gcc 432 ttc agc cgg agc tcc ttg gtt agc tta atg gaa aca ttc aac gag aag 480 gct gag cag ctg gtg gag att cta gaa gcc aag gca gat ggg cag acc 528 cca gtg tcc atg cag gac atg ctg acc tac acc gcc atg gac atc ctg 576 gcc aag gca gct ttt ggg atg gag acc agt atg ctg ctg ggt gcc cag 624 aag cct ctg tcc cag gca gtg aaa ctt atg ttg gag gga atc act gcg 672 tcc cgc aac act ctg gca aag ttc ctg cca ggg aag agg aag cag ctc 720 cgg gag gtc cgg gag agc att cgc ttc ctg cgc cag gtg ggc agg gac 768 tgg gtc cag cgc cgc cgg gaa gcc ctg aag agg ggc gag gag gtt cct 816 gcc gac atc ctc aca cag att ctg aaa gct gaa gag gga gcc cag gac 864 gac gag ggt ctg ctg gac aac ttc gtc acc ttc ttc att gct ggt cac 912 gag acc tct gcc aac cac ttg gcg ttc aca gtg atg gag ctg tct cgc 960 cag cca gag atc gtg gca agg ctg cag gcc gag gtg gat gag gtc att 1008 ggt tct aag agg tac ctg gat ttc gag gac ctg ggg aga ctg cag tac 1056 ctg tcc cag gtc ctc aaa gag tcg ctg agg ctg tac cca cca gca tgg 1104 ggc acc ttt cgc ctg ctg gaa gag gag acc ttg att gat ggg gtc aga 1152 gtc ccc ggc aac acc ccg ctc ttg ttc agc acc tat gtc atg ggg cgg 1200 atg gac aca tac ttt gag gac ccg ctg act ttc aac ccc gat cgc ttc 1248 ggc cct gga gca ccc aag cca cgg ttc acc tac ttc ccc ttc tcc ctg 1296 ggc cac cgc tcc tgc atc ggg cag cag ttt gct cag atg gag gtg aag 1344 gtg gtc atg gca aag ctg ctg cag agg ctg gag ttc cgg ctg gtg ccc 1392 ggg cag cgc ttc ggg ctg cag gag cag gcc aca ctc aag cca ctg gac 1440 ccc gtg ctg tgc acc ctg cgg ccc cgc ggc tgg cag ccc gca ccc cca 1488 cca ccc ccc tgc tga 1503 <210> 2 <211> 500 <212> PRT <213> Homo sapiens <400> 2 Met Ser Pro Gly Leu Leu Leu Leu Gly Ser Ala Val Leu Leu Ala Phe 1 5 10 15 Gly Leu Cys Cys Thr Phe Val His Arg Ala Arg Ser Arg Tyr Glu His 20 25 30 Ile Pro Gly Pro Pro Arg Pro Ser Phe Leu Leu Gly His Leu Pro Cys 35 40 45 Phe Trp Lys Lys Asp Glu Val Gly Gly Arg Val Leu Gln Asp Val Phe 50 55 60 Leu Asp Trp Ala Lys Lys Tyr Gly Pro Val Val Arg Val Asn Val Phe 65 70 75 80 His Lys Thr Ser Val Ile Val Thr Ser Pro Glu Ser Val Lys Lys Phe 85 90 95 Leu Met Ser Thr Lys Tyr Asn Lys Asp Ser Lys Met Tyr Arg Ala Leu 100 105 110 Gln Thr Val Phe Gly Glu Arg Leu Phe Gly Gln Gly Leu Val Ser Glu 115 120 125 Cys Asn Tyr Glu Arg Trp His Lys Gln Arg Arg Val Ile Asp Leu Ala 130 135 140 Phe Ser Arg Ser Ser Leu Val Ser Leu Met Glu Thr Phe Asn Glu Lys 145 150 155 160 Ala Glu Gln Leu Val Glu Ile Leu Glu Ala Lys Ala Asp Gly Gln Thr 165 170 175 Pro Val Ser Met Gln Asp Met Leu Thr Tyr Thr Ala Met Asp Ile Leu 180 185 190 Ala Lys Ala Ala Phe Gly Met Glu Thr Ser Met Leu Leu Gly Ala Gln 195 200 205 Lys Pro Leu Ser Gln Ala Val Lys Leu Met Leu Glu Gly Ile Thr Ala 210 215 220 Ser Arg Asn Thr Leu Ala Lys Phe Leu Pro Gly Lys Arg Lys Gln Leu 225 230 235 240 Arg Glu Val Arg Glu Ser Ile Arg Phe Leu Arg Gln Val Gly Arg Asp 245 250 255 Trp Val Gln Arg Arg Arg Glu Ala Leu Lys Arg Gly Glu Glu Val Pro 260 265 270 Ala Asp Ile Leu Thr Gln Ile Leu Lys Ala Glu Glu Gly Ala Gln Asp 275 280 285 Asp Glu Gly Leu Leu Asp Asn Phe Val Thr Phe Phe Ile Ala Gly His 290 295 300 Glu Thr Ser Ala Asn His Leu Ala Phe Thr Val Met Glu Leu Ser Arg 305 310 315 320 Gln Pro Glu Ile Val Ala Arg Leu Gln Ala Glu Val Asp Glu Val Ile 325 330 335 Gly Ser Lys Arg Tyr Leu Asp Phe Glu Asp Leu Gly Arg Leu Gln Tyr 340 345 350 Leu Ser Gln Val Leu Lys Glu Ser Leu Arg Leu Tyr Pro Pro Ala Trp 355 360 365 Gly Thr Phe Arg Leu Leu Glu Glu Glu Thr Leu Ile Asp Gly Val Arg 370 375 380 Val Pro Gly Asn Thr Pro Leu Leu Phe Ser Thr Tyr Val Met Gly Arg 385 390 395 400 Met Asp Thr Tyr Phe Glu Asp Pro Leu Thr Phe Asn Pro Asp Arg Phe 405 410 415 Gly Pro Gly Ala Pro Lys Pro Arg Phe Thr Tyr Phe Pro Phe Ser Leu 420 425 430 Gly His Arg Ser Cys Ile Gly Gln Gln Phe Ala Gln Met Glu Val Lys 435 440 445 Val Val Met Ala Lys Leu Leu Gln Arg Leu Glu Phe Arg Leu Val Pro 450 455 460 Gly Gln Arg Phe Gly Leu Gln Glu Gln Ala Thr Leu Lys Pro Leu Asp 465 470 475 480 Pro Val Leu Cys Thr Leu Arg Pro Arg Gly Trp Gln Pro Ala Pro Pro 485 490 495 Pro Pro Pro Cys 500 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 3 tcctgagcgc aagtactctg t 21 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 ctgatccaca tctgctggaa g 21 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> AGA TCA TTG TCA CTC ACT TTC CCT <400> 5 agatcattgt cactcacttt ccct 24 <210> 6 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 6 acgaagtggt aggtgatgtc cag 23 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 7 tcatcaccac gcctcttgaa ac 22 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 8 catcatcact gtcttccacg ctc 23 <210> 9 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 9 ggctaagaag tatggtcctg ttgtaaga 28 <210> 10 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 10 ggtggacatc aggaacttct tgact 25 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 11 gtcacattgc tgacaggatg ccta 24 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 12 gggttggttg ctttgccact c 21 <210> 13 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 13 ccccacattc actcttgacg ctct 24 <210> 14 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 14 gctggcggac gcctgacat 19 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 15 ggtccagcag gtcccagttg t 21 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 16 ctgcagtctt cacggtggct c 21 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 tgttgacgca gacagccaat g 21 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 cgctctctgc tcctcctgtt 20 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 19 ccatggtgtc tgagcgatgt 20 <210> 20 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 20 cgagtcctga gtcggttaag aagtt 25 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty one agtctggagc gcacggtaca t 21 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty two ccacctctcc tatgggcaga 20 <210> twenty three <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty three ccgggccttt tcaatgtct 19

Claims

1. Use of a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase in the preparation of a medicament for treating amyotrophic lateral sclerosis (ALS).

2. The use according to claim 1, wherein The ALS is associated with at least one motor neuron-related disorder.

3. The use according to claim 2, wherein The ALS is associated with frontotemporal dementia.

4. The use according to any one of claims 1 to 3, wherein The amino acid sequence of the cholesterol 24-hydroxylase is shown as SEQ ID NO 2.

5. The use according to claim 4, wherein The nucleotide sequence of the nucleic acid is shown as SEQ ID NO 1.

6. The use according to claim 1, wherein The vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes virus vector and an adeno-associated virus (AAV) vector.

7. The use according to claim 6, wherein The vector is an AAV vector.

8. The use according to claim 7, wherein The AAV vector is an AAV9 vector, an AAV10 vector or an AAVPHP.eB vector.

9. The use according to claim 1, wherein The vector will be administered intravenously or directly into the patient's brain.

10. The use according to claim 9, wherein The vector will be administered to the spinal cord and / or motor cortex.

11. The use according to claim 10, wherein The vector is administered by stereotactic microinjection.

12. The use according to claim 1, wherein The vector will be administered by intravascular, intranasal, intracerebroventricular or intrathecal injection.

13. The use according to claim 1, wherein The vector will be administered by intravenous injection.

14. Use of a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase in the preparation of a pharmaceutical composition for treating amyotrophic lateral sclerosis.

15. Use of a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase in the preparation of a medicament for the prophylactic treatment of amyotrophic lateral sclerosis (ALS) in a pre-symptomatic subject.

16. The use according to claim 15, wherein The subject had familial ALS before the onset of symptoms.

17. The use according to claim 15 or 16, wherein The amino acid sequence of the cholesterol 24-hydroxylase is shown as SEQ ID NO 2.

18. The use according to claim 15, wherein The nucleotide sequence of the nucleic acid is shown as SEQ ID NO 1.

19. The use according to claim 15 or 16, wherein The vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes virus vector and an adeno-associated virus (AAV) vector.

20. The use according to claim 19, wherein The vector is an AAV vector.

21. The use according to claim 20, wherein The AAV vector is an AAV9 vector, an AAV10 vector or an AAVPHP.eB vector.

22. The use according to claim 15 or 16, wherein The vector will be administered intravenously or directly into the patient's brain.

23. The use according to claim 22, wherein The vector will be administered to the spinal cord and / or motor cortex.

24. The use according to claim 23, wherein The vector is administered by stereotactic microinjection.

25. The use according to claim 15 or 16, wherein The vector will be administered by intravascular, intranasal, intracerebroventricular or intrathecal injection.

26. The use according to claim 15 or 16, wherein The vector will be administered by intravenous injection.

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