Gene therapy using nucleic acid constructs containing the methyl CPG-binding protein 2 (MECP2) promoter sequence.

Abstract

This invention provides a promoter sequence that can be used to extend the length of a viral vector construct while simultaneously achieving robust central nervous system (CNS) target expression of progranulin (PGRN). [Solution] The present invention relates to a nucleic acid construct comprising a methyl CpG-binding protein 2 (MeCP2) promoter sequence. The present invention further relates to vectors, viral vectors, host cells, and pharmaceutical compositions comprising the nucleic acid construct. The present invention also relates to the therapeutic use of the nucleic acid construct, vectors, viral vectors, and pharmaceutical compositions.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid construct comprising a methyl CpG-binding protein 2 (MeCP2) promoter sequence. The present invention further relates to vectors, viral vectors, host cells, and pharmaceutical compositions comprising the nucleic acid construct. The present invention also relates to the therapeutic use of the nucleic acid construct, vectors, viral vectors, and pharmaceutical compositions. [Background technology]

[0002] Frontotemporal dementia (FTD) is the second most common type of dementia after Alzheimer's disease (Olney et al. Neurol. Clin. 2017 May;35(2):339-374). Mutations in one allele of the GRN gene, which encodes the protein progranulin (PGRN), are associated with the development of FTD (Baker et al., Nature. 2006 Aug 24;442(7105):916-919). Homozygous mutations in GRN are associated with neuronal ceroid lipofuscinosis 11 (NCL11), which is characterized by cerebellar ataxia, seizures, retinitis pigmentosa, and cognitive impairment, and usually begins between the ages of 13 and 25 (Faber et al. Brain. 2020;143(1):303-31).

[0003] Various mutations can lead to loss of PGRN function. In PGRN-deficient mouse models, driving neuronal expression of PGRN using an AAV gene therapy approach has been shown to modify behavioral disorders associated with FTD (Arrant et al. Brain. 2017;140.5:1447-1465). Therefore, there is strong biological evidence for therapeutic approaches that increase PGRN levels in central nervous system (CNS) tissues and cells to treat neurological disorders associated with PGRN deficiency.

[0004] Adeno-associated virus (AAV) vectors are commonly used vehicles for delivering molecular therapeutics to treat clinical disorders. Many AAV-based therapies are gene replacement therapies. However, to achieve robust AAV generation and transgene expression, the AAV construct containing the transgene of interest should be 4.1kb–4.7kb in size to allow for optimal AAV packaging. So-called "stuffer sequences" or inactive DNA can be added to the transgene or vector backbone to extend the full length of the construct. However, since vectors are sensitive to stuffer sequences, they must be carefully selected to avoid adverse effects on transgene expression, patient immune response, and AAV packaging efficiency. Another approach to incorporating the full length into the AAV construct is to modify the transgene sequence itself. However, this approach may not be suitable when it is desirable to use the natural (wild-type) transgene nucleotide sequence.

[0005] A further approach to extending the full length of the AAV construct is to include engineered promoter sequences. Such promoters must be carefully selected to ensure appropriate in vivo transgene expression levels. Furthermore, when site-specific transgene expression is required to treat neurological disorders, as in PGRN gene therapy, the selection of promoters that achieve targeted expression of the transgene of interest in the desired tissue or cell type is crucial.

[0006] Typically, the nucleotide sequence encoding the PGRN coding sequence is about 1.8 kb long, which is considerably shorter than the optimal 4.1–4.7 kb length for packaging the nucleic acid construct in AAV. Therefore, there is still a need for a promoter sequence that can be used to extend the length of the viral vector construct while simultaneously achieving robust CNS target expression of the PGRN. [Overview of the project]

[0007] Promoters derived from the methyl-CpG binding protein 2 (MeCP2) gene have been found to be highly effective in driving CNS-targeted expression of PGRNs in gene therapy. Such promoters were observed to achieve higher PGRN expression and transduction efficiency than equivalent promoters, including alternative CNS-specific promoters such as those derived from the neuron-specific enolase 1 (NSE1) gene.

[0008] Furthermore, the inventors generated an engineered MeCP2 promoter exceeding 2000 bp. In addition to the minimal MeCP2 promoter sequence, the engineered MeCP2 promoter includes an additional intron. The nucleotide sequence of this intron was either derived from a naturally occurring stretch of the MECP2 gene (natural intron) or constructed by combining different sequences derived from the MECP2 gene (synthetic intron). Gene therapy constructs containing the engineered MeCP2 promoter of the present invention were found to achieve higher expression levels and / or improved transduction efficiency in CNS cells compared to constructs containing the minimal promoter. Furthermore, MeCP2 promoters containing synthetic introns were found to achieve the highest expression levels and transduction efficiency.

[0009] Therefore, the present invention provides a nucleic acid construct comprising a methyl CpG-binding protein 2 (MeCP2) promoter operably linked to a nucleotide sequence encoding a progranulin (PGRN) protein.

[0010] Furthermore, the present invention provides a nucleic acid construct comprising an engineered methyl CpG-binding protein 2 (MeCP2) promoter operably linked to a nucleotide sequence encoding a protein of interest (POI), wherein the engineered MeCP2 promoter comprises a minimal promoter sequence and at least one intron.

[0011] The present invention further provides a vector comprising the nucleic acid construct of the present invention. The vector may be a plasmid or a viral vector.

[0012] The present invention further provides a host cell comprising the nucleic acid construct and / or the vector of the present invention and / or a host cell that produces the viral vector of the present invention, the host cell being optionally HEK293 cell or HEK293T cell.

[0013] Further provided by the present invention are pharmaceutical compositions comprising the nucleic acid construct of the present invention, the vector of the present invention, and / or the viral vector of the present invention together with a pharmaceutically acceptable carrier, excipient, or diluent.

[0014] Furthermore, the present invention provides nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention, which are used in methods for treating or preventing diseases characterized by progranulin (PGRN) deficiency in patients requiring treatment or prevention of such diseases.

[0015] The present invention further provides a method for treating or preventing a disease characterized by logranulin (PGRN) deficiency in a patient requiring treatment or prevention of said disease, comprising administering to the patient a therapeutically effective amount of the nucleic acid construct of the present invention, the vector of the present invention, the viral vector of the present invention, and / or the pharmaceutical composition of the present invention.

[0016] Furthermore, the present invention provides for the use of the nucleic acid constructs of the present invention, the vectors of the present invention, the viral vectors of the present invention, and / or pharmaceutical compositions of the present invention for the manufacture of pharmaceuticals for treating or preventing diseases characterized by progranulin (PGRN) deficiency in patients requiring treatment or prevention of such diseases. [Brief explanation of the drawing]

[0017] [Figure 1]A. Schematic diagram showing the composition of constructs pAK169, pPG21, pPG35, and pPG36. MeCP2 (250 bp) shows the minimal MeCP2 promoter sequence. GFP shows the gene encoding green fluorescent protein. 5'MeCP2 (2100 bp) shows a native intron of approximately 2100 bp on the 5' side of MeCP2 (250 bp). PGRN (1800 bp) shows the polynucleotide sequence encoding PGRN. Intron (2100 bp) shows a synthetic intron sequence of approximately 100 bp. B. Images of Western blot analysis of promoter activity. PGRN expression was evaluated for each of pAK169, pPG21, pPG35, and pPG36. [Figure 2] A. Schematic diagram showing the composition of pAK168, pPG20, pPG33, and pPG34. NSE1 (1300 bp) shows the minimal NSE1 promoter sequence. GFP shows the gene encoding green fluorescent protein. 5'NSE1 (1100 bp) shows a native intron of approximately 1100 bp on the 5' side of NSE1 (250 bp). PGRN (1800 bp) shows the polynucleotide sequence encoding PGRN. Intron (900 bp) shows a synthetic intron sequence of approximately 900 bp. B. Image of Western blot analysis of promoter activity. PGRN expression was evaluated for each of pAK168, pPG20, pPG33, and pPG34. [Figure 3] Evaluation of PGRN expression in primary neurons and astrocytes for constructs pPG20, pPG33, pPG34, pPG21, pPG21, pPG35, and pPG36. A. Bar graph showing (A) transduction efficiency in neurons; (B) PGRN expression level in transduced neurons; (C) transduction efficiency in astrocytes; and (D) PGRN expression level in transduced astrocytes. [Figure 4] Evaluation of PGRN secretion by primary neurons and astrocytes. The bar graph shows the concentration of PGRN secreted by neuron-astrocyte co-cultures transduced with constructs pPG21, pPG35, pPG36, pPG20, and pPG26. A control without transduction is also shown. [Figure 5] Codon optimization of nucleic acid constructs encoding PGRN. A. Bar graph showing the expression levels of PGRN in GRN− / − HAP-1 cells transfected with a lentiviral vector encoding PGRN, determined by ELISA. Vectors containing codon-optimized nucleotide sequences encoding PGRN (designated CpG0, 4, 9, 17, 25, 40, 71, and 90) were compared with vectors containing the wild-type nucleotide sequence encoding PGRN (designated WT). Also shown are control transfection with an empty vector and PGRN expression levels in WT HAP-1 cells (GRN+ / +). B. Image of Western blot analysis of PGRN expression levels in GRN− / − HAP-1 cells transfected with a lentiviral vector containing a codon-optimized nucleotide sequence encoding PGRN (designated CpG25, 40, 71, and 90) and a vector containing the wild-type nucleotide sequence encoding PGRN (designated WT). Also shown are PGRN expression levels for control transfection with an empty vector (designated mock), non-transfected wild-type GRN+ / + HAP-1 cells (designated WT), and non-transfected GRN− / − HAP-1 cells (designated KO). [Figure 6] Expression of human PGRN corrects lysosomal defects in GRN− / − mouse primary neurons. A. Image of Western blot analysis performed to quantify the levels of the lysosomal protein cathepsin D in WT (GRN+ / +) and KO (GRN− / −) primary neurons transduced with a lentiviral vector containing the pPG36 construct. B. Bar graph showing the levels of cathepsin D protein (immature, mature heavy chain, and mature light chain, respectively). Values for cathepsin D expression are normalized to the expression levels of actin and GADPH. [Figure 7]ELISA analysis and FRET analysis of human progranulin (hPGRN) CNS expression in WT mice and GRN− / − mice after striatal injection of AAVTT-p1PG36. A. Bar graph showing CSF levels and plasma levels of hPGRN (ng / ml) measured by ELISA. High levels of hPGRN were detected in CSF (1:100 dilution) of both WT mice and GRN− / − mice in animals injected with AAVTT-p1PG36 (AAVTT vector containing the pPG36 construct). Also, hPGRN was detected in mouse plasma (1:10 dilution). B. Bar graph showing the results of FRET measurements of hPGRN concentration (ng / mg) in various brain regions of WT mice or GRN− / − mice injected with AAVTT-p1PG36. The highest expression of hPGRN was detected near the injection sites (striatum and midbrain). Also, moderate levels of hPGRN expression were detected in the cortex and hippocampus. Low levels of hPGRN expression were detected in distant brain regions such as the brainstem, olfactory bulb, and cerebellum. C. Bar graph showing CSF levels (ng / ml) of hPGRN measured by ELISA after striatal injection of AAVTT-p1PG36 and AAVTT-p2PG36 in WT mice. High levels of hPGRN were detected in CSF (1:100 dilution) in animals injected with both AAV constructs. [Figure 8] Images of IHC analysis of human progranulin (hPGRN) CNS expression in GRN− / − mice after striatal injection of AAVTT-p1PG36. IHC staining of hPGRN was observed in the brains of GRN− / − KO mice that received striatal administration of AAVTT-p1PG36. The immunoreactive signal was specific to human progranulin as no signal was observed in mice that received vehicle or control AAV-GFP. High levels of hPGRN were detected mainly throughout the forebrain of GRN− / − KO mice, particularly in the striatum, thalamus, hypothalamus, cerebral cortex, and hippocampus, as well as in the midbrain and substantia nigra. [Figure 9]Human PGRN expression affects cathepsin D activity in vivo. The bar graph shows the measurement of cathepsin D enzyme activity in midbrain lysates of WT (GRN+ / +) mice treated with vehicle (indicated by black circles) and GRN- / - KO mice treated with vehicle (black circles) or AAVTT-p1PG36 (black triangles). Increased cathepsin D enzyme activity was observed in 4-month-old GRN- / - mice. Decreased cathepsin D activity was observed in GRN- / - mice injected with AAVTT-p1PG36 compared to mice injected with vehicle. [Figure 10] A schematic diagram showing the composition of the constituent nucleic acid sequences in the AAVTT-pPG36 construct (SEQ ID NO: 17). [Figure 11] A schematic diagram showing the location of the constituent region of the MeCP2_2 intron (SEQ ID NO: 2) within the full-length mouse MECP2 gene.

[0018] A brief explanation of arrays Sequence ID 1 is the nucleotide sequence of the MeCP2 minimal promoter.

[0019] Sequence ID 2 is the nucleotide sequence of the MeCP2_2 intron.

[0020] Sequence ID 3 is the nucleotide sequence of the MeCP2_2 promoter.

[0021] Sequence ID 4 is the nucleotide sequence of exon 1 of the MeCP2_2 intron.

[0022] Sequence ID 5 is the nucleotide sequence of the 5' intron of the MeCP2_2 intron.

[0023] Sequence ID 6 is the nucleotide sequence of the 3' intron of the MeCP2_2 intron.

[0024] Sequence ID 7 is the nucleotide sequence of exon 2 of the MeCP2_2 intron.

[0025] Sequence ID 8 is the nucleotide sequence of the MeCP2_1 promoter.

[0026] Sequence ID 9 is the nucleotide sequence of the MeCP2_1 intron.

[0027] Sequence IDs 10 and 11 are the nucleotide sequences of constructs pPG35 and pPG36, respectively.

[0028] Sequence IDs 12 and 13 correspond to the human PGRN nucleotide sequence and amino acid sequence, respectively.

[0029] Sequence ID 14 is the nucleotide sequence of the Age1 restriction site (5'-ACCGGT-3').

[0030] Sequence ID 15 is the nucleotide sequence of the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0031] Sequence ID 16 is the nucleotide sequence of the SV40 polyadenylated (poly(A) signaling) sequence.

[0032] Sequence ID 17 is the nucleotide sequence of the AAVTT-pPG36 construct.

[0033] Sequence ID 18 is the nucleotide sequence of the AAVTT-p1PG36 plasmid.

[0034] Sequence ID 19 is the nucleotide sequence of the AAVTT-p2PG36 plasmid.

[0035] Sequence ID 20 is the nucleotide sequence of the 5'ITR used in the AAVTT-pPG36 construct.

[0036] Sequence ID 21 is the nucleotide sequence of the 5' adjacent fragment used in the AAVTT-pPG36 construct.

[0037] Sequence ID 22 is the nucleotide sequence of the 3' adjacent fragment used in the AAVTT-pPG36 construct.

[0038] Sequence ID 23 is the nucleotide sequence of the 3'ITR used in the AAVTT-pPG36 construct.

[0039] Sequence ID 24 is the nucleotide sequence of the Kozak sequence used in the AAVTT-pPG36 construct. [Modes for carrying out the invention]

[0040] In this specification, all publications, patents, and patent applications cited, whether above or below, are incorporated by reference in their entirety.

[0041] definition In this specification and the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to "a nucleic acid" includes "multiple nucleic acids."

[0042] The term "comprise" or "comprising" should be understood to have its usual meaning in the art, that is, it includes the features or group of features described, but does not exclude the existence of any other features or groups of features described. For example, a promoter containing a minimal promoter sequence may contain one or more other components, such as introns. Similarly, the term "consists of" should be understood to have its usual meaning in the art, that is, it includes the features or group of features described, excluding any further features. For example, a promoter consisting of a minimal promoter sequence contains a minimal promoter sequence and does not contain any other components. For all embodiments in which "comprises" or "comprising" is used, further embodiments in which "consists of" or "consisting of" is used are expected. Therefore, all disclosures of "comprises" should be considered disclosures of "consists of".

[0043] The terms “protein” and “polypeptide” are used interchangeably herein and, in their broadest sense, refer to compounds of two or more subunit amino acids, amino acid analogs, or other peptide mimetic compounds. Thus, the term “protein” includes both short peptide sequences and longer polypeptides. As used herein, the term “amino acid” refers to natural and / or unnatural amino acids, or synthetic amino acids, including both D and L optical isomers, as well as amino acid analogs and peptide mimetic compounds.

[0044] The terms "patient" and "subject" are used interchangeably within this specification. Typically, a patient is a human being.

[0045] Sequence homology / identity Furthermore, while sequence homology may be considered in terms of functional similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of this document, homology is preferred to be expressed in terms of sequence identity.

[0046] Sequence comparison can be performed visually or, more commonly, with the help of readily available sequence comparison programs. These publicly or commercially available computer programs can calculate the percentage of homology (or identity, etc.) between two or more sequences.

[0047] The percentage of identity can be calculated over the entire sequence, that is, by aligning one sequence with the other and directly comparing each amino acid in one sequence with the corresponding amino acid in the other, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are performed only over a relatively small number of residues (e.g., fewer than 50 consecutive amino acids). For comparisons over longer sequences, gap scoring is used to generate the best alignment that accurately reflects the level of identity in related sequences that have insertions or deletions relative to each other. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Other software capable of performing sequence comparisons includes, but is not limited to, the BLAST package, FASTA (Altschul et al., 1990, J.Mol.Biol.215:403-410), and the GENEWORKS comparison tools suite.

[0048] Typically, sequence comparison is performed over the entire length of the reference sequence. For example, if a user wants to determine if a given sequence is 70% identical to sequence number 2, then sequence number 2 is the reference sequence. For example, to evaluate whether a sequence is at least 90% identical to sequence number 2 (an example of a reference sequence), a person skilled in the art would perform an alignment over the entire length of sequence number 2 to identify how many positions in the test sequence were identical to the positions in sequence number 2. If at least 70% of the positions are identical, then the test sequence is at least 70% identical to sequence number 2. If the sequence is shorter than sequence number 27, any gaps or missing positions should be considered non-identical positions.

[0049] Those skilled in the art will be aware of the various computer programs available for determining homology or identity between two sequences. For example, the comparison of two sequences and the determination of their identity percentage can be achieved using mathematical algorithms. In one embodiment, the identity percentage between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm, which is incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM 250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0050] As used herein, the term "fragment" refers to a contiguous portion of a reference sequence. For example, a 50-nucleotide fragment of SEQ ID NO: 2 refers to the 50 consecutive nucleotides of SEQ ID NO: 2.

[0051] As used herein, the term "functional variant" refers to a nucleic acid or amino acid sequence that is modified compared to a reference sequence but retains the function of the reference sequence. For example, a functional variant of the MeCP2 promoter retains the ability to drive the expression of nucleotide sequences encoding POI in CNS cells such as neurons or astrocytes. Similarly, a functional variant of a PGRN protein retains the activity of the reference PGRN protein.

[0052] nucleic acid The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or substantially isolated forms. Substantially isolated means that the polypeptide may be substantially isolated, but not completely isolated, from any surrounding medium. Polynucleotides may be mixed with a carrier or diluent that does not interfere with their intended use and may still be considered substantially isolated. The nucleic acid sequence that “encodes” the selected polypeptide is a nucleic acid molecule that is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA) under the control of an appropriate regulatory sequence, for example, when placed in an expression vector. The coding sequence boundary is determined by the 5' (amino) terminal start codon and the 3' (carboxy) terminal translation termination codon. For the purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, or synthetic DNA sequences. The transcription termination sequence may be located on the 3' side of the coding sequence.

[0053] Polynucleotides can be synthesized according to methods well known in the art, such as those described, for example, in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0054] As used herein, the term “nucleic acid construct” refers to an artificial nucleic acid (e.g., produced or synthesized by recombination) comprising at least one regulatory sequence (e.g., a promoter) and at least one nucleotide sequence encoding a protein of interest (POI). Thus, the nucleic acid construct of the present invention can be considered an expression cassette. The nucleic acid construct of the present invention may be isolated or substantially isolated. Typically, the nucleic acid construct of the present invention includes a regulatory sequence (e.g., a MeCP2 promoter) operably linked to a nucleotide sequence encoding a protein of interest (e.g., PGRN), thereby enabling in vivo expression of the protein of interest. The nucleic acid construct of the present invention may also include appropriate promoters, enhancers, initiators, and other elements, such as polyadenylation (polyA) signaling and / or woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequences. Furthermore, the nucleic acid construct of the present invention may include nucleotide sequences that facilitate genetic manipulation, such as restriction sites (e.g., an Age1 restriction site having the nucleotide sequence of SEQ ID NO: 14).

[0055] As used herein, the term “operatably linked” refers to the juxtaposition of two or more nucleotide sequences such that each of the two or more sequences is able to perform its normal function. Typically, the term “operatably linked” is used to refer to the juxtaposition of a regulatory element (e.g., a promoter, enhancer, poly(A) signaling sequence, WPRE sequence, etc.) and a nucleotide sequence encoding the protein of interest (POI). For example, an operable linkage between a promoter and a protein-coding nucleotide sequence allows the promoter to function to drive the expression of the POI in vivo.

[0056] In addition to the MeCP2 promoter, the nucleic acid construct of the present invention may include one or more further regulatory elements. Preferred regulatory elements function to stabilize mRNA transcribed from the nucleic acid construct and / or to enhance the expression of a protein of interest (POI), such as PGRN, from the nucleic acid construct.

[0057] A preferred regulatory element that can be used in the nucleic acid construct of the present invention is the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). A WPRE is a DNA sequence that, when transcribed to mRNA, enhances mRNA stability and the expression of POI encoded by the nucleic acid construct by introducing a tertiary structure to the mRNA transcript. In the nucleic acid construct of the present invention, the WPRE may be located at the 3' end of the nucleotide sequence encoding the POI or PGRN protein. The WPRE may include the nucleotide sequence of SEQ ID NO: 15, or a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of SEQ ID NO: 15. The functional variant or fragment of the WPRE retains the characteristics of the corresponding non-variant or full-length WPRE. Therefore, variants or fragment WPREs may be able to impart tertiary structure to mRNA transcripts, and / or enhance the stability of mRNA transcripts, and / or enhance the expression of POIs encoded by the nucleic acid construct. The enhancement is compared to mRNA without the variant or fragment WPRE.

[0058] A preferred regulatory element that can be used in the nucleic acid construct of the present invention is a polyadenylation (poly(A)) signal sequence. In eukaryotic cells, the polyadenylation signal sequence in the mRNA transcript is recognized and processed to add a poly(A) tail consisting of multiple adenosine monophosphates to the 3' end of the mRNA transcript. The poly(A) tail functions to enhance the expression of POI encoded by the nucleic acid construct by promoting the transport of mRNA from the nucleus to the cytoplasm and preventing mRNA degradation. In the nucleic acid construct of the present invention, the polyadenylation signal sequence may be located on the 3' side of the nucleotide sequence encoding the POI or PGRN protein. The polyadenylation signal sequence may include the nucleotide sequence of SEQ ID NO: 16, or a functional variant or fragment thereof having at least 90% identity with the nucleotide sequence of SEQ ID NO: 16. The functional variant or fragment of the polyadenylation sequence retains the characteristics of the corresponding non-variant or full-length polyadenylation signal sequence.

[0059] The nucleic acid construct of the present invention may include, in the 5' to 3' direction, a nucleotide sequence encoding a MeCP2 promoter, a POI or PGRN protein, a WPRE, and a polyadenylation signal sequence.

[0060] The nucleic acid constructs of the present invention may be provided within a vector (e.g., a plasmid or recombinant viral vector). A suitable vector may be any vector that has a sufficient amount of genetic information and can enable in vivo expression of the POI. A vector containing the nucleic acid constructs of the present invention may be administered directly to a patient in need. Such vectors are routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid DNA and suitable initiators, promoters, enhancers, and other elements, such as a polyadenylation signal that may be essential and is oriented correctly to enable the expression of the peptide of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples therein, see Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0061] Methyl CpG-binding protein 2 (MeCP2) promoter Methyl CpG-binding protein 2 (MeCP2) is a transcriptional repressor, and it is hypothesized that it silences gene transcription by binding to methylated cytosine nucleotides within the gene's promoter and then recruiting a co-repressor protein complex. In addition, MeCP2 binds to DNA methyltransferase 1 and regulates histone methyltransferase activity. This helps maintain DNA methylation and promotes the methylation of Lys9 in histone H3. Therefore, by binding to methylated DNA, MeCP2 enhances its repressive function by maintaining DNA methylation and by promoting multiple epigenetic modifications such as histone deacetylation and methylation.

[0062] MeCP2 is highly expressed in the brain, lungs, and spleen, and moderately expressed in the heart and kidneys. In particular, within the central nervous system (CNS), MeCP2 is highly expressed in neurons.

[0063] The human MECP2 gene (gene ID: 4204) is approximately 122 kbp long, located on the long arm of the X chromosome (Xq28), and contains four coding exons (Singh et al. Nucleic Acids Research. (2008) Vol.36, No.19 6035-6047). The mouse MECP2 gene (gene ID: 17257) is approximately 59 kbp long, located on the mouse X chromosome at position ChrX:73070198-73129296 bp (-strand).

[0064] Two MeCP2 isoforms, MeCP2_e1(e1) and MeCP2_e2(e2), have been identified. The e1 isoform is 498 amino acids long and encoded by exons 1, 3, and 4. The e2 isoform is 486 amino acids long and encoded by exons 2, 3, and 4. The promoter regions of the mouse and human MECP2 genes have been characterized, in particular, by Adachi et al. (Hum.Mol.Genetics.2005;14(23):3709-3722). The MECP2 gene segment (-677 / +56) was found to exhibit strong promoter activity in neuronal cell lines and cortical neurons, but was inactive in non-neuronal cells and glial cells. The region essential for neuron-specific promoter activity (called the MR element) was observed to be located within a 19 bp region (-63 / -45).

[0065] As described by Adachi et al. (Hum.Mol.Genetics.2005;14(23):3709-3722), the sequence of the mouse (-677 / +56) region of the MECP2 gene is 68% similar to the corresponding human MeCP2 promoter. In particular, the human and mouse sequences are 92% identical between nucleotide positions -87 and +56, which contain the MR element.

[0066] The MeCP2 sequences (e.g., SEQ ID NOs: 1-9) used in the constructs described and illustrated herein are derived from the mouse MECP2 gene. However, as described above, there is a high level of sequence similarity between the minimal promoter regions of the mouse MECP2 gene and the human MECP2 gene. Furthermore, there is a very high degree of sequence identity between the mouse MR elements and the human MR elements, which is responsible for neuron-specific expression. Therefore, embodiments of the present invention, which include one or more mouse MeCP2 nucleotide sequences, are also provided in which the one or more mouse MeCP2 nucleotide sequences are replaced by corresponding human MeCP2 nucleotide sequences.

[0067] Therefore, as used herein, the term “MeCP2 promoter” refers to a nucleotide sequence of the MECP2 gene (e.g., mouse or human MECP2 gene) that can function as a promoter, that is, can drive the expression of a protein encoded by a nucleotide sequence to which the MeCP2 promoter is operably linked, by driving the transcription of the nucleotide sequence to which the MeCP2 promoter is operably linked. Typically, the MeCP2 promoter sequences used in the present invention are specific to a particular tissue or cell type. Preferably, the MeCP2 promoters used in the present invention are specific to CNS cells. More preferably, the MeCP2 promoters used in the present invention specifically drive the expression of a protein of interest (POI), such as PGRN, in neurons and / or astrocytes.

[0068] The MeCP2 promoter used in the nucleic acid construct of the present invention may be a functional variant or fragment of the MeCP2 promoter described herein. The functional variant or fragment of the MeCP2 promoter described herein may be functional in the sense that it retains the characteristics of the corresponding non-variant or full-length MeCP2 promoter. Thus, the functional variant or fragment of the MeCP2 promoter described herein retains the ability to drive the expression of a protein encoded by a nucleotide sequence by driving the transcription of the nucleotide sequence to which the functional variant or fragment is operably linked. The functional variant or fragment of the MeCP2 promoter described herein may retain specificity for a particular tissue type. For example, the functional variant or fragment of the MeCP2 promoter described herein may be specific to cells of the CNS. The functional variant or fragment of the MeCP2 promoter described herein may specifically drive the expression of a protein of interest (POI), such as PGRN, in neurons and / or astrocytes.

[0069] The MeCP2 promoter used in the present invention may include a "minimum promoter sequence," which should be understood as a nucleotide sequence of sufficient length from the promoter region of the MECP2 gene, and includes elements necessary to function as a MeCP2 promoter, that is, to drive the expression of the protein encoded by the nucleotide sequence by driving the transcription of the nucleotide sequence to which the MeCP2 promoter is operably linked.

[0070] The minimal MeCP2 promoter used in the nucleic acid construct of the present invention may be a functional variant or fragment of the minimal MeCP2 promoter described herein. The functional variant or fragment of the minimal MeCP2 promoter described herein may be functional in the sense that it retains the characteristics of the corresponding non-variant or full-length minimal MeCP2 promoter. Thus, the functional variant or fragment of the minimal MeCP2 promoter described herein may be of sufficient length, contain the elements necessary to function as a MeCP2 promoter, and drive the expression of the protein encoded by the nucleotide sequence to which the functional variant or fragment is operably linked by driving the transcription of the nucleotide sequence.

[0071] A preferred minimal promoter sequence that can be used in the MeCP2 promoter described herein may include, or consist of, the nucleotide sequence of SEQ ID NO: 1, or a functional variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of SEQ ID NO: 1. Alternatively, a fragment of SEQ ID NO: 1 or the functional variant of any length may be used as the minimal promoter sequence in the nucleic acid construct of the present invention. The minimal promoter sequence may be 160-300 bp, 170-290 bp, 180-280 bp, 190-270 bp, 200-260 bp, 210-250 bp, 220-240 bp, or about 230 bp.

[0072] The MeCP2 promoter used in the present invention may contain one or more introns. As used herein, the term "intron" refers to a non-coding nucleotide sequence within a gene. Typically, introns are transcribed from DNA to messenger RNA (mRNA) during gene transcription, but are excised from the mRNA transcript by pretranslational splicing.

[0073] The MeCP2 promoter used in the present invention may include functional variants or fragments of introns described herein. Functional variants or fragments of introns described herein may be functional in the sense that they retain the characteristics of the corresponding non-variant or full-length intron. Therefore, functional variants or fragments of introns described herein are non-coding. Furthermore, functional variants or fragments of introns described herein may retain the ability to be transcribed from DNA to mRNA and / or excised from mRNA by splicing.

[0074] A MeCP2 promoter including a minimum promoter array and introns is referred to herein as an "operated MeCP2 promoter."

[0075] The introns that can be incorporated into the MeCP2 promoter used in this invention may originate from the native non-coding regions of the MECP2 gene. Therefore, the term intron encompasses nucleotide sequences corresponding to naturally occurring contiguous nucleotide sequences in the MECP2 gene. Such introns are referred to herein as “native” introns.

[0076] A preferred intron that can be used in the MeCP2 promoter described herein includes or comprises the nucleotide sequence of SEQ ID NO: 9, or a functional variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 9. Alternatively, fragments of the intron may be used. Such fragments may be 1000-2107 bp, 1200-2100 bp, 1400-2000 bp, 1600-1900 bp, or 1700-1800 bp. Alternatively, longer nucleotide sequences containing the intron may be used.

[0077] A preferred MeCP2 promoter that can be used in the nucleic acid construct of the present invention is named MeCP2_1 (SEQ ID NO: 8). This MeCP2 promoter contains an intron having the nucleotide sequence of SEQ ID NO: 9. Therefore, the MeCP2 promoter used in the nucleic acid construct of the present invention may contain, or consist of, the nucleotide sequence of SEQ ID NO: 8, or a functional variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 8. Alternatively, a fragment of the MeCP2 promoter may be used. Such a fragment may be 1000-2336 bp, 1200-2300 bp, 1400-2200 bp, 1600-2100 bp, 1700-2000 bp, or 1800-1900 bp. Furthermore, a longer nucleotide sequence including the MeCP2 promoter may be used.

[0078] The MeCP2 promoter used in the nucleic acid construct of the present invention may include a "synthetic intron." A synthetic intron should be understood as being constructed from two or more different (e.g., distinct and discontinuous) sequences of, for example, the MECP2 gene. The two or more sequences used to prepare the synthetic intron may originate from any position in the MECP2 gene. Therefore, the synthetic intron may include the nucleotide sequence of an intron in the MECP2 gene, which is hence referred to as the "intron sequence."

[0079] In addition, the constituent nucleotide sequences of a synthetic intron do not necessarily have to originate from the intron of the MECP2 gene, but may instead originate from the exons (i.e., protein-coding nucleotides) of the MECP2 gene. Typically, the nucleotide sequences of the MECP2 exon gene are modified (e.g., by truncation, deletion, substitution, etc.) and / or placed within the synthetic intron so that the exon sequence is not expressed. Therefore, such nucleotide sequences cannot produce transcripts that can be translated into polypeptides (e.g., the MeCP2 protein or fragments thereof). Accordingly, the synthetic introns used in the MeCP2 promoters described herein may include, for example, one or more "unexpressed exon sequences" of the MECP2 gene. Appropriately, the unexpressed exon sequences may be located on the side of the intron sequence to provide a splice site. This splice site allows the synthetic intron to be excised by splicing from mRNA produced by the transcription of the nucleic acid construct containing the synthetic intron.

[0080] The synthetic introns used in the MeCP2 promoters described herein may include functional variants or fragments of the non-expressed exon sequences described herein. These functional variants or fragments of the non-expressed exon sequences may be functional in the sense that they retain the characteristics of the corresponding non-variant or full-length exon sequence. Therefore, these functional variants or fragments of the non-expressed exon sequences may retain the ability to occupy the lateral aspect of the intron sequence and may include splice sites. These can be joined together (or spliced) by exon removal.

[0081] The synthetic introns used in the MeCP2 promoters described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 intron sequences and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unexpressed exon sequences. Preferably, the synthetic intron comprises 2 intron sequences and 2 unexpressed exon sequences.

[0082] A preferred non-expressed exon sequence that can be used in the MeCP2 promoter described herein includes or comprises the nucleotide sequence of SEQ ID NO: 4, or a functional variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4. Alternatively, a fragment of the non-expressed exon sequence may be used. Alternatively, a longer nucleotide sequence containing the non-expressed exon sequence may be used.

[0083] A preferred non-expressed exon sequence that can be used in the MeCP2 promoter described herein includes or comprises the nucleotide sequence of SEQ ID NO: 7, or a functional variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7. Alternatively, a fragment of the non-expressed exon sequence may be used. Alternatively, a longer nucleotide sequence containing the non-expressed exon sequence may be used.

[0084] A preferred intron sequence that can be used in the MeCP2 promoter described herein includes or comprises the nucleotide sequence of SEQ ID NO: 5, or a functional variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 5. Alternatively, a fragment of the intron sequence may be used. Alternatively, a longer nucleotide sequence containing the intron sequence may be used.

[0085] A preferred intron sequence that can be used in the MeCP2 promoter described herein includes or comprises the nucleotide sequence of SEQ ID NO: 6, or a functional variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 6. Alternatively, a fragment of the intron sequence may be used. Alternatively, a longer nucleotide sequence containing the intron sequence may be used.

[0086] Therefore, the nucleic acid construct of the present invention is: (a) The nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; (b) The nucleotide sequence of SEQ ID NO: 5, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 5; (c) the nucleotide sequence of SEQ ID NO: 6, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 6; and / or (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 A MeCP2 promoter may include, or may consist of, a MeCP2 promoter containing at least one synthetic intron containing .

[0087] A composite intron may contain (a), (b), (c), and / or (d) in any order from 5' to 3'. A composite intron may contain (a), (b), (c), and / or (d) in the order listed above. For example, from 5' to 3', the composite intron is: i. (a) and (b); ii. (a) and (c); iii. (a) and (d); iv.(b) and (c); v.(b) and (d); vi.(c) and (d); vii. (a), (b), and (c); viii. (a), (b), and (d); ix. (b), (c), and (d); or x may include (a), (b), (c), and (d).

[0088] Synthetic introns may contain an unexpressed exon sequence at their 5' end. For example, a synthetic intron may have: (a) the nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; or (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 It may include.

[0089] Synthetic introns have a non-expressed exon sequence at their 3' end: (a) the nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; or (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 It may include.

[0090] Synthetic introns may contain unexpressed exon sequences at their 5' and 3' ends. For example, a synthetic intron may have: (a) the nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; or (d) The nucleotide sequence of SEQ ID NO: 7, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7; It may include, and Synthetic introns have the following at their 3' end: (a) the nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; or (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 It may include.

[0091] The unexpressed exon sequences at the 5' and 3' ends may be located on the sides of one or more intron sequences, for example, one or more intron sequences described herein. For example, in the 5' to 3' direction, the synthetic intron used in the MeCP2 promoter described herein is: i. (a), (b), and (d); ii. (a), (c), and (d); iii. (a), (b), (c), and (d); iv. (a), (c), (b), and (d); v.(a), (b), and (a); vi. (a), (c), and (a); vii. (a), (b), (c), and (a); viii. (a), (c), (b), and (a); ix. (d), (b), and (d); x.(d), (c), and (d); xi. (d), (b), (c), and (d); xii. (d), (c), (b), and (d) xiii. (d), (b), and (a); xiv. (d), (c), and (a); xv.(d), (b), (c), and (a); or xvi. (d), (c), (d), and (a) may include: (a) corresponds to the nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; (b) corresponds to the nucleotide sequence of SEQ ID NO: 5, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 5; (c) corresponds to the nucleotide sequence of SEQ ID NO: 6, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 6; and (d) corresponds to the nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7.

[0092] Preferred synthetic introns that may be used in the MeCP2 promoter described herein are in the 5' to 3' direction: (a) The nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; (b) The nucleotide sequence of SEQ ID NO: 5, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 5; (c) the nucleotide sequence of SEQ ID NO: 6, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 6; and / or (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 It includes or consists of.

[0093] Preferred synthetic introns that may be used in the MeCP2 promoter described herein are in the 5' to 3' direction: (a) The nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; (b) The nucleotide sequence of SEQ ID NO: 5, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 5; (c) the nucleotide sequence of SEQ ID NO: 6, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 6; and (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 It includes or consists of.

[0094] Preferred synthetic introns that may be used in the MeCP2 promoter described herein are in the 5' to 3' direction: (a) The nucleotide sequence of SEQ ID NO: 4, or an unexpressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 4; (b) The nucleotide sequence of SEQ ID NO: 5, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 5; (c) the nucleotide sequence of SEQ ID NO: 6, or an intron sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 6; and (d) The nucleotide sequence of SEQ ID NO: 7, or a non-expressed exon sequence containing a functional variant or fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 7 It consists of.

[0095] Preferred synthetic introns that can be used in the MeCP2 promoters described herein include or consist of the nucleotide sequence of SEQ ID NO: 2, or a functional variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 2. Alternatively, fragments of the synthetic intron may be used. Such fragments may be 1000-2005 bp, 1200-2000 bp, 1400-1900 bp, 1600-1800 bp, or 1700-1800 bp. Longer nucleotide sequences containing the synthetic intron may also be used.

[0096] A preferred MeCP2 promoter that can be used in the nucleic acid construct of the present invention is named MeCP2_2 (SEQ ID NO: 3). This promoter region includes a synthetic intron having the nucleotide sequence of SEQ ID NO: 2. Therefore, the MeCP2 promoter used in the nucleic acid construct of the present invention may include, or consist of, the nucleotide sequence of SEQ ID NO: 3, or a functional variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 3. Alternatively, a fragment of the MeCP2 promoter may be used. Such a fragment may be 1000-2234 bp, 1200-2200 bp, 1400-2100 bp, 1600-2000 bp, or 1700-1900 bp. Alternatively, a longer nucleotide sequence containing the MeCP2 promoter may be used.

[0097] Nucleic acid constructs comprising the MeCP2 promoter described herein enhance the expression of the target protein (POI) encoded by it, such as PGRN. Furthermore, the constructs enhance transduction efficiency. Therefore, in certain embodiments, the expression of POI or PGRN proteins from the nucleic acid constructs of the present invention comprising the MeCP2 promoter may be increased compared to otherwise identical constructs lacking the MeCP2 promoter. In certain embodiments, the nucleic acid constructs of the present invention comprising the MeCP2 promoter exhibit increased transduction efficiency compared to otherwise identical constructs lacking the MeCP2 promoter.

[0098] Nucleic acid constructs comprising the manipulated MeCP2 promoter described herein enhance the expression of the target protein (POI) it encodes, such as PGRN. Furthermore, the constructs enhance transduction efficiency. Therefore, in certain embodiments, the expression of POI or PGRN proteins from the nucleic acid constructs of the present invention comprising the manipulated MeCP2 promoter may be increased compared to constructs lacking the manipulated MeCP2 promoter, such as equivalent constructs containing a minimal MeCP2 promoter. In certain embodiments, nucleic acid constructs of the present invention comprising the manipulated MeCP2 promoter exhibit increased transduction efficiency compared to constructs lacking the manipulated MeCP2 promoter, such as equivalent constructs containing a minimal MeCP2 promoter.

[0099] Nucleic acid constructs comprising an engineered MeCP2 promoter containing synthetic introns as described herein enhance the expression of the protein of interest (POI) encoded by it. Furthermore, the constructs enhance transduction efficiency. Therefore, in certain embodiments, the expression of POI or PGRN proteins from the nucleic acid constructs of the present invention comprising an engineered MeCP2 promoter containing synthetic introns may be increased compared to constructs lacking the engineered MeCP2 promoter containing synthetic introns, such as constructs containing a minimal MeCP2 promoter, or constructs containing an engineered MeCP2 promoter lacking synthetic introns. In certain embodiments, the nucleic acid constructs of the present invention comprising an engineered MeCP2 promoter containing synthetic introns exhibit increased transduction efficiency compared to constructs lacking the engineered MeCP2 promoter containing synthetic introns, such as constructs containing a minimal MeCP2 promoter, or constructs containing an engineered MeCP2 promoter lacking synthetic introns.

[0100] Progranulin (PGRN) Progranulin (PGRN; also known as granulin-epiterin precursor, proepiterin, prostate cancer (PC) cell-derived growth factor, and acrogranin) is a secreted glycoprotein expressed by many cell types throughout the body. Encoded by a single gene (GRN; Gene ID: 2896) on chromosome 17q21, PGRN is a 593-amino acid cysteine-rich protein with an estimated molecular weight of 68.5 kDa. It contains 7.5 granulin-like domains, each consisting of a highly conserved tandem repeat of 12 cysteinyl motifs. Proteolytic cleavage of PGRN by extracellular proteases such as elastase yields smaller peptide fragments called granulins or epitherins (e.g., granulin A, granulin B, granulin C, etc.). These fragments range in size from 6 to 25 kDa and are involved in a variety of biological functions.

[0101] PGRN deficiency is strongly associated with the pathogenesis of frontotemporal dementia (FTD), also known as frontotemporal dementia. Mutations in one allele of the GRN gene, which encodes the protein progranulin (PGRN), are associated with the development of FTD (Baker et al., Nature. 2006 Aug 24;442(7105):916-919). The GRN-associated form of FTD is a proteinopathy characterized by the appearance of neuronal inclusions containing ubiquitinated and fragmented TDP-43 (encoded by TARDBP). In a PGRN-deficient mouse model, driving neuronal expression of PGRN using an AAV gene therapy approach has been shown to modify behavioral disorders associated with FTD (Arrant et al. Brain. 2017;140.5:1447-1465).

[0102] Furthermore, PGRN deficiency is also associated with neuronal ceroid lipofuscinosis 11 (NCL11). In particular, homozygous mutations in GRN are associated with neuronal ceroid lipofuscinosis 11 (NCL11), which is characterized by cerebellar ataxia, seizures, retinitis pigmentosa, and cognitive impairment, and usually begins between the ages of 13 and 25 (Faber et al. Brain. 2020;143(1):303-31).

[0103] Therefore, there is strong biological evidence for therapeutic approaches that increase PGRN levels in the central nervous system to treat neurological disorders associated with PGRN deficiency. The association between PGRN deficiency and CNS disorders (including FTD and NCL11) is discussed in detail in Mole and Cotman, Biochimica et Biophysica Acta. 2015;1852:2237-2241, Chitramuthu et al. Brain. 2017;140:3081-3104, and Huin et al., Brain. 2020;143:303-319.

[0104] The nucleotide sequence encoding the PGRN protein used in the nucleic acid construct of the present invention may encode human PGRN protein. The nucleotide sequence encoding the PGRN protein used in the nucleic acid construct of the present invention may encode wild-type PGRN protein. The nucleotide sequence encoding the PGRN protein used in the nucleic acid construct of the present invention may encode wild-type human PGRN protein.

[0105] The inventors have found that codon optimization of the nucleotide sequence encoding the PGRN protein in nucleic acid constructs and vectors containing the MeCP2 promoter results in lower PGRN expression levels compared to the wild-type nucleotide sequence encoding PGRN (see Example 5 and Figure 5). Therefore, in some embodiments of the present invention, the nucleotide sequence encoding the PGRN protein is not codon optimized.

[0106] Preferred nucleotide sequences encoding PGRN proteins that can be used in the nucleic acid constructs of the present invention include, or consist of, the nucleotide sequence of SEQ ID NO: 12, or a functional variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of SEQ ID NO: 12. Alternatively, fragments of the nucleotide sequence may be used. Such fragments may be 1000-1781 bp, 1200-1750 bp, 1400-1700 bp, or 1500-1600 bp in length.

[0107] Preferred nucleotide sequences encoding a PGRN protein that can be used in the nucleic acid construct of the present invention include the amino acid sequence of SEQ ID NO: 13, or a functional variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the amino acid sequence of SEQ ID NO: 13, or encoding a PGRN protein consisting of such a variant. Alternatively, nucleotides encoding a fragment of the PGRN protein may be used. Such fragments may have amino acid residue lengths of 300-592, 350-490, 400-480, or 450-475.

[0108] In any protein or polypeptide described herein, the amino acid sequence may be modified by addition, deletion, or substitution, provided that the polypeptide having the modified sequence exhibits the same activity as the polypeptide having the unmodified sequence. “Same” should be understood as the polypeptide having the modified sequence not exhibiting significantly reduced activity compared to the polypeptide having the unmodified sequence. Such a modified protein or nucleotide sequence encoding such a modified protein may be considered a “functional variant.”

[0109] The nucleic acid constructs of the present invention may include functional variants or fragments of nucleotide sequences encoding the PGRN proteins described herein. Functional variants or fragments of nucleotide sequences encoding the PGRN proteins described herein may be functional in the sense that they retain the characteristics of the corresponding non-variant or full-length nucleotide sequence encoding the PGRN protein.

[0110] The nucleic acid constructs of the present invention may include nucleotide sequences encoding functional variants or fragments of the PGRN proteins described herein. Functional variants or fragments of the PGRN proteins described herein may be functional in the sense that they retain the characteristics of the corresponding non-variant or full-length PGRN protein.

[0111] Research characterizing the function and intracellular interactions of PRRNs is still ongoing. Nevertheless, PGRNs have been observed to co-localize with the lysosomal marker protein LAMP-1 (lysosomal membrane protein 1) and to play a role in regulating lysosomal function and biosynthesis through lysosome acidification (Tanaka et al., Human Molecular Genetics. 2017;26(5):969-988).

[0112] In certain embodiments, a functional variant or fragment of a nucleotide sequence encoding a PGRN protein encodes a PGRN protein that can colocalize with LAMP-1. The colocalization of the PGRN protein encoded by the functional variant or fragment and LAMP-1 may be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the colocalization of the PGRN protein encoded by the corresponding non-variant or full-length nucleotide sequence and LAMP-1 under the same conditions. The colocalization of the PGRN protein encoded by the functional variant or fragment and LAMP-1 may be substantially the same as, or greater than, the colocalization of the PGRN protein encoded by the corresponding non-variant or full-length nucleotide sequence and LAMP-1 under the same conditions.

[0113] In certain embodiments, functional variants or fragments of the PGRN protein can co-localize with LAMP-1. The co-localization of a PGRN protein fragment variant with LAMP-1 may be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the co-localization of the corresponding non-variant or full-length PGRN protein under the same conditions. The co-localization of a PGRN protein fragment variant may be substantially the same as, or greater than, the co-localization of the corresponding non-variant or full-length PGRN protein under the same conditions.

[0114] The co-localization of PGRN protein and LAMP-1 can be evaluated and / or quantified using any appropriate technique known in the art. For example, in cultured cells lacking PGRN (e.g., GRN - / -Cells (or cells in which PGRN expression is downregulated by siRNA) may be transfused using a vector containing a nucleic acid construct that includes a functional variant or fragment of a nucleotide sequence encoding the PGRN protein. The cells may then be immunostained using a first fluorescently labeled (e.g., green) antibody specific to PGRN and a second fluorescently labeled (e.g., red) antibody specific to LAMP-1. Co-localization of red and green staining can then be evaluated using a fluorescence microscope (see Tanaka et al., Human Molecular Genetics. 2017;26(5):969-988).

[0115] In certain embodiments, a functional variant or fragment of a nucleotide sequence encoding a PGRN protein encodes a PGRN protein capable of regulating lysosomal acidification. The regulation of lysosomal acidification by the PGRN protein encoded by the functional variant or fragment may be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the regulation of lysosomal acidification by the PGRN protein encoded by the corresponding non-variant or full-length nucleotide sequence under the same conditions. The regulation of lysosomal acidification by the PGRN protein encoded by the functional variant or fragment may be substantially the same as, or greater than, the regulation of lysosomal acidification by the PGRN protein encoded by the corresponding non-variant or full-length nucleotide sequence under the same conditions.

[0116] In certain embodiments, functional variants or fragments of the PGRN protein can modulate lysosomal acidification. The modulation of lysosomal acidification by a variant or fragment of the PGRN protein may be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modulation of lysosomal acidification by the corresponding non-variant or full-length PGRN protein under the same conditions. The modulation of lysosomal acidification by a variant or fragment of the PGRN protein may be substantially the same as, or greater than, the modulation of lysosomal acidification by the corresponding non-variant or full-length PGRN protein under the same conditions.

[0117] In certain embodiments, a functional variant or fragment of a nucleotide sequence encoding a PGRN protein encodes a PGRN protein capable of increasing lysosomal acidification. The PGRN protein encoded by the functional variant or fragment can increase lysosomal acidification to at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the increase in lysosomal acidification achieved by the PGRN protein encoded by the corresponding non-variant or full-length nucleotide sequence, under the same conditions. The PGRN protein encoded by the functional variant or fragment can increase lysosomal acidification to substantially the same extent as, or exceeding, the increase in lysosomal acidification provided by the PGRN protein encoded by the corresponding non-variant or full-length nucleotide sequence, under the same conditions.

[0118] In certain embodiments, functional variants or fragments of the PGRN protein can increase lysosomal acidification. The variants or fragments of the PGRN protein can increase lysosomal acidification to at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the increase in lysosomal acidification provided by the corresponding non-variant or full-length PGRN protein under the same conditions. The variants or fragments of the PGRN protein can increase lysosomal acidification to substantially the same extent as, or exceeding, the regulation of lysosomal acidification by the corresponding non-variant or full-length PGRN protein under the same conditions.

[0119] The effect of PGRN on lysosomal acidification can be evaluated using any appropriate technique in the field. For example, cultured cells lacking PGRN (e.g., GRN - / - Cells (or cells in which PGRN expression is downregulated by siRNA) may be transfused using a vector containing a nucleic acid construct that includes a functional variant or fragment of the nucleotide sequence encoding the PGRN protein. Lysosomal acidification in the transfused cells can then be evaluated using a cell-permeable dye such as LysoSensor DND-189 or acridine orange (see Tanaka et al., Human Molecular Genetics. 2017;26(5):969-988). The fluorescence of LysoSensor DND-189 increases in a lysosomal acidity-dependent manner. The acridine orange monomer emits green fluorescence, which is formed when its dimer and oligomer are protonated. Therefore, the red / green fluorescence ratio indicates the relative acidity of the lysosome. The fluorescence signal produced by the dye can be measured using a fluorescence microscope or a fluorescence plate reader.

[0120] Any comparison of activity between sequences should be performed using the same assay. Unless otherwise specified, modifications to polypeptide sequences are preferably conservative amino acid substitutions. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid it substitutes for. In addition, conservative substitutions may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 major amino acids defined in Table A1 below. If the amino acids have similar polarity, this can be determined by referring to the hydrophobicity scale of the amino acid side chains in Table A2. [Table 1]

[0121] vector The present invention provides vectors comprising the nucleic acid constructs of the present invention. The vectors may be of any type. For example, the vector may be a plasmid vector or minicircle DNA. However, typically, the vectors of the present invention are viral vectors. The viral vectors may be herpes simplex virus, adenovirus, or lentivirus-based. The viral vectors may be adeno-associated virus (AAV) vectors or derivatives thereof. Viral vector derivatives may be chimeric, shuffled, or capsid-modified derivatives.

[0122] The viral vector may contain a naturally occurring serotype of AAV, an isolate, or an AAV genome derived from a clade. The AAV serotype determines the tissue specificity of the AAV virus's infection (or tropism). Preferably, the AAV used in the present invention can be transduced into cells of the CNS, such as nerve cells, astrocytes, and / or oligodendrocytes. For example, the AAV serotype may be AAV2, AAV5, or AAV8, preferably AAV2.

[0123] The effectiveness of gene therapy generally depends on the proper and efficient delivery of the donated DNA. This method is usually mediated by a viral vector. Adeno-associated virus (AAV), a member of the parvovirus family, is commonly used in gene therapy. Wild-type AAV, containing the viral gene, inserts its genomic material into chromosome 19 of the host cell (Kotin, et al. PNAS USA 1990.87:2211-2215). The AAV single-stranded DNA genome contains two reverse-ended repeats (ITRs) and two open reading frames, containing the structural (cap) gene and the packaging (rep) gene (Hermonat et al., J. Virol 1984.51:329-339). For therapeutic purposes, the only sequences required for cis are the therapeutic gene and the ITRs. Therefore, the AAV virus is modified: the viral gene is removed from the genome, resulting in recombinant AAV (rAAV), which contains only the therapeutic gene and the two ITRs. Removal of the viral genes prevents rAAV from actively inserting its genome into host cell DNA. Instead, the rAAV genome fuses via ITR to form a circular episomal structure or is inserted into an existing chromosomal break. For viral generation, the structural and packaging genes that are now removed from rAAV are supplied trans in the form of a helper plasmid. The AAV vector is limited by a relatively small packaging capacity of approximately 4.8 kb.

[0124] Most gene therapy vector constructs are based on AAV serotype 2 (AAV2). AAV2 binds to target cells via the heparan sulfate proteoglycan receptor (Summerford and Samulski J. Virol, 1998, 72:1438-1445). The AAV2 genome, like the genomes of all AAV serotypes, can be encapsulated within several different capsid proteins. AAV2 can be packaged within its native AAV2 capsid (AAV2 / 2) or pseudotyped by other capsids (e.g., AAV2 genome in the AAV1 capsid; AAV2 / 1, AAV2 genome in the AAV5 capsid; AAV2 / 5, and AAV2 genome in the AAV8 capsid; AAV2 / 8).

[0125] The vector of the present invention may include an adeno-associated virus (AAV) genome or a derivative thereof.

[0126] The AAV genome is a polynucleotide sequence that encodes functions necessary for the generation of AAV virus particles. These functions include those that operate in the AAV replication and packaging cycle within host cells, and include the capsid formation of the AAV genome onto the AAV virus particle. Naturally occurring AAV viruses are replication-deficient and rely on the provision of helper functions in the trans for the completion of the replication and packaging cycle. Therefore, and by further removal of the AAV rep and cap genes, the AAV genome of the vector of the present invention is replication-deficient.

[0127] The AAV genome can be either a single-stranded or double-stranded form, either positive-sense or negative-sense. The use of the double-stranded form allows for bypassing the DNA replication process in target cells, thus accelerating transgene expression. The AAV genome can originate from any naturally occurring serotype, or from an isolated AAV or clade. As is known to those skilled in the art, naturally occurring AAV viruses can be classified according to various biological systems.

[0128] Generally, AAV viruses are referred to in terms of their serotypes. Serotypes correspond to variant subspecies of AAV that have characteristic reactivity, which can be used to distinguish them from other variant subspecies based on the expression profile of the capsid surface antigen. Typically, viruses with a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific to any other AAV serotype. Examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrH10), and AAV11, as well as recombinant serotypes, such as Rec2 and Rec3, identified from primate brains. In the vector of the present invention, the genome may be derived from any AAV serotype. The capsid may also be derived from any AAV serotype. The genome and capsid may be derived from the same serotype or from different serotypes. In the vector of the present invention, the genome is preferably derived from AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), or AAV serotype 8 (AAV8). More preferably, the genome is derived from AAV2.

[0129] It is even more preferable that the AAV is an AAV-TT. The AAV-TT is described in detail in Tordo et al., Brain. 2018;141(7):2014-2031 and International Publication No. 2015 / 121501 (these are incorporated herein by reference in their entirety).

[0130] A review of AAV serotypes can be found in Choi et al (Curr Gene Ther. 2005; 5(3); 299-310) and Wu et al (Molecular Therapy. 2006; 14(3), 316-327). The ITR sequence, the sequence of the AAV genome including the rep or cap gene, or the sequence of an element of the AAV genome used in the present invention may be derived from the following accession numbers for the whole AAV genome sequence: adeno-associated virus 1 NC_002077, AF063497; adeno-associated virus 2 NC_001401; adeno-associated virus 3 NC_001729; adeno-associated virus 3B NC_001863; adeno-associated virus 4 NC_001829; adeno-associated virus 5 Y18065, 5 AF085716; adeno-associated virus 6 NC_001862; avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; avian AAV strain DA-1 NC_006263, AY629583; bovine AAV NC_005889, AY388617.

[0131] Furthermore, AAV viruses may be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally occurring AAV viruses and, typically, to a group of AAV viruses that can be traced back to a common ancestor and include all of its descendants. In addition, AAV viruses may be referred to in terms of specific isolates, i.e., genetic isolates of specific AAV viruses found in nature. The term genetic isolate describes a population of AAV viruses that, due to limited genetic mixing with other naturally occurring AAV viruses, define a population that is recognizably distinct at the genetic level. Examples of AAV clades and isolates that may be used in this invention include: ·Clade A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu.48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609; ·Creed B:Hu.19 AY530584, Hu.20 AY530586, Hu 23 AY530589, Hu22 AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63 AYS30624, Hu64 AY530625, Hu3 AY530578, Hu56 AY530618, Hu57 AY530619, Hu49 AY530612, Hu58 AY530620, Hu34 AY530598, Hu35 AY530599, AAV2 NC_001401, Hu45 AY530608, Hu47 AY530610, Hu51 AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17 AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 AY695372, Hu T32 AY695371, Hu T17 AY695370, Hu LG15 AY695377; ·Clored C:Hu9 AY530629, Hulo AY530576, Hull AY530577, Hu53 AY530615, Hu55 AY530617, Hu54 AY530616, Hu7 AY530628, Hul8 AY530583, Hul5 AY530580, Hul6 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595, Hul AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 AY530623; ·Creed D:Rh62 AY530573,Rh48 AY530561,Rh54 AY530567,Rh55 AY530568,Cy2 AY243020,AAV7 AF513851,Rh35 AY243000,Rh37 AY242998,Rh36 AY242999,Cy6 AY243016,Cy4 AY243018,Cy3 AY243019,Cy5 AY243017,Rhl3 AY243013; ·Clade E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627, Hu40 AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2 AY243007, Bbl AY243023, Bb2 AY243022, RhlO AY243015, Hul7 AY530582, Hub AY530621, Rh25 AY530557, Pi2 AY530554, Pil AY530553, Pi3 AY530555, Rh57 AY530569, Rh50 AY530563, Rh49 AY530562, Hu39 AY530601, Rh58 AY530570, Rhbl AY530572, Rh52AY530565, Rh53 AY530566, Rh51 AY530564, Rh64 AY530574, Rh43 AY530560, AAV8 AF513852, Rh8 AY242997, Rhl AY530556; and Clade F: Hu 14(AAV9)AY530579, Hu31 AY530596, Hu32 AY530597; Clone isolates AAV5 Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4 15 NC_001829, Rh34 AY243001, Rh33 AY243002, Rh32 AY243003.

[0132] Those skilled in the art can, based on common general knowledge, select an appropriate serotype, clade, clone, or isolate of AAV used in the present invention. However, it should be understood that the present invention also encompasses the use of AAV genomes of other serotypes that may not yet be identified or characterized.

[0133] Typically, the AAV genome of a naturally occurring serotype of AAV, or an isolate or clade of AAV, contains at least one reverse-terminal repeat (ITR). The vector of the present invention may contain two ITRs, preferably one ITR at each end of the genome. The ITR sequences act cis-in to provide a functional origin for replication and enable vector integration and excision from the cell genome. Preferred ITR sequences are those of AAV2 and its variants. The AAV genome typically contains packaging genes such as rep and / or cap genes that encode the packaging function of the AAV virus particle. The rep genes encode one or more of the proteins Rep78, Rep68, Rep52, and Rep40, or their variants. The cap genes encode one or more capsid proteins, e.g., VP1, VP2, and VP3, or their variants. These proteins constitute the capsid of the AAV virus particle. Capsid variants are discussed below.

[0134] Preferably, the AAV genome will be derivatized for administration to patients. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivatives of the AAV genome, and derivatives that can be produced using techniques known in the art. Derivatization of the AAV genome and AAV capsid has been reviewed in Coura and Nardi (Virology Journal. 2007;4:99) and Choi et al. mentioned above.

[0135] Examples of AAV genome derivatives include any truncated or modified form of the AAV genome that enables in vivo expression of the Rep-1 transgene from the vector of the present invention. Typically, the AAV genome can be truncated considerably to contain minimal viral sequences while retaining the aforementioned functions. This is preferred for safety reasons, as it reduces the risk of recombination of the vector with wild-type virus and also avoids triggering a cellular immune response due to the presence of viral gene proteins in target cells. Typically, the derivative will contain at least one reverse terminal repeat sequence (ITR), preferably multiple ITRs, e.g., two or more ITRs. One or more ITRs may be derived from AAV genomes with different serotypes, or they may be chimeric or mutant ITRs. A preferred mutant ITR is one having a deletion of trs (terminal segregation sites). This deletion allows for continued replication of the genome, generating a single-stranded genome containing both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows for bypassing DNA replication in target cells, thus enabling accelerated transgene expression.

[0136] Preferably, one or more ITRs will be located on the side of the nucleic acid construct of the present invention, i.e., the nucleotide sequence containing the MeCP2 promoter and the nucleotide sequence encoding the PGRN protein. Including one or more ITRs is preferable to help package the vector of the present invention into viral particles. In a preferred embodiment, the ITR element will be the only sequence retained from the native AAV genome within the derivative. Therefore, the derivative will preferably not contain rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferable for the reasons mentioned above, and also because it reduces the possibility of incorporating the vector into the host cell genome. In addition, reducing the size of the AAV genome makes it possible to increase the flexibility in incorporating other sequence elements (such as regulatory elements) into the vector in addition to the transgene.

[0137] Therefore, with reference to the AAV2 genome, the following portions can be removed from the derivative of the present invention: one reverse-terminal repeat (ITR) sequence, replication (rep), and capsid (cap) genes. However, in some embodiments, including in vitro embodiments, the derivative may also include one or more rep and / or cap genes or other viral sequences from the AAV genome. The derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAV viruses. The present invention encompasses the use of capsid protein sequences derived from different serotypes, clades, clones, or isolates of AAV within the same vector. The present invention also encompasses packaging the genome of one serotype into the capsid of another serotype, i.e., pseudotyping. The chimeric, shuffled, or capsid-modified derivative may be selected to provide one or more desired functional groups to the viral vector. Therefore, these derivatives may exhibit improved gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism range, and / or improved targeting of specific cell types compared to AAV viral vectors containing naturally occurring AAV genomes (such as those of AAV2). Improved gene delivery efficiency can be achieved through improved receptor or co-receptor binding on the cell surface, improved internalization, improved transport into the cell and nucleus, improved uncoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Furthermore, improved efficiency may be associated with altered tropism range or targeting of specific cell populations, such that the vector dose is not diluted by administration to tissues that do not require it.

[0138] Chimeric capsid proteins can be generated by recombination between two or more capsid-coding sequences of naturally occurring AAV serotypes. This can be performed, for example, by a marker rescue approach in which a non-infectious capsid sequence of one serotype is simultaneously transfused with a capsid sequence of a different serotype, and targeted selection is used to select a capsid sequence with desired properties. The capsid sequences of different serotypes can be modified by intracellular homologous recombination to generate novel chimeric capsid proteins. Chimeric capsid proteins can also be generated by manipulating capsid protein sequences to transpose specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes. Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of relevant AAV genes, such as genes encoding capsid proteins of multiple different serotypes, and then reassembling the fragments in a self-priming polymerase reaction, which can also induce cross-reactivity in regions of sequence homology. By shuffling several serotype capsid genes, a library of hybrid AAV genes thus created can be screened to identify viral clones with desired functionality. Similarly, AAV capsid genes can be randomly mutated using error-prone PCR to create a diverse library of variants, which can then be selected for desired properties.

[0139] Furthermore, the sequence of the capsid gene can be genetically modified to introduce deletions, substitutions, or insertions specific to the natural wild-type sequence. In particular, the capsid gene can be modified by inserting a sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence, or at the N-terminus and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide may advantageously confer improved binding to target cells by acting as a ligand for a particular cell type, or improve the specificity of the vector's targeting to a particular cell population. Alternatively, the unrelated protein may assist in the purification of viral particles as part of the production method, i.e., it may be an epitope or an affinity tag. The insertion site is typically selected so as not to interfere with other functions of the viral particle, such as internalization or viral particle transport. Those skilled in the art can identify suitable insertion sites based on general knowledge. Specific sites are disclosed in Choi et al. above.

[0140] The present invention also includes the use of AAV genome sequences in an order and configuration different from that of the native AAV genome. Furthermore, the present invention includes replacing one or more AAV sequences or genes with a chimeric gene composed of sequences from another virus or multiple viruses. Such a chimeric gene may consist of sequences from two or more related viral proteins from different virus species.

[0141] Furthermore, the present invention provides AAV virus particles comprising the vector of the present invention. The AAV particles of the present invention include a transcapsidized form in which an AAV genome or derivative having one serotype ITR is packaged within a capsid of a different serotype. The AAV particles of the present invention also include a mosaic form in which a mixture of unmodified capsid proteins derived from two or more different serotypes constitutes the viral envelope. The AAV particles also include a chemically modified form having a ligand adsorbed to the capsid surface. For example, such a ligand may include an antibody for targeting a specific cell surface receptor.

[0142] The vectors and AAV virus particles of the present invention, including the AAV vector, can be prepared by standard means known in the art for providing vectors for gene therapy. Therefore, suitable vector preparations can be prepared using well-established public domain translocation, packaging, and purification methods.

[0143] The nucleic acid constructs and vectors of the present invention, comprising nucleotide sequences encoding PGRN proteins, have the ability to rescue loss of PGRN function that may result from, for example, mutations in one or both alleles of a patient's GRN gene. "Rescue" generally means any improvement or delay of progression of phenotypes associated with PRGN deficiency, such as the restoration of the presence of PGRN proteins in the brain and / or mitigation of neurological pathology.

[0144] The properties of the nucleic acid constructs and vectors of the present invention may be tested using techniques known to those skilled in the art. For example, the nucleic acid constructs of the present invention can be assembled into the vectors of the present invention, delivered to PRGN-deficient test animals such as mice or primates, and their effects can be observed and compared with a control.

[0145] Sequence ID 10 corresponds to the nucleotide sequence of construct pPG36 containing the MeCP2_2 promoter. In certain embodiments, the nucleic acid construct or viral vector of the present invention comprises or consists of the nucleotide sequence of Sequence ID 10, or a functional variant or fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of Sequence ID 10.

[0146] Sequence ID 11 corresponds to the nucleotide sequence of construct pPG35 containing the MeCP2_1 promoter. In certain embodiments, the nucleic acid construct or viral vector of the present invention comprises or consists of the nucleotide sequence of Sequence ID 11, or a functional variant or fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of Sequence ID 11.

[0147] Sequence ID 17 corresponds to the nucleotide sequence of AAVTT-pPG36, i.e., the AAVTT vector genome containing the nucleotide sequence of constructor pPG36. In certain embodiments, the viral vector of the present invention, for example, the AAV vector or AAVTT vector, contains or consists of the nucleotide sequence of Sequence ID 17, or a functional variant or fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity thereto with respect to the nucleotide sequence of Sequence ID 17.

[0148] Sequence IDs 18 and 19 correspond to nucleotide sequences of two alternative AAVTT vector genomes, referred to as AAVTT-p1PG36 and AAVTT-p2PG36, respectively. Both AAVTT-p1PG36 (SEQ ID 18) and AAVTT-p2PG36 (SEQ ID 19) contain the nucleotide sequence of Sequence ID 17. In certain embodiments, the viral vector of the present invention, for example, the AAV vector or AAVTT vector, contains or consists of the nucleotide sequence of Sequence ID 18, or a functional variant or fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with respect to the nucleotide sequence of Sequence ID 18. In certain embodiments, the viral vector of the present invention, for example, the AAV vector or the AAVTT vector, comprises or consists of the nucleotide sequence of SEQ ID NO: 19, or a functional variant or fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of SEQ ID NO: 19.

[0149] Pharmaceutical composition and dosage The nucleic acid constructs and vectors of the present invention can be formulated into pharmaceutical compositions. Therefore, the present invention provides pharmaceutical compositions comprising the nucleic acid constructs, vectors, and / or viral vectors of the present invention together with a pharmaceutically acceptable carrier, excipient, or diluent.

[0150] The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other materials can be determined by those skilled in the art, depending on the route of administration.

[0151] Pharmaceutical compositions may be provided in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, magnesium chloride, dextrose, or a solution of other sugars, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol. Optionally, surfactants such as 0.001% pluronic acid (PF68) may be used.

[0152] For injection at the site of the disease, the active ingredient is pyrogen-free and in the form of an aqueous solution with appropriate pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer injection, Lactringer injection, or Hartmann's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. For delayed release, the vector may be contained in a pharmaceutical composition formulated for sustained release according to methods known in the art, for example, in a microcapsule formed from a biocompatible polymer or in a liposome carrier system.

[0153] The dosage and administration regimen can be determined within the scope of the normal skills of the physician administering the composition.

[0154] Treatment methods and medical use Furthermore, the present invention encompasses the use of nucleic acid constructs, vectors, viral vectors, and pharmaceutical compositions described herein in treating or preventing diseases or symptoms in patients.

[0155] Therefore, the present invention provides nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention for use in methods of treating or preventing a disease or symptom in a patient who requires treatment or prevention of the disease or symptom. The present invention further provides a method of treating or preventing a disease or symptom in a patient who requires treatment or prevention of the disease or symptom, comprising administering to the patient a therapeutically effective amount of the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention. The present invention also provides the use of the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention for manufacturing pharmaceuticals for treating or preventing a disease or symptom in a patient who requires treatment or prevention of the disease or symptom.

[0156] The disease or symptoms may be characterized by PGRN deficiency. This PGRN deficiency may result from the loss of functional mutations in one or both alleles of the GRN gene in the patient being treated.

[0157] Therefore, the present invention provides nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention for use in methods of treating or preventing diseases characterized by progranulin (PGRN) deficiency in patients requiring treatment or prevention of said disease. The present invention further provides a method of treating or preventing diseases characterized by progranulin (PGRN) deficiency in patients requiring treatment or prevention of said disease, comprising administering to a patient a therapeutically effective amount of the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention. The present invention also provides the use of the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention for manufacturing pharmaceuticals for treating or preventing diseases characterized by progranulin (PGRN) deficiency.

[0158] Diseases characterized by PGRN deficiency, which will be treated with the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention, may be diseases of the central nervous system (CNS).

[0159] A disease characterized by PGRN deficiency to be treated with the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention may be frontotemporal dementia (FTD).

[0160] A disease characterized by PGRN deficiency to be treated with the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention may be neuronal ceroid lipofuscinosis type 11 (NCL11).

[0161] Diseases characterized by PGRN deficiency, which will be treated with the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention, may be further characterized by lysosomal dysfunction, such as dysregulation of lysosomal acidification. Such lysosomal dysfunction may be characterized by increased expression levels and / or activity of cathepsin D, preferably mature heavy chain and / or light chain cathepsin D.

[0162] Patients requiring treatment with the nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention may be male or female. Such patients may be at risk of or have been previously identified as having a disease characterized by PGRN deficiency. Such patients may be at risk of or have been previously identified as having FTD. Such patients may be at risk of or have been previously identified as having NCL11.

[0163] The dosage of the vector of the present invention can be determined according to various parameters, in particular the age, weight, and condition of the patient to be treated; the route of administration; and the required regimen. A physician can determine the required route of administration and dosage for any particular patient.

[0164] The nucleic acid constructs, vectors, viral vectors, or pharmaceutical compositions of the present invention may be administered to the brain and / or cerebrospinal fluid (CSF) of a patient. Delivery to the brain may be selected from intracerebral delivery, intraparenchymal delivery, intraputaminal delivery, and combinations thereof. Further target regions within the brain may include the thalamus, cerebellum, subthalamic nucleus, and combinations thereof. Delivery to the CSF may be selected from intracisional delivery, intrathecal delivery, intraventricular (ICV) delivery, and combinations thereof.

[0165] Delivery to the patient's brain and / or cerebrospinal fluid (CSF) may be by injection. Injections to the brain may be selected from intracerebral injection, intraparenchymal injection, intraputaminal injection, and combinations thereof. Delivery to the CSF may be selected from intracisional injection, intrathecal injection, intraventricular (ICV) injection, and combinations thereof.

[0166] Injections into the brain and / or cerebrospinal fluid may include convection-enhanced delivery (CED). The CED procedure involves minimally invasive surgical exposure of the brain followed by direct placement of a small-diameter catheter into a target area of ​​the brain. CED is described, for example, by Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27.

[0167] The doses of the nucleic acid constructs, vectors, viral vectors, or pharmaceutical compositions of the present invention may be provided as single doses, but may be repeated if the vector does not target the correct region. The treatment is preferably a single injection, but repeated injections with different AAV serotypes, for example, over the next few years, may be considered.

[0168] host cell The present invention also provides a host cell containing the nucleic acid construct of the present invention, the vector of the present invention, the viral vector of the present invention, and / or the AAV virus particles of the present invention. Furthermore, the present invention provides a host cell that produces the viral vector of the present invention and / or the AAV particles of the present invention.

[0169] Any suitable host cell may contain the nucleic acid construct of the present invention, the vector of the present invention, the viral vector of the present invention, and / or the AAV viral particles of the present invention. Furthermore, the viral vector and / or AAV particles of the present invention can be produced using any suitable host cell. Generally, such cells are mammalian cells into which translocation occurs, but other cell types, such as insect cells, may also be used. With regard to mammalian cell production systems, HEK293 and HEK293T are preferred for AAV vectors. BHK cells or CHO cells may also be used.

[0170] kit The present invention further provides a kit comprising the nucleic acid construct of the present invention, the vector of the present invention, the viral vector of the present invention, and / or the pharmaceutical composition of the present invention.

[0171] The present invention will be further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples, individually and in any combination thereof, can serve as materials for realizing the present invention in its various forms.

[0172] example Example 1 - Materials and Methods cell culture HEK293T cells were obtained from the American Tissue Collection Center (ATCC, Manassas, VA, USA) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. A549 cells were obtained from Sigma Aldrich (St. Louis, MO, USA) and maintained in Kaighn-modified Ham's F-12 medium (F-12K) supplemented with 10% FBS and 1% penicillin / streptomycin. CaSki cells were obtained from ATCC (Manassas, VA, USA) and maintained in Roswell Park Memorial Laboratory's 1640 medium (RPMI-1640) supplemented with 10% FBS and 1% penicillin / streptomycin. COS-7 cells were obtained from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. VERO cells were obtained from ATCC (Manassas, VA, USA) and maintained in Eagle's minimal essential medium (EMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. Neuro-2A cells were obtained from ATCC (Manassas, VA, USA) and maintained in Eagle's minimal essential medium (EMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. NIH3T3 cells were obtained from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. HAP1 cells and HAP1 GRN knockout cells were obtained from Horizon Discovery (Waterbeach, United Kingdom) and maintained in Iscove's modified Dulbecco's medium (IMEM) supplemented with 10% FBS and 1% penicillin / streptomycin.

[0173] Codon optimization We generated codon-optimized nucleotide sequences encoding PGRNs with reduced CpG content. These codon-optimized sequences were named "CpG X," where X represents the percentage of wild-type CpG sites retained within the codon-optimized sequence. For example, a nucleotide sequence named CpG 90 contains 90% of the CpG sites of the corresponding wild-type sequence. The resulting sequences were cloned into an expression vector.

[0174] lentivirus generation All lentiviral vectors used were second-generation and were generated using the standard virus generation method previously described in Salmon, P. and D. Trono ("Production and titration of lentiviral vectors," Curr Protoc Neurosci, 2006, Chapter 4: Unit 4.21). Briefly, 5.7 million HEK293T cells were plated per 10 cm dish. The following day, the cells were transfused using lipofectamine 2000 (ThermoFisher) containing 10 μg of transfer vector, 3 μg of pMD2G, and 8 μg of psPAX2. The culture medium was changed 12–14 hours after transfusion. At 24 and 48 hours after this medium change, the viral supernatant was collected from a total of 20 mL of virus and filtered through a 0.45 μm filter. The viral supernatant was concentrated to 20× in PBS using a Lenti-X® concentrator (CloneTech) before rapid freezing.

[0175] lentivirus titration All lentiviruses were titrated using the Lenti-X qRT-PCR titration kit (Takara).

[0176] Lentiviral phenotype introduction Cells were resuspended and plated in equal concentrations of viral supernatant supplemented with 4 μg / ml polyblen. After 12–24 hours, the viral supernatant was replaced with fresh medium. Cos-8, NIH3T3, A549, CaSKi, HEK293T, and SK-N-SH cells were transduced at a MOI of 200. VERO and Neuro-2A cells were transduced at a MOI of 1000.

[0177] Western blot analysis The construct of interest was transfused into HEK293T cells using Lipofectamine 2000 (ThermoFisher) according to the manufacturer's instructions. Two days after transfusion, the cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were measured using BCA protein assay reagent (ThermoFisher) and a Varioskab LUX Microplate Reader (ThermoFisher). The lysates were mixed with loading buffer; an equal volume of protein was electrophoresed on Mini-PROTEAN TGX 4-15% precast polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane using a Trans-Blot Turbo System (Bio-Rad). Nonspecific antibody binding was blocked with Intercept TBS blocking buffer (Li-Cor) at room temperature for 1 hour. The membranes were incubated with the following primary antibodies: anti-PGRN (1:200 dilution, AF2420, R&D Systems) (in Intercept T20 TBS (Li-Cor)) overnight at 4°C; anti-actin (1:5000 dilution, Sigma-Aldrich, A 2066) (in Intercept T20 TBS (Li-Cor)) overnight at 4°C. The membranes were washed with TBST for 15 minutes, incubated with donkey anti-goat 680 RD (Li-Cor, 1:5000) and donkey anti-rabbit 800 CW (Li-Cor, 1:5000) antibodies (in Intercept T20 TBS) for 45 minutes, and then washed with TBST for 15 minutes. The membranes were visualized using Odyssey CLx (Li-Cor).

[0178] Using Lipofectamine 2000 (ThermoFisher), the construct of interest was processed according to the manufacturer's instructions, and then GRN was added. + / + HAP-1 (wild-type) cells and GRN - / - HAP-1 (KO) cells were transfused. Two days after transfusion, the cells were lysed in RIPA buffer (Sigma-Aldrich). Protein concentrations were measured using Pierce BCA protein assay reagent (ThermoFisher) and a SpectraMax i3X Multiplate Reader (Molecular Devices). The lysates were mixed with loading buffer; an equal amount of protein was electrophoresed on Mini-PROTEAN TGX 4-15% precast polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane using a Trans-Blot Turbo System (Bio-Rad). Nonspecific antibody binding was blocked with Intercept TBS blocking buffer (Li-Cor) at room temperature for 1 hour. The membranes were incubated with the following primary antibodies: anti-PGRN (1:200 dilution, AF2420, R&D Systems) (in Intercept T20 TBS (Li-Cor)) overnight at 4°C; anti-actin (1:10000 dilution, Sigma-Aldrich, A 2066) (in Intercept T20 TBS (Li-Cor)) overnight at 4°C. The membranes were washed with TBST for 15 minutes, incubated with donkey anti-goat 680 RD (Li-Cor, 1:5000) and donkey anti-mouse 800 CW (Li-Cor, 1:5000) antibodies (in Intercept T20 TBS) for 1 hour, and then washed with TBST for 15 minutes. The membranes were visualized using Odyssey CLx (Li-Cor).

[0179] Co-culture and transduction of neurons and astrocytes Primary neuron-astrocytic cell cocultures were prepared from 17-day embryos of C57BL / 6J mice (Janvier Labs, France). Freshly incised cortical tissue was first dissociated using papain solution (Sigma Aldrich, P4762). Cells were diluted in neuronal attachment medium and plated onto 96-well plates pre-coated with poly-D-lysine (CORNING 356692) (10,000 cells / well). The neuronal attachment medium consisted of Neurobasal plus medium (ThermoFisher Scientific, A3582901) supplemented with 2.5% heat-inactivated fetal bovine serum (ThermoFisher Scientific, A3840002), 1 mM sodium pyruvate (ThermoFisher Scientific, 11360070), 2 mM Glutamax-100X (ThermoFisher Scientific, 35050061), B27 Plus Supplement (ThermoFisher Scientific, 17504044), and 50 units / ml penicillin / streptomycin (ThermoFisher Scientific, 15070063). Cells were maintained by weekly supplementation with fresh serum-free neurobasal medium. Lentivirus-mediated transduction was performed on DIV3 (in vitro day). Lentivirus stocks were diluted in culture medium and applied to the top of cells at a given MOI (Multiple Infection Intake) as shown in the figure caption. At DIV14, i.e., 10 days after transduction, the cells were fixed and immunocytochemistry was performed.

[0180] Immunolabeling and imaging Immunocytochemistry was performed after transduction in primary neurons and astrocytes. Cells were washed three times (1×PBS) and fixed with 4% PFA (ThermoFischer Scientific, 28908) at room temperature for 10 minutes. The cells were then permeabilized for 10 minutes with a 0.25% Triton-X / 3% BSA / 1X PBS solution (BSA: bovine serum albumin, VWR, 1005-30-1L; Sigma Aldrich Triton-X-100, T8787). After permeabilization, the cells were blocked with a 3% BSA / 1X PBS solution for 30 minutes. The cells were then labeled with a primary antibody (60 minutes) and then with a fluorescently conjugated secondary antibody (45 minutes) (see below for a list of antibodies). Imaging was performed using Zeiss LSM 880 (SH-SY5Y cells) and Perkin Elmer OperaPhenix (neurons / astrocytes) instruments. Threshold setting and quantification were performed using ImageJ or Perkin Elmer Harmony software. [Table 2]

[0181] ELISA The levels of secreted human PGRN in mouse neuron-astrocytic cell co-cultures after transduction were quantified using a human progranulin ELISA kit (AG-45A-0018YEK-KI 01, Adipogen). Cell culture media were collected 10 days after transduction. Samples were diluted 1:100 and ELISA measurements were performed according to the supplier's instructions. Colorimetric reactions were measured using a standard plate reader (Flex Station 3, Molecular Devices).

[0182] Using the Human Programurin ELISA Kit (DPGRN0, RD Systems), GRN + / + HAP-1 (wild-type) cells and GRN - / -The levels of secreted human progranulin after transfusion of HAP-1 (KO) cells were quantified. Cell culture medium was collected 24 hours after transfusion. ELISA analysis was performed according to the supplier's instructions. Colorimetric reactions were measured using a SpectraMax i3X Multiplate Reader (Molecular Devices).

[0183] Brain sectioning, immunohistochemistry, and acquisition Brain sectioning was performed at Neuroscience Associates (TN, USA). First, the brain was treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent freezing artifacts, and then embedded in a gelatin matrix using MultiBrain® Technology. After curing, the block was rapidly frozen by immersion in isopentane cooled to -70°C with crushed dry ice and placed on the freezing stage of an AO860 slide microtome. The MultiBrain® block was sectioned into 40 μm sections in the coronal plane. All sections were collected sequentially into 24 containers per block filled with antigen preservation solution (49% PBS pH 7.0, 50% ethylene glycol, 1% polyvinylpyrrolidone). Sections that were not immediately stained were stored at -20°C.

[0184] Free-floating sections were immunostained with an antibody against human progranulin (R&D-AF2420) (diluted to 1:15.000). All incubation solutions after blocking serum were supplied using Tris-buffered saline (TBS) containing Triton X-100 as the vehicle; all rinsing was performed with TBS. Endogenous peroxidase activity was blocked by 0.9% hydrogen peroxide treatment, and nonspecific binding was blocked with 1.26% normal whole serum. After rinsing, sections were stained overnight at room temperature with the primary antibody. 0.3% Triton X-100 was added to the vehicle solution for permeabilization. After rinsing, sections were incubated with avidin-biotin-HRP complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) at room temperature for 1 hour. After rinsing, the sections were treated with diaminobenzidine tetrahydrochloride (DAB) and 0.0015% hydrogen peroxide to produce a visible reaction product. The product was then placed on a gelled (subbed) glass slide, air-dried, lightly stained with thionine, dehydrated in alcohol, cleared in xylene, and covered with Permount mounting medium. Digital images of the stained sections were obtained using an AxioScan Z1 slide scanner (Zeiss) equipped with a 20× objective lens.

[0185] AAV Vector AAV vectors were obtained from two different sources. The corresponding plasmid sequences are shown in SEQ ID NO: 18 (AAVTT-p1PG36) and SEQ ID NO: 19 (AAVTT-p2PG36). AAV vectors were generated using a triple plasmid translocation method, as previously described in Grieger et al. ("Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector" (Molecular Therapy vol.24 no.2, 287-297 feb.2016), using HEK 293T cells or HEK293 cells, respectively, containing a helper plasmid, a Rep / Cap coding plasmid, and a plasmid containing SEQ ID NO: 17 (AAVTT-pPG36). Figure 10 provides a schematic diagram showing the constituent parts of the nucleotide sequence of sequence number 17.

[0186] Example 2 - Generation and evaluation of manipulated promoter constructs Lentiviral vector constructs pAK169, pPG21, pPG35, and pPG36 were generated as described above. All of these constructs contain neuron-specific MeCP2 promoter sequences, as shown in Figure 1A. Constructs pAK169 and pPG21 each contain a minimal 229 bp MeCP2 promoter sequence (SEQ ID NO: 1).

[0187] The construct pPG35 (SEQ ID NO: 10) contains an engineered promoter region called MeCP2_1 (SEQ ID NO: 8). The MeCP2_1 promoter contains a minimal promoter sequence (SEQ ID NO: 1) and a native intron. The native intron is a 2108 bp nucleotide sequence (SEQ ID NO: 9) from the mouse MeCP2 gene, located 5' to the minimal promoter sequence. The MeCP2_1 promoter sequence is 2337 bp long.

[0188] The construct pPG36 (SEQ ID NO: 11) contains an engineered promoter region called MeCP2_2 (SEQ ID NO: 3). The MeCP2_2 promoter contains a minimal MeCP2 promoter sequence (SEQ ID NO: 1) and a 2006 bp synthetic intron (SEQ ID NO: 2) located 3' to the minimal promoter sequence. The MeCP2_2 promoter is 2235 bp in length. The synthetic intron (MeCP2_2 intron; SEQ ID NO: 2) was constructed from two intron sequences and two silenced (i.e., unexpressed) exons of the mouse MECP2 gene. The exons were silenced by directional mutations that removed the start codon. See Figure 11 for a schematic diagram illustrating the construction of the MeCP2_2 intron (SEQ ID NO: 2).

[0189] The manipulated MeCP2_2 promoter includes the 5'-3':MeCP2 minimal promoter sequence (SEQ ID NO: 1), Age1 restriction site (ACCGGT; SEQ ID NO: 14), exon 1 (SEQ ID NO: 5), 5' intron (SEQ ID NO: 6), 3' intron (SEQ ID NO: 7), and exon 2 (SEQ ID NO: 8).

[0190] Furthermore, control lentiviral vector constructs pAK168, pPG20, pPG33, and pPG34 were generated. Each of these constructs contains a neuron-specific NSE1 promoter sequence, as shown in Figure 2A. Constructors pAK168 and pPG20 contain a minimal NSE1 promoter sequence of approximately 1300 bp. Constructors pAK168 and pPG20 were used as equivalent controls to pAK169 and pPG21, respectively.

[0191] Constructor pPG33 contains an engineered promoter region called NSE1_1, which is the minimal promoter sequence, and an 1100 bp naturally occurring sequence of the human NSE1 gene located 5' to the minimal promoter sequence. Constructor pPG34 contains an engineered promoter region called NSE_2, which includes a synthetic intron approximately 0.9 kb long. Constructors pPG33 and pPG34 were used as equivalent controls to pPG35 and pPG36, respectively.

[0192] HEK293T cells were transfused using MeCP2 and NSE1 vectors, respectively. PGRN expression was evaluated by Western blotting. The results of these experiments are shown in Figures 1B and 2B.

[0193] Constructs containing the MeCP2 promoter, namely pPG21, pPG35, and pPG36, achieved higher PGRN expression levels than constructs containing the NSE1 promoter (i.e., pPG20, pPG33, and pPG34).

[0194] Furthermore, it was observed that constructor pPG36 (containing the MeCP2_2 promoter) achieved higher PGRN expression levels compared to constructors pPG21 (containing the minimal MeCP2 promoter sequence) and pPG35 (containing the MeCP2_1 promoter).

[0195] Example 3 - Evaluation of transgene expression by NSE1 promoter and MeCP2 promoter in primary neurons and astrocytes Wild-type mouse primary cortical neurons and astrocytes were transduced to express human progranulin protein using lentiviruses. Lentiviruses were applied at various MOIs (memories of infection) as shown in Figure 3. Ten days after lentiviral transduction, the cells were fixed and immunolabeled with NeuN (neuron marker), GFAP (astrocytocyte marker), and human progranulin antibody. The percentage of transduced cells is shown in Figure 3A (neurons) and Figure 3C (astrocytes), and the expression level (fluorescence intensity / cell) is shown in Figure 3B (neurons) and Figure 3D (astrocytes).

[0196] Constructs containing manipulated NSE1 promoters (pPG33 and pPG34) were found to have no alteration in transduction efficiency or expression levels compared to construct pPG20 containing the minimum NSE1 promoter. In contrast, constructs containing promoters MeCP2_1 and MeCP2_2 (pPG35 and pPG36, respectively) increased transduction efficiency or expression levels compared to construct pPG21 containing the minimum MeCP2 promoter. In particular, the MeCP2_2 promoter (pPG36) performed best among all promoters tested in terms of transduction efficiency and PGRN expression levels.

[0197] Example 4 - Evaluation of PGRN secretion by neurons and astrocytes transfected with a vector containing a MeCP2 promoter. Wild-type mouse primary cortical neuron-astrocytocyte co-cultures were transduced to express human programurin protein using a lentiviral vector. Lentiviruses were applied at 20 multiples of infection (MOI). Ten days after lentiviral transduction, the culture medium was collected and ELISA was performed. The results of this experiment are shown in Figure 4. Constructs containing the manipulated promoters MeCP2_1 and MeCP2_2 (pPG35 and pPG36, respectively) were found to increase secreted PGRN compared to the construct containing the minimal MeCP2 promoter (pPG21). Promoter MeCP2_2 (pPG36) provided the highest expression level of secreted PGRN among all promoters tested.

[0198] Example 5 - PGRN Codon Optimization The unmethylated CpG site can induce an innate immune response mediated by Toll-like receptor 9 (TLR9). Therefore, codon-optimized nucleotide sequences encoding human PGRNs with reduced CpG content were generated and cloned into expression vectors labeled CpG 0, 4, 9, 17, 25, 40, 71, and 90. Each of these vectors contains a codon-optimized human PGRN nucleotide sequence with reduced CpG content compared to the corresponding WT sequence.

[0199] HAP-1 GRN knockout (GRN) transfected with codon-optimized vectors and WT vectors. - / - PGRN expression levels in the cells were evaluated by ELISA and Western blotting. Expression level data are shown in Figure 5. Vectors containing the WT PGRN nucleotide sequence were observed to achieve higher levels of PGRN expression than all codon-optimized vectors tested.

[0200] Example 6 - Human PGRN expression is GRN - / - Correcting lysosome deficiencies in mouse primary neurons. Western blot analysis was performed to quantify the level of the lysosomal protein cathepsin D. Cathepsin D is a soluble lysosomal aspartic endopeptidase. The immature form is proteolytically cleaved to yield a mature active lysosomal protease composed of a heavy chain (approximately 30 kDa) and a light chain (14 kDa) that are linked by non-covalent interactions. Cathepsin D is a marker of lysosomal dysfunction, and an increase in the level of cathepsin D suggests impairment of proteolysis and accumulation of autophagic cargo.

[0201] Primary mouse cortical neurons were prepared from WT mice or GRN− / − (KO) mice. Three days after plating, the neurons were transduced with a lentiviral construct (20 MOI) to express human PGRN protein. Ten days after transduction, the cells were harvested and the protein was extracted using RIPA buffer. The protein lysates were then subjected to Western blotting to detect cathepsin D protein. The Western blot levels of cathepsin D were normalized to the expression levels of actin and GAPDH. The data are from three independent experiments.

[0202] An increase in the level of mature cathepsin D was observed in untransduced KO neurons compared to WT neurons. Lentiviral-mediated expression of hPGRN (pPG36) inhibited the maturation of cathepsin D. These results are shown in Figure 6.

[0203] In WT mice and GRN after striatal injection of Example 7 - AAVTT - p1PG36 - / - CNS expression of human PGRN (hPGRN) in mice Adult (4 - month - old) WT mice or GRN - / - The striatum of mice was injected with AAVTT - p1PG36 (AAVTT containing the MeCP2_2 promoter + human PGRN transgene construct; SEQ ID NO: 18) or AAVTT - GFP (MeCP2_2 promoter + GFP transgene) or vehicle at 2 10The total dose of vector genome (vg) was injected bilaterally. Four weeks later, animals were sacrificial, and CSF, plasma, and brain tissue were collected and analyzed. Transcardiac perfusion with 1×PBS was performed before dissection. One half of the brain was fixed for immunohistochemical analysis, and the other half was used for biochemical analysis (FRET). Different brain regions were dissected and frozen.

[0204] ELISA analysis and FRET analysis The CSF and plasma levels of hPGRN (ng / ml) were measured using ELISA (Adipogen). High levels of hPGRN were observed in WT mice and GRN in animals injected with AAVTT-p1PG36 (SEQ ID NO: 18) and AAVTT-p2PG36 (SEQ ID NO: 19). - / - Both mouse CSF (1:100 dilution) samples were detected. These results are shown in Figures 7A and 7C. Additionally, low levels of hPGRN were detected in mouse plasma (1:10 dilution). These results are shown in Figure 7A.

[0205] WT mice or GRN mice injected with AAVTT-p1PG36 using FRET (Cisbio) - / - hPGRN concentrations (ng / mg) were measured in various brain regions of mice. The highest levels of hPGRN expression were detected near the injection site (striatum and midbrain). Intermediate levels of hPGRN expression were detected in the cortex and hippocampus. Low levels of hPGRN expression were detected in distal brain regions such as the brainstem, olfactory bulb, and cerebellum. These results are shown in Figure 7B.

[0206] immunohistochemistry IHC staining of hPGRNs is observed in GRNs treated with intrastriatal administration of AAV-p1PG36 (SEQ ID NO: 18). - / -This was observed in the brains of KO mice. Specific human PGRN signaling was detected only in AAVTT-p1PG36 injected mice and not in GFP injected mice. Similar to FRET results, strong immunoreactivity was observed in the striatum of injected mice. hPGRN immunoreactivity was also observed in brain regions distant from the injection site, namely the thalamus, midbrain, substantia nigra, cortex, and hippocampus. Cellular (cell body) immunoreactivity was mainly observed near the injection site, namely the striatum, parts of the cortex, parts of the hippocampus, thalamus, and midbrain. This suggests transduction of cells by AAVTT-p1PG36 in these regions. Diffusion staining could be observed in most other brain regions, with decreasing intensity from the injection site. This indicates that hPGRN is secreted into the extracellular space and diffuses to distal brain regions via ISF and CSF flows. GRN - / - The results obtained for mice are shown in Figure 8, and similar images were obtained for WT mice injected with AAV-p1PG36.

[0207] Immunofluorescence The MeCP2 promoter has been shown to drive neuronal-specific expression of hPGRN in vitro in primary mixed astrocyte-neuron cultures (see Examples 3 and 4 above). To determine whether this neuronal specificity is maintained in vivo in mice, sections obtained from mice injected with AAVTT-p1PG36 (SEQ ID NO: 18) were subjected to double immunofluorescence labeling to label human PGRN and NeuN (neuron marker) according to the following protocol (all steps performed at room temperature): Sections were incubated overnight in a humidified chamber with the neuronal marker NeuN (1:2,000; Abcam, ab 177487) and human progranulin (1:1,000; R&D-AF2420) primary antibody, diluted in PBS containing 0.3% Triton X-100. After incubation, sections were washed three times with PBS and incubated for 1 hour with anti-rabbit Alexa 488 and anti-goat Alexa 647 secondary antibodies (all diluted 1:1,000 in PBS; all Thermo Fisher). Sections were then counterstained with DAPI to label the cell nuclei and washed three times with PBS. Finally, sections were mounted with Prolong Gold anti-fading mounting medium (Life Technologies) and coverslips were applied. Digital images of the stained sections were obtained using an AxioScan Z1 slide scanner (Zeiss) with a 20× objective lens.

[0208] Almost all cells expressing human PGRNs within the cell body were observed to be NeuN-positive. This demonstrates that AAVTT-p1PG36-mediated expression of hGRN is neuron-specific in vivo. Neuronal expression of hGRN was observed in all various brain regions, including the striatum, cortex, hippocampus, and thalamus. Importantly, cellularly expressed hGRN was not observed in the cell bodies of astrocytes (identified using GFAP staining) or microglia (identified using Iba1 staining). Therefore, these in vivo data support the conclusion that the MeCP2_2 promoter used is a neuron-specific promoter.

[0209] Example 8 - Human PGRN (hPGRN) expression was observed in WT mice and GRN after striatal injection of AAVTT-p1PG36. - / - It affects cathepsin D activity in vivo in mice. Increased levels of cathepsin D suggest impaired protein degradation and autophagy cargo accumulation. Cathepsin D enzyme activity was measured in WT(GRN) treated with the vehicle. + / +) Mice (indicated by black circles), and GRN treated with vehicle or AAVTT-p1PG36 (SEQ ID NO: 18) - / - Measurements were taken in the midbrain lysate of knockout mice. These results are shown in Figure 9.

[0210] In vitro studies have shown that GRN is overridden by pPG36 expression. - / - Accelerated maturation of cathepsin D in primary neurons was revealed (see Example 6 above). Increased maturation is expected to be associated with increased cathepsin D activity. Compared to WT mice, younger (4-5 months old) GRNs were observed. - / - Cathepsin D enzyme activity is slightly increased in various brain regions of mice. In particular, the increase in cathepsin D activity is more pronounced in older animals (e.g., 1 year old). Therefore, cathepsin D activity can be detected at an early age of 4-5 months, which is a significant factor in GRN (Gross-Resistant Neural Network) testing. - / - It has been proposed to be an early marker of stress in mice.

[0211] In all cases, GRN was 4 months old. - / - In mice, AAVTT-p1PG36-mediated hGRN expression resulted in decreased cathepsin D enzyme activity. This demonstrates that AAV-mediated hPGRN expression, driven by the neuron-specific MeCP2_2 promoter, directly affects cathepsin D activity (a marker of lysosomal dysfunction). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16]

[0212] Further aspects of the present invention: 1. A nucleic acid construct comprising a methyl CpG-binding protein 2 (MeCP2) promoter operably linked to a nucleotide sequence encoding a progranulin (PGRN) protein.

[0213] 2. The nucleic acid construct according to paragraph 1, wherein the MeCP2 promoter is an engineered MeCP2 promoter comprising a minimal promoter sequence and at least one intron.

[0214] 3. A nucleic acid construct comprising an engineered methyl CpG-binding protein 2 (MeCP2) promoter operably linked to a nucleotide sequence encoding a protein of interest (POI), wherein the engineered MeCP2 promoter comprises a minimal promoter sequence and at least one intron.

[0215] 4. The nucleic acid construct according to paragraph 3, wherein the POI is a progranulin (PGRN) protein.

[0216] 5. The nucleic acid construct according to any one of paragraphs 2-4, wherein (a) at least one intron is located 3' to the minimal promoter sequence; or (b) at least one intron is located 5' to the minimal promoter sequence.

[0217] 6. The nucleic acid construct according to any one of paragraphs 2-5, wherein at least one intron is a synthetic intron.

[0218] 7. The nucleic acid construct according to paragraph 6, wherein at least one synthetic intron comprises one or more nucleotide sequences of the MECP2 gene, and optionally, at least one synthetic intron comprises one or more intron sequences of the MECP2 gene and / or one or more non-coding exon sequences of the MECP2 gene, and preferably, the MECP2 gene is the human MECP2 gene.

[0219] 8. The nucleic acid construct according to paragraph or 7, wherein at least one synthetic intron comprises two intron sequences of the human MECP2 gene and two non-coding exon sequences of the human MECP2 gene.

[0220] 9. At least one synthetic intron is: (a) A nucleotide sequence of SEQ ID NO: 4, or a non-expressed exon sequence containing a nucleotide sequence having at least 90% identity with SEQ ID NO: 4; (b) an intron sequence containing the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 5; (c) an intron sequence containing the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 6; and / or (d) A non-expressed exon sequence containing the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 7. A nucleic acid construct as described in any of paragraphs 6-8, including the following:

[0221] In the 10.5' to 3' direction, at least one synthetic intron is: (a) A non-expressed exon sequence containing the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 4; (b) an intron sequence containing the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 5; (c) an intron sequence comprising the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 6; and (d) A non-expressed exon sequence containing the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 7. A nucleic acid construct as described in any of paragraphs 6-9, including the following:

[0222] 11. A nucleic acid construct according to any one of paragraphs 6 to 10, wherein at least one synthetic intron comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 2.

[0223] 12. A nucleic acid construct according to any of paragraphs 2-5, wherein at least one intron is a natural intron.

[0224] 13. The nucleic acid construct described in paragraph 12, wherein at least one natural intron comprises a nucleotide sequence of the MeCP2 gene, preferably the human MeCP2 gene.

[0225] 14. The nucleic acid construct according to paragraph 13, wherein at least one natural intron comprises the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 9.

[0226] 15. A nucleic acid construct according to any of paragraphs 2 to 14, wherein the minimum promoter sequence comprises the nucleotide sequence of SEQ ID NO: 1, or a functional variant or fragment thereof having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO: 1.

[0227] 16. A nucleic acid construct according to any of paragraphs 1 to 11, wherein the manipulated MeCP2 promoter comprises the nucleotide sequence of SEQ ID NO: 3, or a functional variant or fragment thereof having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO: 3.

[0228] 17. A nucleic acid construct according to any of paragraphs 1-5 or 12-14, wherein the manipulated MeCP2 promoter comprises the nucleotide sequence of SEQ ID NO: 8, or a functional variant or fragment thereof having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO: 8.

[0229] 18. The MeCP2 promoter is at least about 1000 bp, 1500 bp, 2000 bp, 2100 bp, 2150 bp, 2175 bp, 2200 bp, 2210 bp, 2220 bp, 2230 bp, 2240 bp, 2250 bp, 2260 bp, 2280 bp, 2290 bp, 2300 bp, 2310 bp, 2320 bp, 2330 bp, and preferably the MeCP2 promoter is about 2200-2350 bp, as described in any of paragraphs 1-17.

[0230] 19. (a) PGRN proteins are human PGRN proteins; (b) The PGRN protein is a wild-type protein; (c) The nucleotide sequence encoding the PGRN protein is a human nucleotide sequence; (d) The nucleotide sequence encoding the PGRN protein is the wild-type nucleotide sequence; (e) The nucleotide sequence encoding the PGN protein is not codon-optimized; and / or (f) The nucleotide sequence encoding the PGRN protein is at least about 1600 bp, 1700 bp, 1750 bp, 1760 bp, 1770 bp, or 1780 bp, preferably about 1780 bp. A nucleic acid construct as described in any of paragraphs 1, 2, or 4-18.

[0231] 20. The nucleotide sequence encoding the PGRN protein includes the nucleotide sequence of SEQ ID NO: 12, or a functional variant or fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 12; and / or The PGRN protein contains the amino acid sequence of SEQ ID NO: 13, or a functional variant or fragment having at least 70% identity to the amino acid sequence of SEQ ID NO: 13. A nucleic acid construct as described in any of paragraphs 1, 2, or 4-19.

[0232] 21. (a) A Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) sequence, optionally, the WPRE is located on the 3' side of the nucleotide sequence encoding the POI or PGRN protein, and / or the nucleotide sequence of SEQ ID NO: 15, or a functional variant or fragment thereof having at least 90% identity to the nucleotide sequence of SEQ ID NO: 15, the WPRE sequence; (b) A polyadenylation signal sequence, optionally, located on the 3' side of the nucleotide sequence encoding the POI or PGRN protein, and / or the nucleotide sequence of SEQ ID NO: 16, or a functional variant or fragment thereof having at least 90% identity to the nucleotide sequence of SEQ ID NO: 16, the polyadenylation signal sequence; or (c) The nucleic acid construct according to any one of paragraphs 1 to 20, further comprising the above (a) and (b) wherein optionally, in the 5' to 3' direction, the nucleic acid construct comprises a MeCP2 promoter, a nucleotide sequence encoding the POI or PGRN protein, a WPRE, and a polyadenylation signal sequence.

[0233] 22. The nucleic acid construct according to any one of paragraphs 1 to 21, which is 3700 - 4700 bp, 3800 - 4800 bp, 3900 - 4700 bp, 4000 - 4600 bp, 4000 - 4500 bp, 4000 - 4400 bp, 4000 - 4300 bp, or 4000 - 4200 bp.

[0234] 23. A vector comprising the nucleic acid construct defined in any one of paragraphs 1 to 22.

[0235] 24. The vector according to paragraph 23, which is a plasmid or a viral vector.

[0236] 25. (a) SEQ ID NO: 11, or a functional variant or fragment thereof having at least 70% identity to the nucleotide sequence of SEQ ID NO: 11; or ​(b) SEQ ID NO: 10, or a functional variant or fragment having at least 70% identity with the nucleotide sequence of SEQ ID NO: 10. The vector described in paragraph 23 or 24, which is a viral vector containing the nucleotide sequence of the following:

[0237] 26. A viral vector, selected from the following, as described in any of paragraphs 23-25: (a) Adeno-associated virus (AAV) vector, or A viral vector containing the AAV genome or a derivative thereof, wherein the derivative may be a chimeric, shuffled, or capsid-modified derivative; or (b) Lentiviral vector, or A viral vector containing a lentiviral genome or its derivatives. (The vector of paragraph any one of paragraphs 23-25, which is a viral vector selected from: (a) an adeno-associated virus (AAV) vector or which comprises an AAV genome or a derivative thereof, optionally said derivative is a chimeric, shuffled or capsid modified derivative; or (b) a lentiviral vector or which comprises a lentivirus genome or a derivative thereof.)

[0238] 27. An AAV vector comprising a genome derived from AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or AAV serotype rh10 (AAVrh10), preferably the viral vector described in paragraph 26, wherein the AAV comprises a genome derived from AAV2, AAV9, or AAVrH10.

[0239] 28. The AAV vector described in paragraph 27, comprising a genome derived from AAV2, preferably the AAV being AAV-TT.

[0240] 29. A host cell that comprises a nucleic acid construct described in any of paragraphs 1 to 22 and / or a vector described in any of paragraphs 23 to 28, and / or produces a viral vector described in any of paragraphs 25 to 28, and is optionally a HEK293 cell or a HEK293 T cell.

[0241] 30. A pharmaceutical composition comprising a nucleic acid construct described in any of paragraphs 1 to 22, a vector described in paragraph 23 or 24, and / or a viral vector described in any of paragraphs 25 to 28, together with a pharmaceutically acceptable carrier, excipient, or diluent.

[0242] 31. Nucleic acid constructs as defined in any one of paragraphs 1 to 22, vectors as defined in paragraph 23 or 24, viral vectors as defined in any one of paragraphs 25 to 28, and / or pharmaceutical compositions as defined in paragraph 30, used in methods for treating or preventing diseases characterized by progranulin (PGRN) deficiency in patients requiring treatment or prevention of such diseases.

[0243] 32. A method for treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient requiring treatment or prevention of such disease, comprising administering to the patient a therapeutically effective dose of a nucleic acid construct as defined in any one of paragraphs 1 to 22, a vector as defined in paragraph 23 or 24, a viral vector as defined in any one of paragraphs 25 to 28, and / or a pharmaceutical composition as defined in paragraph 30.

[0244] 33. Use of a nucleic acid construct as defined in any one of paragraphs 1 to 22, a vector as defined in paragraph 23 or 24, a viral vector as defined in any one of paragraphs 25 to 28, and / or a pharmaceutical composition as defined in paragraph 30, for the manufacture of a medicine for treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient requiring treatment or prevention of such disease.

[0245] 34. Diseases characterized by PGRN deficiency are diseases of the central nervous system; Diseases characterized by PGRN deficiency are characterized by a deficiency of PGRNs in the patient's neurons and / or astrocytes; The patient has a loss-of-function mutation in at least one allele of the GRN gene; and / or The patient has loss-of-function mutations in both alleles of the GRN gene. A nucleic acid construct, vector, viral vector, or pharmaceutical composition used in accordance with the methods described in paragraphs 31 and 32, or the use described in paragraph 33.

[0246] 35. Diseases characterized by PGRN deficiency are frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis type 11 (NCL11), nucleic acid constructs, vectors, viral vectors, or pharmaceutical compositions used in accordance with the methods described in paragraphs 31 or 34, 32 or 34, or 33 or 34.

[0247] 36. The nucleic acid construct, vector, viral vector, or pharmaceutical composition is administered to the patient by delivery to the patient's brain and / or cerebrospinal fluid (CSF), and optionally, delivery is by injection: (i) The patient's brain; and / or (ii) The patient's CSF was treated, Preferably, the injection into the brain is selected from intracerebral injection, intraparenchymal injection, intrahypothalamic injection, and combinations thereof; Preferably, the injection into the CSF is selected from intracisional injection, intrathecal injection, intraventricular (ICV) injection, and combinations thereof. A nucleic acid construct, vector, viral vector, or pharmaceutical composition used in accordance with the method described in paragraphs 31, 34, or 35, 32, 34, or 35, or any one of the uses described in paragraphs 33 to 35. [Sequence Listing Free Text]

[0248] Sequence Listing 1 <223> MeCP2 Smallest Promoter Sequence Listing 2 <223> MeCP2_2 intron Sequence Listing 3 <223> MeCP2_2 Promoter Sequence Listing 4 <223> MeCP2_2 intron-exon 1 Sequence Listing 5 <223> MeCP2_2 intron-5' intron Sequence Listing 6 <223> MeCP2_2 intron-3' intron Sequence Listing 7 <223> MeCP2_2 intron-exon 2 Sequence Listing 8 <223> MeCP2_1 Promoter Sequence Listing 9 <223> MeCP2_1 intron Sequence Listing 10 <223> pPG35 Sequence Listing 11 <223> pPG36 Sequence Listing 14 <223> Age 1 Restricted Areas Sequence Listing 16 <223> PolyA signal sequence Sequence Listing 17 <223> AAVTT-pPG36 Sequence Listing 18 <223> AAVTT-p1PG36 Sequence Listing 19 <223> AAVTT-p2PG36 Sequence Listing 20 <223> 5'ITR Sequence Listing 21 <223> 5' Adjacent fragment Sequence Listing 22 <223> 3' Adjacent fragment Sequence Listing 23 <223> 3'ITR Sequence Listing 24 <223> Kozak Array

Claims

1. A nucleic acid construct comprising a methyl CpG-binding protein 2 (MeCP2) promoter operably linked to a nucleotide sequence encoding a progranulin (PGRN) protein.

2. The nucleic acid construct according to claim 1, wherein the MeCP2 promoter is an engineered MeCP2 promoter comprising a minimal promoter sequence and at least one intron.

3. A nucleic acid construct comprising an engineered methyl CpG-binding protein 2 (MeCP2) promoter operably linked to a nucleotide sequence encoding a protein of interest (POI), wherein the engineered MeCP2 promoter comprises a minimal promoter sequence and at least one intron.

4. The nucleic acid construct according to claim 3, wherein POI is a progranulin (PGRN) protein.

5. (a) at least one intron is located on the 3' side of the minimal promoter sequence; or (b) at least one intron is located on the 5' side of the minimal promoter sequence, according to any one of claims 2 to 4.

6. A nucleic acid construct according to any one of claims 2 to 5, wherein at least one intron is a synthetic intron.

7. The nucleic acid construct according to claim 6, wherein at least one synthetic intron comprises one or more nucleotide sequences of the MECP2 gene, and at least one synthetic intron may comprise one or more intron sequences of the MECP2 gene and / or one or more unexpressed exon sequences of the MECP2 gene, preferably the MECP2 gene is a mouse or human MECP2 gene, and more preferably the MECP2 gene is a mouse MECP2 gene.

8. The nucleic acid construct according to claim 6 or 7, wherein at least one synthetic intron comprises two intron sequences of the mouse MECP2 gene and two unexpressed exon sequences of the mouse MECP2 gene.

9. A nucleic acid construct according to any one of claims 6 to 8, wherein at least one synthetic intron comprises: (a) A non-expressed exon sequence containing the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 90% identity with SEQ ID NO: 4; (b) an intron sequence comprising the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 5; (c) an intron sequence comprising the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 6; and / or (d) A non-expressed exon sequence containing the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:

7.

10. A nucleic acid construct according to any one of claims 6 to 9, wherein at least one synthetic intron in the 5' to 3' direction includes the following: (a) an unexpressed exon sequence containing the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 4; (b) an intron sequence comprising the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 5; (c) an intron sequence comprising the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 6; and (d) A non-expressed exon sequence containing the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:

7.

11. A nucleic acid construct according to any one of claims 6 to 10, wherein at least one synthetic intron comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO:

2.

12. A nucleic acid construct according to any one of claims 2 to 5, wherein at least one intron is a natural intron.

13. The nucleic acid construct according to claim 12, wherein at least one natural intron comprises a nucleotide sequence of a MECP2 gene, preferably a mouse or human MECP2 gene.

14. The nucleic acid construct according to claim 13, wherein at least one natural intron comprises a nucleotide sequence of the mouse MECP2 gene.

15. The nucleic acid construct according to claim 14, wherein at least one natural intron comprises the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO:

9.

16. A nucleic acid construct according to any one of claims 2 to 15, wherein the minimum promoter sequence comprises the nucleotide sequence of SEQ ID NO: 1, or a functional variant or fragment thereof having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO:

1.

17. A nucleic acid construct according to any one of claims 1 to 11, wherein the manipulated MeCP2 promoter comprises the nucleotide sequence of SEQ ID NO: 3, or a functional variant or fragment thereof having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO:

3.

18. A nucleic acid construct according to any one of claims 1 to 5 and 12 to 15, wherein the manipulated MeCP2 promoter comprises the nucleotide sequence of SEQ ID NO: 8, or a functional variant or fragment thereof having at least 90% identity with respect to the nucleotide sequence of SEQ ID NO:

8.

19. The nucleic acid sequence according to any one of claims 1 to 18, wherein the MeCP2 promoter has a length of at least about 1000 bp, 1500 bp, 2000 bp, 2100 bp, 2150 bp, 2175 bp, 2200 bp, 2210 bp, 2220 bp, 2230 bp, 2240 bp, 2250 bp, 2260 bp, 2280 bp, 2290 bp, 2300 bp, 2310 bp, 2320 bp, and 2330 bp, preferably the MeCP2 promoter has a length of about 2200 to 2350 bp.

20. The nucleic acid construct according to any one of claims 1, 2, and 4 to 19 is as follows: (a) The PGRN protein is human PGRN protein; (b) The PGRN protein is a wild-type protein; (c) The nucleotide sequence encoding the PGRN protein is a human nucleotide sequence; (d) The nucleotide sequence encoding the PGRN protein is the wild-type nucleotide sequence; (e) The nucleotide sequence encoding the PGN protein is not codon-optimized; and / or (f) The nucleotide sequence encoding the PGRN protein is at least about 1600 bp, 1700 bp, 1750 bp, 1760 bp, 1770 bp, or 1780 bp, preferably the nucleotide sequence encoding the PGRN protein is about 1780 bp in length.

21. The nucleotide sequence encoding the PGRN protein is the nucleotide sequence of SEQ ID NO: 12, or includes a functional variant or fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 12; and / or The PGRN protein contains the amino acid sequence of SEQ ID NO: 13, or a functional variant or fragment thereof that has at least 70% identity with the amino acid sequence of SEQ ID NO:

13. A nucleic acid construct according to any one of claims 1, 2, and 4 to 20.

22. Furthermore, the nucleic acid construct according to any one of claims 1 to 21 includes the following: (a) Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) sequence, wherein the WPRE is located at the 3' end of a nucleotide sequence encoding a POI or PGRN protein, and / or comprises the nucleotide sequence of SEQ ID NO: 15, or a functional variant or fragment thereof having at least 90% identity to the nucleotide sequence of SEQ ID NO: 15; (b) Polyadenylated signal sequence, which may be: A polyadenylated signal sequence located at the 3' end of the nucleotide sequence encoding a POI or PGRN protein, and / or containing the nucleotide sequence of SEQ ID NO: 16, or a functional variant or fragment thereof having at least 90% identity to the nucleotide sequence of SEQ ID NO: 16; or (c) The above (a) and (b), The nucleotide sequence encoding the MeCP2 promoter, POI, or PGRN protein, the WPRE, and the polyadenylation signal sequence may be included in the 5' to 3' direction.

23. A nucleic acid construct according to any one of claims 1 to 22, wherein the bp is 3700-4700 bp, 3800-4800 bp, 3900-4700 bp, 4000-4600 bp, 4000-4500 bp, 4000-4400 bp, 4000-4300 bp, or 4000-4200 bp.

24. A vector comprising a nucleic acid construct according to any one of claims 1 to 23.

25. The vector according to claim 24, which is a plasmid or a viral vector.

26. (a) SEQ ID NO: 11, or a functional variant or fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 11; (b) SEQ ID NO: 10, or a functional variant or fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO:

10. The vector according to claim 24 or 25, which is a viral vector containing the nucleotide sequence.

27. A viral vector, selected from the following, according to any one of claims 24 to 26: (a) Adeno-associated virus (AAV) vector, A viral vector containing the AAV genome or a derivative thereof, wherein the derivative may be a chimeric, shuffled, or capsid-modified derivative; or (b) Lentiviral vector, or A viral vector containing a lentiviral genome or its derivatives.

28. The viral vector according to claim 27, comprising a genome derived from AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or AAV serotype rh10 (AAVrh10), preferably wherein the AAV comprises a genome derived from AAV2, AAV9, or AAVrH10.

29. The AAV vector according to claim 28, wherein the AAV vector comprises a genome derived from AAV2, and preferably the AAV is AAV-TT.

30. The AAV vector is in the direction from 5' to 3': (a) 5'ITR; (b) 5' adjacent fragment; (c) Minimum MeCP2 promoter array; (d) at least one synthetic intron; (e) Kozak sequence; (f) Polynucleotide sequence encoding the PGRN protein; (g) SV40 poly(A) sequence; (h) 3' adjacent fragment; and (i) 3'ITR The AAV vector according to claim 28 or 29, comprising a nucleotide sequence containing one or more of the following.

31. The AAV vector according to claim 30 is as follows: (a) The 5'ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 20, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO: 20; (b) The 5' adjacency fragment comprises or consists of the nucleotide sequence of SEQ ID NO: 21, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO: 21; (c) The minimum MeCP2 promoter sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 1, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO: 1; (d) At least one synthetic intron comprises or consists of the nucleotide sequence of SEQ ID NO: 2, or a functional variant or fragment thereof having at least 70% identity with respect to SEQ ID NO: 2; (e) The Kozak sequence contains or consists of the nucleotide sequence of Sequence ID No. 24; (f) The polynucleotide sequence encoding the PGRN protein comprises or consists of the nucleotide sequence of SEQ ID NO: 12, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO: 12; (g) The SV40 poly(A) sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 16, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO: 16; (h) The 3' adjacent fragment contains or consists of the nucleotide sequence of SEQ ID NO: 22, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO: 22; and / or (i) The 3'ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 23, or a functional variant or fragment thereof having at least 70% identity with SEQ ID NO:

23.

32. The AAV vector according to any one of claims 29 to 31, wherein the AAV vector comprises the nucleotide sequence of SEQ ID NO: 17, or a functional variant or fragment thereof having at least 70% identity with respect to the nucleotide sequence of SEQ ID NO:

17.

33. AAV vectors: (a) SEQ ID NO: 18, or a functional variant or fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 18; or (b) SEQ ID NO: 19, or a functional variant or fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO:

19. An AAV vector according to any one of claims 29 to 32, comprising or consisting of a nucleotide sequence.

34. A host cell, which may be a HEK293 cell or a HEK293 T cell, that produces a nucleic acid construct according to any one of claims 1 to 23 and / or a vector according to any one of claims 24 to 33, and / or a viral vector according to any one of claims 26 to 33.

35. A pharmaceutical composition comprising a nucleic acid construct according to any one of claims 1 to 23, a vector according to claim 24 or 25, and / or a viral vector according to any one of claims 26 to 33, together with a pharmaceutically acceptable carrier, excipient, or diluent.

36. A nucleic acid construct according to any one of claims 1 to 23, a vector according to claim 24 or 25, a viral vector according to any one of claims 26 to 33, and / or a pharmaceutical composition according to claim 35, for use in a method of treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient requiring treatment or prevention of the said disease.

37. A method for treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient requiring treatment or prevention of the said disease, comprising administering to the patient a therapeutically effective amount of a nucleic acid construct according to any one of claims 1 to 23, a vector according to claim 24 or 25, a viral vector according to any one of claims 26 to 33, and / or a pharmaceutical composition according to claim 35.

38. Use of a nucleic acid construct according to any one of claims 1 to 23, a vector according to claim 24 or 25, a viral vector according to any one of claims 26 to 33, and / or a pharmaceutical composition according to claim 35, for manufacturing a pharmaceutical for treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient requiring treatment or prevention of said disease.

39. The following are nucleic acid constructs, vectors, viral vectors or pharmaceutical compositions for use according to claim 36, the method according to claim 37, or the use according to claim 38: Diseases characterized by PGRN deficiency are central nervous system disorders; Diseases characterized by PGRN deficiency are characterized by a deficiency of PGRN in the patient's neurons and / or astrocytes; The patient has a loss-of-function mutation in at least one allele of the GRN gene; and / or The patient has loss-of-function mutations in both alleles of the GRN gene.

40. A nucleic acid construct, vector, viral vector or pharmaceutical composition for use according to claim 36 or 39, the method according to claim 37 or 39, or the use according to claim 38 or 39, wherein the disease characterized by PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis type 11 (NCL11).

41. The following are nucleic acid constructs, vectors, viral vectors or pharmaceutical compositions for use according to claim 36, 39, or 40, the method according to claim 37, 39, or 40, or the use according to any one of claims 38 to 40: The nucleic acid construct, vector, viral vector, or pharmaceutical composition is administered to a patient by delivery to the patient's brain and / or cerebrospinal fluid (CSF). Here, the delivery may be carried out by injection as follows: (i) the patient's brain; the injection into the brain is preferably selected from intracerebral injection, intraparenchymal injection, intrahypothalamic injection, and combinations thereof; and / or (ii) The patient's CSF; the injection into the CSF is preferably selected from intracisional injection, intrathecal injection, intraventricular (ICV) injection, and a combination thereof.

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