vector

Abstract

The present invention relates to the field of recombinant viral vectors suitable for in vivo delivery of therapeutic genes. Described is an adeno-associated viral (AAV) vector comprising (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synapsin promoter sequence, and / or (iii) a progranulin 3' untranslated region (UTR) sequence operably linked to a polynucleotide sequence encoding a polypeptide of interest. A particular use of such vectors is to enhance expression of a polypeptide of interest, such as progranulin (PGRN), to treat subjects who have a genetic mutation or whose endogenous polypeptide levels are lower than physiologically normal levels.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to the field of gene recombinant viral vectors. In particular, the present invention relates to gene recombinant viral vectors suitable for in vivo delivery of therapeutic genes.

Background Art

[0002] (Background of the Invention) Frontotemporal dementia (FTD) is the second most common form among dementia patients showing symptoms under 65 years old. This dementia is characterized by changes in behavior, personality, and language, and causes great distress to the patient himself / herself, his / her family, and friends. Approximately 30% of FTD patients have a family history of dementia, and among them, nearly 20% have a loss-of-function (LoF) mutation in the GRN gene encoding the protein progranulin. Children with homozygous GRN mutations develop neuronal ceroid lipofuscinosis (NCL11), a lysosomal storage disorder characterized by neurodegeneration and blindness (J.D. Rohrer et al., (2015b) Lancet Neurol. 14:253 - 262). Adults with heterozygous LOF mutations develop adult-onset FTD with lysosomal storage lesions and TDP-43 inclusion formation in the cortical neurons of the frontal and temporal lobes. MRI scans of pre-onset GRN mutation carriers show structural differences in the temporal and parietal lobes approximately 15 years before the predicted clinical onset (Rohrer et al., supra).

[0003] Currently, there is no effective treatment method that can significantly change the disease progression of any form of FTD, amyotrophic lateral sclerosis (ALS), or any form of neurodegenerative disease. The only drugs prescribed for FTD are drugs for reducing the effects of depressive states (antidepressants) or drugs for reducing the effects of problematic behaviors (major tranquilizers), and these often have serious and harmful short-term and long-term side effects.

[0004] Progranulin (PGRN) is a secreted glycoprotein that has important nutrient effects on neurons, suppresses inflammation, and plays a crucial role in autophagy (BPChitramuthu et al., (2017) Brain. 140(12):3081-3104). Progranulin is produced by microglia and neurons, but is taken up by all cell types. When progranulin binds to its receptor, sorbitol, on the cell membrane, it is transported by endosomes to lysosomes, where it is broken down into several granulins (CJHoller et al., (2017) eNeuro. 18:4). One of these granulins acts as a chaperone for the protease cathepsin D, promoting lysosomal acidification and effective proteolysis. Lysosomal defects inhibit autophagy, leading to the accumulation of TDP-43 in the nucleus and cytoplasmic inclusions.

[0005] GRN- / - mice exhibit subtle behavioral phenotypes (reduced social dominance) and do not show TDP-43 proteinopathy or neuronal loss, even when they develop lipofuscinosis, reactive gliosis, and lysosomal abnormalities (AWArrant et al., (2018) J. Neurosci. 38(9):2341-2358). Selective knockout of GRN from neurons in mice (TLPetkau et al., (2017) Neurobiol Dis. 106:14-22) or partial knockout from microglia (TLPetkau et al., (2017) J. Neuroinflammation. 14(1):225) reduced PGRN levels by approximately 50%, but did not result in any phenotype or detectable disease state. This indicates that both cell types are capable of secreting sufficient progranulin to provide cross-collection throughout the tissue.

[0006] Despite the lack of a suitable animal model, previous studies using viral vectors to deliver PGRN have shown promise. Studies showing that mouse GRN genes delivered via adeno-associated virus 1 (AAV1) and intraparenchymally injected into 12-month-old GRN- / - mice reduced lipofuscinosis and inflammation suggest that progranulin supplementation may reverse the disease progression (AEArrant et al., (2017) Brain. 140(5):1447-1465; AEArrant et al., (2018) cited above). However, another study using human GRN delivered via AAV9 and AAV4 at approximately 33 times higher doses reported significant hippocampal degeneration with T cell infiltration in GRN- / - mice and non-transgenic mice after 3 months (DAAmado et al., doi: https: / / doi.org / 10.1101 / 308692).

[0007] Recent, remarkable successes in trials of AAV9-SMN in type I spinal muscular atrophy (SMA) have spurred the search for gene therapy for neurological disorders. SMA is caused by homozygous deletion of the survival motor neuron gene (SMN). These children are born with muscle weakness, and 95% require mechanical ventilation before the age of two, becoming progressively weaker and unable to sit, crawl, stand, or speak. In 15 patients who received a single infusion of AAV9-SMN between 1 and 7 months of age, none required mechanical ventilation, 14 of the 15 showed significant muscle strength, were able to stand or walk without assistance, and all were able to speak (JRMendell et al., (2017) N. Engl. J. Med. 377(18):1713-1722).

[0008] However, high doses of intravenous AAV9-SMN have been reported to be toxic in non-human primates and miniature pigs (C. Hinderer et al., (2018) Hum. Gene Ther. 29:285-298), and therefore careful consideration is needed to minimize the vector dose in any clinical trial. Furthermore, the creation of AAV vectors is an extremely expensive process, with current estimates reaching up to $1 million per patient.

[0009] Overexpression of GRN can double the survival rate of transgenic mice expressing TDP-43 mutants, which are models of amyotrophic lateral sclerosis (ALS) and FTD (S. Beel et al., (2018) Mol. Neurodegener. 13(1):55). AAV-GRN therapy may be successful in treating ALS and FTD caused by TDP-43 accumulation, which are unrelated to GRN mutations. The removal of amyloid-beta deposits in the brain was also enhanced by administering a lentivirus that delivers GRN to a transgenic mouse model of Alzheimer's disease (SSMinami et al., (2014) Nat. Med. 20(10):1157-64), and the delivery of the progranulin gene was shown to be neuroprotective in an MTPT toxin model of Parkinson's disease (JMVan Kampen et al., (2014) PLoS One. 9(5):e97032). Because GRN can enhance autophagy and reduce inflammation, AAV-GRN may also offer therapeutic effects against many other neurodegenerative disorders in which misfolded proteins accumulate.

[0010] Many research groups are already investigating various approaches to find appropriate treatments for FTD-GRN patients, including the delivery of progranulin using AAV vectors. For example, Passage Bio has received substantial funding to develop AAV-delivered therapies to treat rare monogenic CNS diseases, including FTD (https: / / www.passagebio.com / investors-and-media / news-and-events / press-releases / press-release-details / 2019 / Passage-Bio-Launches-with-1155-Million-Series-A-to-Develop-AAV-Delivered-Therapeutics-to-Treat-Rare-Monogenic-CNS-Diseases / default.aspx). International patent application publication WO2017 / 151884 also describes the use of a vector containing a nucleic acid encoding progranulin, in which the vector transduces cells into which it comes into contact with cerebrospinal fluid (CSF) and expresses progranulin. Specifically, the vector contains an AAV capsid protein, and its progranulin nucleic acid is inserted between a pair of AAV inverted terminal repeats.

[0011] However, high-dose AAV vectors are known to be toxic, and from this perspective, there is a practical need to optimize the expression of progranulin proteins introduced by such vectors so that administration of these vectors can restore brain progranulin levels to physiological levels in subjects with loss-of-function (LoF) mutations in the GRN gene, and in subjects with reduced, suppressed, or decreased progranulin levels. In fact, the optimization of therapeutic protein expression will be applied to and expanded for many diseases in which the cause of the disease is a genetic defect or disruption of a physiological system that produces proteins at levels lower than physiologically normal. Therefore, there is a need for improved AAV vectors that can increase the expression of heterologous genes, such as progranulin, in the brain. [Overview of the project]

[0012] (Summary of the invention) In one aspect, the present invention provides an adeno-associated virus (AAV) vector comprising a nucleic acid including a human growth hormone intron 3 (hGHi3) sequence operably linked to a polynucleotide sequence encoding a polypeptide of interest.

[0013] In a further embodiment, the present invention provides an adeno-associated virus (AAV) vector comprising a synapsin promoter sequence operably ligated to a polynucleotide sequence encoding a polypeptide of interest. Preferably, the polypeptide of interest is progranulin.

[0014] In a further embodiment, the present invention provides an adeno-associated virus (AAV) vector comprising a progranulin 3' untranslated region (UTR) sequence operably ligated to a polynucleotide sequence encoding a polypeptide of interest. Preferably, the polypeptide of interest is progranulin.

[0015] In a further embodiment, the present invention provides an adeno-associated virus (AAV) vector comprising a polynucleotide sequence encoding progranulin, the polynucleotide sequence having at least 95% sequence identity with SEQ ID NO: 4.

[0016] In one embodiment, the hGHi3 sequence may include the sequence of SEQ ID NO: 7, or a variant, fragment, or homolog thereof. For example, a preferred variant may include a sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 7, for example, over at least 30, 50, 70, 80, or 90 residues of the full length of SEQ ID NO: 7, or over its entire length.

[0017] Preferably, the polynucleotide sequence encodes a heterologous polypeptide or transgenic protein, such as a non-AAV protein. In some embodiments, the polynucleotide sequence may encode a mammalian polypeptide, such as a human polypeptide or protein. In one embodiment, the polynucleotide sequence may include the complete coding sequence of the polypeptide of interest or a portion of the coding sequence of that polypeptide. For example, the coding sequence may be derived from a GRN and may encode a progranulin (PGRN), such as human progranulin. It is preferable that the polynucleotide sequence includes the complete coding sequence of a PGRN.

[0018] Based on the above, the polynucleotide sequence preferably encodes an amino acid sequence defined as SEQ ID NO: 16, or a fragment, homolog, or variant thereof. In one preferred embodiment, the encoded amino acid sequence may have at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 16, for example, over at least 50, 100, 200, 300, or 500 residues of the full length of SEQ ID NO: 16, or over its entire length.

[0019] In one embodiment, the polynucleotide sequence may include the wild-type coding sequence of human programurin (SEQ ID NO: 1), or a variant, fragment, or homolog thereof.

[0020] In one preferred embodiment, the polynucleotide sequence may be codon-optimized, for example, for expression in humans. In one preferred embodiment, the codon-optimized sequence has a human codon adaptation index (CAI) of at least 0.8, more preferably at least 0.9. Most preferably, the codon-optimized sequence has a CAI of at least 0.9 for both humans and mice. Preferably, the codon-optimized sequence has a GC content of 30-70%, most preferably 60-65%. In one embodiment, the codon-optimized sequence has sequence similarity or sequence identity of at least 50%, 60%, 70%, 75%, or most preferably at least 80% with the wild-type progranulin sequence.

[0021] Codon optimization may introduce novel or different regulatory elements that enhance protein production. Such regulatory elements may further restrict expression in specific tissues and / or organs, such as the brain. In one example, the codon-optimized sequence may include the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or its variants, fragments, or homologs. In a particularly preferred embodiment, the codon-optimized sequence may include the sequence of SEQ ID NO: 4, or its variants, fragments, or homologs, i.e., based on Candidate III (PGRN-GS) described in the examples herein.

[0022] A preferred variant may include a sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, for example, over at least 100, 200, 300, 500, 1000, or 1500 residues of the full length of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or over its entire length. In a particularly preferred embodiment, the polynucleotide sequence may include a sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 4, for example, over at least 100, 200, 300, 500, 1000, or 1500 residues of the full length of SEQ ID NO: 4, or over its entire length.

[0023] Generally, a preferred fragment may comprise at least 20, 30, 50, 100, 200, 300, 500, 1000 or 1500 residues of any one of the polynucleotide sequences or amino acid sequences described herein, for example, at least 20, 30, 50, 100, 200, 300, 500, 1000 or 1500 residues of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

[0024] In another embodiment, the AAV vector may further comprise an exonic splicing element (ESE). The ESE may be upstream of the polynucleotide coding sequence, for example, within the 5' flanking sequence. It will be recognized that the ESE may be inserted as part of or within the flanking sequence. Such a 5' flanking sequence may be a guide sequence, for example, a 5' flanking sequence derived from a wild-type polynucleotide sequence. For example, the 5' flanking sequence may be a 5' guide sequence derived from the wild-type GRN gene. In one example of this case, the wild-type GRN 5' guide sequence comprises about 300 to 500 base pairs, such as 350 to 450 base pairs, or about 392 base pairs.

[0025] In one preferred embodiment, the AAV vector may further comprise a 3' untranslated region (UTR) derived from progranulin. The 3' UTR of PGRN functions as an exonic enhancer element and may enhance and potentially regulate PGRN expression. The 3' UTR of PGRN may be located, for example, in a cassette or vector downstream of the polynucleotide coding sequence, i.e., in the 3' region. Preferably, the cassette or vector comprises the 3' UTR of human PGRN, or a variant, fragment or homolog thereof. For example, the cassette or vector may comprise the sequence of SEQ ID NO: 14, or a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 14, for example, over at least 100, 200, 250 residues or over the full length of SEQ ID NO: 14.

[0026] In a further embodiment, the polynucleotide sequence may include a signal sequence derived from human growth hormone (hGH) (i.e., a sequence encoding a signal peptide). This hGH signal sequence may include the sequence of SEQ ID NO: 9, or a variant or homolog thereof. In some embodiments, the signal sequence may replace the signal sequence present in the sequence encoding the polypeptide of interest. In a specific example, the hGH signal sequence replaces residues 1-51 of GRN.

[0027] In a still further embodiment, the AAV vector may further include a sequence that confers tissue-specific or cell-type-specificity to the expression of its protein. For example, a neuron-specific promoter such as a promoter derived from synapsin may be included in the expression cassette. The promoter may include, for example, the sequence of SEQ ID NO: 15 or a variant, fragment or homolog thereof. For example, the cassette or vector may include the sequence of SEQ ID NO: 15, or a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 15, over at least 50, 100, 200, 300 or 400 residues of the full length of SEQ ID NO: 15, or over its full length.

[0028] In another embodiment, the present invention provides an AAV vector comprising a human synapsin promoter, hGHi3 and a progranulin 3'UTR, operably linked to a polynucleotide sequence encoding a polypeptide of interest. Preferably, this polynucleotide sequence may be codon-optimized, for example, codon-optimized for expression in humans. More preferably, the polypeptide of interest is progranulin. The AAV vector may include, for example, the sequence of SEQ ID NO: 17 or a variant, fragment or homolog thereof. For example, the AAV vector may include the sequence of SEQ ID NO: 17, or a sequence having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 17, over at least 100, 200, 300, 500, 1000, 1500, 2000, 2500 or 3000 residues of the full length of SEQ ID NO: 17, or over its full length.

[0029] In another example, this sequence may additionally or alternatively restrict the expression of the target polypeptide to either dividing or non-dividing cells, such as non-dividing neurons. In this case, the risk of carcinogenicity in either case would be reduced.

[0030] Accordingly, in a specific example, the present invention provides an AAV vector cassette for GRN gene delivery for efficient expression and secretion of progranulin to replenish PGRN levels in the brains of subjects suffering from ALS, FTD, NCL11, and related neurodegenerative disorders. Such subjects may have progranulin levels lower than normal physiological levels, and may have GRN mutations or be in a state of partial or complete deficiency of this protein.

[0031] If it is recognized that the present invention encompasses the use of any suitable serotype of AAV, serotype AAV9 is a preferred example.

[0032] In a second aspect, the present invention relates to a pharmaceutical composition or drug comprising (i) an AAV vector as described herein, and (ii) one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0033] In one embodiment, the pharmaceutical composition or drug may be formulated to be administered or delivered directly or indirectly to the brain, or to specific areas of the brain such as the frontal lobe, temporal lobe and / or parietal lobe.

[0034] In a third embodiment, the pharmaceutical compositions or medicinal products described herein may be used for the treatment of a disease, preferably a neurological disease or neurological disorder. Typically, the disease being treated is a neurodegenerative disease. In specific embodiments, neurological disorders include frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease.

[0035] A pharmaceutical composition or drug may be used to treat a patient suffering from this disease, or any subgroup of such patients. In some embodiments, the pharmaceutical compositions or drugs described herein are used to treat (i) subjects with heterozygous, homozygous, or compound heterozygous GRN mutations, (ii) subjects suffering from sporadic neurological disorders, and / or (iii) subjects with PGRN levels lower than physiologically normal levels. “Sporadic disorder” means a subject who is not considered to have another family member suffering from the disorder, or a subject who does not have a loss-of-function GRN mutation associated with the disorder.

[0036] In other words, the use of the pharmaceutical compositions or medicinal products described herein may also be for the manufacture of medicinal products for the treatment of neurological disorders, for example, for the treatment of patients with frontotemporal dementia (FTD-GRN) with heterozygous GRN mutations, or for the treatment of patients with neuronal ceroid lipofuscinosis (NCL11) with homozygous or compound heterozygous GRN mutations. The medicinal products may also be used for other neurological disorders in persons who do not have PGRN levels lower than physiologically normal levels, for example, patients with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease without GRN mutations, and subjects suffering from other sporadic neurological disorders.

[0037] The pharmaceutical compositions or drugs of the present invention may also be used in therapeutic methods, in which a therapeutically effective amount of a pharmaceutically or physiologically acceptable composition comprising the AAV vector described herein may be administered to subjects in need of treatment for neurological disorders, such as frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease, including, for example, subjects suffering from sporadic neurological disorders and / or subjects having PGRN levels lower than physiologically normal. For example, the pharmaceutical compositions or drugs of the present invention may be administered as gene therapy to FTD and ALS patients who have recently shown symptoms, thereby inhibiting the degenerative process. Such treatment may even have the potential to save neurons that are functionally impaired but still alive. Even moderate improvement of dementia symptoms and prevention of progression would dramatically reduce the care burden on the patient's family and community.

[0038] It will be recognized that this composition or pharmaceutical product may be administered prophylactically, particularly to those at greatest risk, to prevent or limit the onset of disease.

[0039] In a further embodiment, the present invention provides nucleic acids (e.g., polynucleotides) operably linked to a heterogeneous polynucleotide sequence encoding a polypeptide of interest (e.g., a polypeptide other than human growth hormone), for example, a nucleic acid (e.g., polynucleotide) comprising, for example, (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synapsin promoter sequence, and / or (iii) a progranulin 3' untranslated region (UTR) sequence. Preferably, the polypeptide of interest comprises progranulin. The coding sequence of the polypeptide may be codon-optimized, for example, as described herein.

[0040] In a further embodiment, the present invention provides a nucleic acid sequence comprising, for example, at least 100, 200, 300, 500, 1000, or 1500 residues of the entire length of SEQ ID NO: 4, or the entire length thereof, a sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 4.

[0041] This nucleic acid sequence may further include one or more regulatory elements (such as promoters) or other elements with respect to an AAV vector containing nucleic acids, etc., as described above. Therefore, in one embodiment, this nucleic acid includes a neuron-specific promoter (e.g., a synapsin promoter). The nucleic acid may include, for example, an expression cassette suitable for use in an AAV vector, and therefore may include, for example, one or more AAV inverted terminal repeats. [Brief explanation of the drawing]

[0042] (Brief explanation of the drawing) [Figure 1] Figure 1: Screening of secreted PGRN expression for codon-optimized candidates. Figure 1a: Twelve codon-optimized PGRN sequences were synthesized under the CMV promoter. HEK-293 cells were used for transfection (250 ng / 2E+10 cells). After 48 hours, the culture medium was collected and Western blot analysis was performed. Figure 1b: PGRN expression intensity was measured for each candidate using ImageJ. Figure 1c: CAI scores before and after double codon optimization (human and other species). [Figure 2] Figure 2: hPGRN fusion cloning strategy. Figure 2a: Schematic diagrams of candidate hPGRNs I, II, and III, codon-optimized using different algorithms. Figure 2b: The 5' sequence of wild-type human PGRN was cleaved using BamH1 and BstB1. The digested DNA fragments were replaced with codon-optimized PGRN candidates to create fusion plasmids.

[0043] [Figure 3]Figure 3: Quantification of PGRN secreted from codon-optimized constructs in cell culture medium. Figure 3a: HEK-293 cells were transfected with PGRN-optimized codons and fusion constructs. After 48 hours, cell culture medium was sampled and PGRN levels were analyzed by Western blotting. Figure 3b: Secreted PGRN levels were measured for each candidate using ImageJ, and the values ​​were standardized first by cell number (protein GAPDH) and then by cassette copy number (CMV / GAPDH qPCR). The results from three biological replicas and four independent experiments were plotted on a scatter plot and subjected to one-way ANOVA statistical analysis (*<0.05).

[0044] [Figure 4] Figure 4: Quantification of PGRN expressed from codon-optimized constructs in cell culture lysates. Figure 4a: HEK-293 cells were transfected with PGRN-optimized codons and fusion constructs. After 48 hours, the cell cultures were lysed and PGRN levels were analyzed by Western blotting. Figure 4b: Expressed PGRN levels were measured for each candidate using ImageJ, and the values ​​were standardized first by cell number (protein GAPDH) and then by cassette copy number (CMV / GAPDH qPCR). The results from three biological replicas and four independent experiments were plotted on a scatter plot and subjected to one-way ANOVA statistical analysis (*<0.05).

[0045] [Figure 5] Figure 5: Intron-mediated enhancement of human growth hormone in PGRN protein expression. Figure 5a: Schematic diagram of the human growth hormone intron sequence located in the genome sequence. Figure 5b: hGH introns 1, 2, or 3 (Int-1, Int-2, or Int-3) were cloned into the 5'UTR of human wild-type (wt) PGRN. Figure 5c: The human PGRN signal sequence (18-amino acid peptide) was replaced with the human growth hormone signal sequence to test which could enhance PGRN secretion.

[0046] [Figure 6]Figure 6: PGRN secretion was enhanced by hGH intron 3 and the hGH signal sequence. Figure 6a: PGRN intron sequence and signal peptide sequence were transfected into HEK-293 cells. After 48 hours, cell culture medium was sampled and PGRN levels were analyzed by Western blotting. Figure 6b: Secreted PGRN levels were measured for each candidate using ImageJ, and the values ​​were standardized first by cell number (protein GAPDH) and then by cassette copy number (CMV / GAPDH qPCR). The results of three biological replicas and four independent experiments were plotted on scatter plots and subjected to one-way ANOVA statistical analysis (*<0.05, ***<0.0005).

[0047] [Figure 7] Figure 7: PGRN expression in cell lysates was enhanced by hGH intron 3, without hGH signaling. Figure 7a: PGRN intron sequences and signal peptide sequences were transfected into HEK-293 cells. After 48 hours, the cell cultures were lysed and PGRN levels were analyzed by Western blotting. Figure 7b: Expressed PGRN levels were measured for each candidate using ImageJ, and the values ​​were standardized first by cell number (protein GAPDH) and then by cassette copy number (CMV / GAPDH qPCR). The results of three biological replicas and four independent experiments were plotted on a scatter plot and subjected to one-way ANOVA statistical analysis (***<0.0001).

[0048] [Figure 8]Figure 8: hGHi3 did not enhance GRN expression from codon-optimized PGRN-GS. Figure 8a: PGRN-GS with hGH intron 3 inserted was transfected into HEK-293 cells. After 48 hours, the culture medium was collected and used for Western blotting for GRN expression. Figure 8b: Expression levels were quantified using ImageJ and Image Studio, and the values ​​were standardized first by cell number (protein GAPDH) and then by cassette copy number (CMV / GAPDH qPCR). Figure 8c: ESE elements of PGRN wild-type sequence and codon-optimized PGRN-GW were analyzed using ESE Finder 2.0. SR protein binding scores were plotted in a bar graph. Each color represents a different type of SR splicing protein.

[0049] [Figure 9] Figure 9: The hGHi3-PGRN fusion construct enhanced GRN expression in primary neurons. Figure 9a: Schematic diagram of hGHi3 cloning for the hGHi3-PGRN fusion construct. Figure 9b: An AAV containing hGHi3-PGRN-GA was created and transduced into primary cortical neurons of 7-day-old rats. After 5 days of incubation, the culture medium was collected and PGRN expression levels were analyzed by Western blot. Figure 9c: Expressed PGRN levels were measured for each candidate using ImageJ, and the values ​​were standardized first by cell number (protein GAPDH) and then by cassette copy number (CMV / GAPDH qPCR). The results of three biological replicas and four independent experiments were plotted on a scatter plot and subjected to one-way ANOVA statistical analysis.

[0050] [Figure 10]Figure 10: The synapsin promoter increased PGRN secretion by rat cortical neurons. Figure 10a: Wild-type PGRN was cloned using the CMV promoter and synapsin (Syn) promoter in an AAV shuttle vector. Plasmids were transfected into rat cortical neurons on day 7 for 5 days, then culture media were sampled and processed by Western blotting. Figure 10b: PGRN expression intensity was measured by ImageJ.

[0051] [Figure 11] Figure 11: AAV9 containing Syn-PGRNwt enhanced PGRN expression in cultured rat neurons. Figure 11a: Viral particles were prepared using a neuron-specific AAV9 capsid. The AAV particles were then transduced into primary rat cortical neurons on day 7, and 1E+6 viral particles were transduced for 5 days. The culture medium was used for Western blot analysis. Figure 11b: PGRN protein levels were measured by ELISA assay (0.001).

[0052] [Figure 12] Figure 12: In vivo distribution of AAV9-PGRN transduction in mouse organs. Figure 12a: AAV9-CMV-PGRN-WT and AAV9-Syn-PGRN-WT were administered by bilateral ICV infusion (1E+12 GC / Kg). Figure 12b: qPCR of human PGRN was performed on genomic DNA to determine its distribution in in vivo tissues.

[0053] [Figure 13]Figure 13: AAV9 containing Syn-PGRNwt enhanced PGRN expression in the mouse cortex. AAV9 vectors containing CMV-PGRN-WT and AAV9 vectors containing Syn-PGRN-WT were delivered by bilateral ICV infusion (1E+12 GC / Kg). Figure 13a: A commercially available ELISA kit (Adipogen) was used for quantitative analysis of PGRN expression levels in serum, CSF, and the cortex. Figure 13b: Serum was diluted 1:300 and used for ELISA (n=3). Figure 13c: CSF was diluted 1:200 and used for ELISA (n=1). Figure 13d: Cortical RIPA lysate was diluted 1:300 and then used for ELISA (n=3).

[0054] [Figure 14] Figure 14: Examples of PGRN vectors suitable for use in gene therapy. Figure 14a: Schematic diagram of a CMV promoter-driven codon-optimized PGRN construct consisting of hGH3 intron 3 in the 5'UTR. Figure 14b: Schematic diagram of a synapsin promoter-driven PGRN codon-optimized construct consisting of hGH3 intron 3 in the 5'UTR.

[0055] [Figure 15] Figure 15: ICV delivery of AAV9-Syn-PGRNwt shows broad distribution of PGRN in the mouse brain. (a) Human GRN expression was detected using IHC on slices of 4-week-old mouse brains. Images were acquired with a Nikon A1R confocal microscope. To reconstruct the whole brain image, scanned images were stitched together with a 20% overlap using the automated settings of the NIC software. Human GRN (green), NeuN (red). b) IHC analysis was performed on slices of brain without injection, but no GRN was detected. c) Cortex, d) Hippocampus, e) Thalamus.

[0056] [Figure 16]Figure 16: AAV9 containing Syn-PGRNwt enhanced PGRN expression in the mouse hippocampus. AAV9 vectors containing CMV-PGRN-WT and AAV9 vectors containing Syn-PGRN-WT were delivered by bilateral ICV infusion (2.5E+10). a) A commercially available ELISA kit (Adipogen) was used for quantitative analysis of PGRN expression levels in serum, CSF, and cortex. b) ELISA of PGRN for AAV9-Syn-EGFP, AAV9-Syn-PGRNwt, AAV9-CMV-EGFP, and AAV9-CMV-PGRNwt (N=3). c) Western blot of PGRN for AAV9-Syn-EGFP and AAV9-Syn-PGRNwt. PGRN was detected at approximately 68 kDa only in hippocampal lysates injected with AAV9-Syn-PGRNwt. d) Western blot of PGRN for AAV9-CMV-EGFP and AAV9-CMV-PGRNwt. [Figure 17] Figure 17: Schematic diagram of Syn-hGHi3-PGRN-GS-UTR. (a) Schematic diagram of a codon-optimized PGRN construct, Syn promoter-driven, consisting of hGH3 intron 3 and 3'UTR. (b) DNA sequence of human PGRN-UTR.

[0057] [Figure 18] Figure 18: AAV9 containing Syn-PGRN-GS, Syn-hGHi3-PGRN-GS, and Syn-hGHi3-PGRN-GS-UTR enhanced PGRN expression in mouse cortex. AAV9 vectors containing Syn-PGRN-GS, Syn-hGHi3-PGRN-GS, and Syn-hGHi3-PGRN-GS-UTR, respectively, were delivered by bilateral IT injection. Cell lysates from cortical tissue were subjected to PGRN ESLISA assay and Western blot analysis. a) A commercially available ELISA kit (Adipogen) was used for quantitative analysis of PGRN expression levels in the cortex. b) Cortical RIPA lysates were diluted 1:300 and then used for ELISA (N=3). c) Cortical RIPA lysates from low (6.2E+10), medium (1.2E+11), and high (2.5E+11) levels were used for ELISA (N=3).

[0058] [Figure 19] Figure 19: Immunohistochemistry of AAV9-PGRN-GS and AAV9-hGHi3-PGRN-GS-UTR. Mice were harvested after IT injection at week 4. Free-frozen tissue was sectioned to a thickness of 30 μM and immunostained overnight with human-specific goat anti-GRN antibody (green). DAPI was used as a counterstain (blue). (a) and (a') AAV-EGFP, (b) and (b') AAV9-PGRN-GS, and (c) and (c') AAV9-PGRN-GS-UTR.

[0059] [Figure 20] Figure 20: High-power 3D rendering images of PGRN in the mouse brain. The distribution of human PGRN was studied in the mouse cortex using immunofluorescence to determine whether PGRN was secreted and taken up by other cells. (a) Many cortical neurons (stained red with NeuN) were positive for human PGRN (green), and this was similarly confirmed in the 3D rendering image (c). Some NeuN-negative cells indicate that PGRN secreted by neurons was taken up by glial cells. (b) Some microglia labeled with IBA1 (red in (b)) showed GRN-positive focus (green), which was similarly confirmed in the 3D rendering image (d), indicating that they were able to take up PGRN secreted by neurons. Brain sections were imaged with a two-photon microscope ((a) and (b)) or sequentially scanned with an A1R confocal microscope. The stacked images were processed for 3D rendering using IMARIS 8.3 to show GRN granules. Image of inside a neuron (c) and inside a microglia (d). Scale bar = 2 μM

[0060] [Figure 21]Figure 21: Plasmid map of the AAV vector for PGRN. The human growth hormone intron is inserted between the synapsin promoter and hPGRN. The native sequence of the hPGRN UTR was added to the 3' end of hPGRN. Cloning was performed from ITR to ITR using a newly synthesized kanamycin resistance skeleton plasmid. A) Linear form. B) Circular plasmid form. AAV2 ITR = adeno-associated virus type 2 inverted terminal repeat; hSyn promoter = human synapsin promoter; hGHi3 = human growth hormone intron 2; PGRN-GS = human programurin codon-optimized with GenScript®; hPGRN-3'UTR = human programurin 3' untranslated region; bGH poly(A) signal = bovine growth hormone polyadenylation signal; f1 ori = F1 origin of replication; KanR = kanamycin resistance gene.

[0061] (Sequence Listing) Sequence ID 1 - Human PGRN wild-type (PGRN-WT) DNA coding sequence; Sequence ID 2 - Candidate DNA coding sequence I for an artificially codon-optimized PGRN (PGRN-IDT); Sequence ID 3 - Candidate DNA coding sequence II for artificially codon-optimized PGRN (PGRN-GA); Sequence ID 4 - Candidate DNA coding sequence III for artificially codon-optimized PGRN (PGRN-GS); Sequence ID 5 - Intron 1 sequence of human growth hormone; Sequence ID 6 - Intron 2 sequence of human growth hormone; Sequence ID 7 - Intron 3 sequence of human growth hormone; Sequence ID 8 - Intron 4 sequence of human growth hormone; Sequence ID 9 - Generic DNA signaling sequence for human growth hormone; SEQ ID NO: 10 - The translated amino acid sequence of the generic signal sequence for human growth hormone; Sequence ID 11-5'ESE adjacent sequence RPL41; Sequence ID 12-5'ESE adjacent sequence UCHL1; Sequence ID 13-5'ESE adjacent sequence RPL38; Sequence ID 14 - Human PGRN3'UTR sequence; Sequence ID 15 - Synapsin promoter sequence; Sequence ID 16 - Human PGRN amino acid sequence; Sequence ID 17-Syn-hGHi3-PGRN-GS-UTR DNA cassette sequence (from ITR to ITR); Sequence ID No. 18 - Bovine growth hormone (bGH) poly(A) signaling; Sequence ID 19-5'AAV2 UTR sequence; Sequence ID 20-3'AAV2 UTR sequence. [Modes for carrying out the invention]

[0062] (Detailed description of the invention) In one embodiment, the present invention relates to an adeno-associated virus (AAV) vector comprising (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synapsin promoter sequence, and / or (iii) a progranulin 3' untranslated region (UTR) sequence, operably linked to a polynucleotide sequence encoding a polypeptide of interest.

[0063] The present invention encompasses specific embodiments in which an AAV vector cassette contains a codon-optimized PGRN gene that significantly enhances the production and secretion of progranulin protein. PGRN secretion will be further increased by placing its sequence under 5' regulatory control of hGH intron 3. The use of synapsin, a neuron-specific promoter, restricts progranulin expression in neurons in vitro and in vivo, thereby reducing the risk of toxicity and carcinogenesis in peripheral organs. To further enhance and regulate PGRN expression, a 3'UTR from PGRN may also be included in the cassette.

[0064] Since the late 1970s, it has been known that intron-containing and intron-deficient genes can exhibit dramatically different expression profiles, even if otherwise identical. hGHi3 is an intronic splicing element (ISE). However, the specific effect of hGHi3 on increasing the expression / secretion of transgenes within AAV vectors had not been previously known. Furthermore, the inventors have found that hGH intron 2 and hGH intron 4 have opposite effects. Therefore, the increased expression / secretion of transgenes from AAV vectors containing the hGHi3 sequence is both surprising and advantageous.

[0065] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly indicates otherwise. As used herein, the terms “contains,” “includes,” and “composed of” are synonymous with “encompassing,” “inclusion,” or “containing,” and are inclusive or open-ended form, not excluding additional unlisted components, elements, or method steps. The terms also encompass “consist of” and “essentially consist of.”

[0066] The detailed description of numerical ranges by endpoints includes all numbers and fractions contained within each range, as well as the endpoints described.

[0067] As used herein, the term "about" refers to a measurable value such as a parameter, quantity, or duration, and means to include variations in and from that value, in particular variations of and from the specified value of + / -10%, preferably + / -5%, more preferably + / -1%, and even more preferably + / -0.1%, to the extent that these variations are appropriate for carrying out the disclosed invention. It should be understood that the values ​​in which the modifier "about" is involved are as specifically and preferably disclosed as the values ​​themselves.

[0068] The term "one or more" in the sense of one or more members of a group of members is self-evident, but to illustrate further, this term encompasses references to any one of the above members or any two or more of the above members, for example, any of the above members ≥3, ≥4, ≥5, ≥6, or ≥7, and up to all of the above members.

[0069] The terms “nucleic acid” or “polynucleotide” refer to a (e.g., polymer) form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. In such cases, modifications to the nucleotide structure may be applied at either a point before or after the polymer assembly. As used herein, the term polynucleotide is interchangeable for both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the polynucleotides of the present invention described herein encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to form the double-stranded form.

[0070] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. These terms also include amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipid modification, phosphorylation, or binding with labeling components. Polypeptides such as anti-angiogenic polypeptides and neuroprotective polypeptides, when discussed in the context of polypeptides for delivering gene products to mammalian targets and compositions for such delivery, refer to intact polypeptides or any fragments thereof or genetically modified derivatives thereof that retain the desired biochemical function of an intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other nucleic acids used for gene product delivery to mammalian targets (sometimes referred to as “transgenes” delivered to recipient cells) include polynucleotides encoding intact polypeptides or any fragments or genetically modified derivatives having the desired biochemical function.

[0071] A polynucleotide or polypeptide is said to have a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when the two sequences are aligned and compared, the percentage of bases or amino acids is the same. Sequence similarity can be determined in various ways. For sequence alignment to determine sequence identity, methods such as the BLAST method (available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / ) and computer programs can be used. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package in Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment techniques are described in Methods in Enzymology, vol. 266: "Computer Methods for Macromolecular Sequence Analysis" (1996), edited by Doolittle, Academic Press, Inc. (a division of Harcourt Brace & Co., San Diego, California, USA). Of particular interest are sorting programs that allow sequence gaps. Smith-Waterman is a type of algorithm that allows gaps in sequence sorting. See Meth. Mol. Biol. 70: 173-187 (1997). Similarly, GAP programs using Needleman and Wunsch sorting methods can be used for sequence sorting. See J. Mol. Biol. 48: 443-453 (1970).

[0072] Of particular interest is the BestFit program, which determines sequence identity using the local homology algorithm described in Smith and Waterman's literature (Advances in Applied Mathematics 2:482-489 (1981)). The gap generation penalty is generally in the range of 1 to 5, usually 2 to 4, and will be 3 in many embodiments. The gap extension penalty is generally in the range of approximately 0.01 to 0.20, and will often be 0.10. This program has default parameters determined by the input sequences for comparison. Preferably, sequence identity is determined using the default parameters determined by this program. This program is also available from the Genetics Computing Group (GCG) package in Madison, Wisconsin, USA.

[0073] Another interesting program is the FastDB algorithm. FastDB is described in "Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications" pp. 127-149, 1988, Alan R. Liss, Inc. Percentage sequence identity is calculated by FastDB based on the following parameters: Mismatch penalty: 1.00; Gap penalty of 1.00; Gap size penalty 0.33; and Joining penalty: 30.0.

[0074] This disclosure provides (recombinant) adeno-associated virus (AAV) vectors. "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise required, this term encompasses all subtypes, as well as both native and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also called recombinant AAV vector (or "rAAV vector"). The term "AAV" includes, for example, AAV1 (AAV-1), AAV2 (AAV-2), AAV3 (AAV-3), AAV4 (AAV-4), AAV5 (AAV-5), AAV6 (AAV-6), AAV7 (AAV-7), AAV8 (AAV-8), AAV9 (AAV-9), AAV10 (AAV-10, including AAVrh10), AAV12 (AAV-12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovid mammals, and so on.

[0075] The genomic sequences of various serotypes of AAV, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are publicly known in this field. Such sequences can be found in the literature or in public databases such as GenBank. For example, see GenBank accessions NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC-006261 (AAV-8), whose disclosures are incorporated herein by reference. Similarly, for example, see the literature by Srivistava et al., (1983) J. Virology 45:555; the literature by Chiorini et al., (1998) J. Virology 71:6823; the literature by Chiorini et al., (1999) J. Virology 73:1309; the literature by Bantel-Schaal et al., (1999) J. Virology 73:939; the literature by Xiao et al., (1999) J. Virology 73:3994; the literature by Muramatsu et al., (1996) Virology 221:208; the literature by Shade et al., (1986) J. Virol. 58:921; the literature by Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; the literature by Moris et al., (2004) Virology See also 33:375-383; International Patent Publications WO00 / 28061, WO99 / 61601, WO98 / 11244; and U.S. Patent No. 6,156,303.

[0076] The AAV vectors described herein are typically recombinant AAV vectors (rAAV). As used herein, “rAAV vector” refers to an AAV vector containing a sequence of polynucleotides not of AAV origin (i.e., polynucleotides heterologous to AAV), typically a target sequence for genetic transformation of cells. In some embodiments, the heterologous polynucleotide may be flanked by at least one, sometimes two, AAV inverted terminal repeat sequences (ITRs). The ITRs are preferably derived from AAV serotype 2, i.e., the rAAV vector contains AAV2 ITRs. In some embodiments, the vector contains one or both of the following AAV2 ITR sequences or their homologs or variants: [ka] .

[0077] Even more suitable AAV ITR sequences are discussed, for example, in Wilmott et al.'s paper, (2019) Human Gene Therapy Methods Vol. 30, No. 6:206-213, and are available from publicly accessible databases.

[0078] The term "rAAV vector" encompasses both rAAV vector particles and rAAV vector plasmids. The rAAV vector may be either single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). A suitable cloning vector containing further sequence elements usable within the vector of the present invention is disclosed as RS540-AAV-ErbB-RASER1C-OFPBidBH3, disclosed in GenBank accession number MK801287.1.

[0079] An "AAV virus," "AAV virus particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (usually all of the capsid proteins of wild-type AAV) and a capsid-encapsulated polynucleotide rAAV vector. If this particle contains heterologous polynucleotides (i.e., polynucleotides different from the wild-type AAV genome, for example, a transgene for delivery to mammalian cells), it is usually called an "rAAV vector particle" or simply an "rAAV vector." Thus, since the vector is contained within the rAAV particle, the creation of an rAAV particle necessarily involves the creation of an rAAV vector.

[0080] As used herein, “recombinant” means that the vector, polynucleotide, polypeptide, or cell is a product obtained by a variety of combinations of cloning steps, restriction steps, or ligation steps (for example, steps relating to the polynucleotide or polypeptide contained therein), and / or other procedures that result in a construct distinctly different from the product found in nature. A recombinant virus or vector is a viral particle containing recombinant polynucleotides. These terms include, respectively, a replica of the original polynucleotide construct and a descendant of the original viral construct.

[0081] In embodiments of the present invention, the AAV vector includes a nucleic acid sequence encoding a gene product, such as a heterogeneous nucleotide sequence encoding a heterogeneous polypeptide. “Genes” refers to polynucleotides comprising at least one open reading frame capable of encoding a specific protein after transcription and translation. “Genes” are molecules resulting from the expression of a specific gene. Genes include, for example, polypeptides, aptamers, interfering RNA, mRNA, and the like.

[0082] "Heterogeneous" means that it originates from an entity whose genotype differs from the remaining entities within the entity being compared. For example, a polynucleotide introduced into a plasmid or vector from a different species using genetic engineering techniques is a heterogeneous polynucleotide. A promoter extracted from its native coding sequence is also a heterogeneous promoter if it is operably linked to a coding sequence to which it is not found to be linked in nature. Therefore, for example, an rAAV containing heterogeneous nucleic acid encoding a heterogeneous gene product is an rAAV containing nucleic acid not normally found in naturally occurring wild-type AAVs, and the heterogeneous gene product it encodes is a gene product not normally encoded by naturally occurring wild-type AAVs.

[0083] In one embodiment, the gene product (the polypeptide of interest) is a therapeutic protein. The "therapeutic" peptide or protein is a peptide or protein that can alleviate or reduce symptoms caused by the absence or defect of a protein in a cell or subject. Alternatively, the "therapeutic" peptide or protein imparts another effect to the subject, such as an anti-degenerative effect.

[0084] When a gene product is a polypeptide, that polypeptide is generally a polypeptide that enhances the function of cells, such as neurons, glial cells, or cells present in neuronal tissues like photoreceptor cells. Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors; VEGF-binding antibodies; VEGF-binding antibody fragments (e.g., single-chain anti-VEGF antibodies); endostatins; tamstatins; angiostatins; soluble Fit polypeptides (see Lai et al., (2005) Mol. Ther. 12:659); Fc fusion proteins containing soluble Fit polypeptides (see Pechan et al., (2009) Gene Ther. 16:10); pigment epithelial-derived factor (PEDF); soluble Tie-2 receptors; etc.); tissue inhibitors of metalloproteinase 3 (TIMP-3); photoresponsive opsins, e.g., rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl), etc.). Suitable polypeptides include, but are not limited to, glial cell line-derived neurotrophic factor (GDNF); fibroblast growth factor 2; neuronurin (NTN); ciliary body neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor; rhodopsin; X-linked apoptosis inhibitors; and sonic hedgehog. Suitable polypeptides are disclosed, for example, in WO2012 / 145601. However, in one preferred embodiment, the encoded polypeptide includes progranulin.

[0085] In embodiments of the present invention, the polynucleotide sequence is operably linked to (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synapsin promoter sequence, and / or (iii) a progranulin 3' untranslated region (UTR) sequence. In some embodiments, the polynucleotide sequence encoding the polypeptide is operably linked to a promoter, such as a constitutive promoter or an inducible promoter. In some cases, the nucleotide sequence encoding the polypeptide of interest is operably linked to a tissue-specific or cell-type-specific regulatory element.

[0086] For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably bound to a neuron-specific regulatory element (e.g., a neuron-specific promoter), such as a regulatory element that selectively expresses a gene operably bound within a neuron. Preferred neuron-specific promoters include, in particular, the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)); the neuronal fibrillary light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:561 1-5 (1991)); and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). However, the neuron-specific promoter is preferably a synapsin promoter.

[0087] A “regulatory element” or “regulatory sequence” is a nucleotide sequence involved in molecular interactions that contribute to the regulation of polynucleotide function, including replication, duplication, transcription, splicing, translation, or degradation. This regulation can affect the frequency, rate, or specificity of a process and may be inherently enhancing or inhibiting. Regulatory elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind to RNA polymerase and initiate transcription of a coding region typically located downstream (3' direction) of the promoter.

[0088] "Operatally linked" or "operably linked" refers to positioning gene elements proximal to each other in a way that enables them to operate in the expected manner. For example, if a promoter assists in the initiation of transcription of a coding sequence, then the promoter is operably linked to its coding region. Residues may be interposed between the promoter and the coding region, as long as this functional relationship is maintained.

[0089] As used herein, the term "(one or more) promoter" may refer to a DNA sequence located adjacent to a DNA sequence encoding a recombinant product. The promoter is preferably operably bound to the adjacent DNA sequence. A promoter typically increases the amount of recombinant product expressed from a DNA sequence compared to the amount expressed in the absence of the promoter. A promoter derived from one organism can be used to enhance the expression of a recombinant product from a DNA sequence originating from another organism. For example, a vertebrate promoter can be used to express jellyfish GFP in a vertebrate. Furthermore, one promoter element can also increase the amount of recombinant product expressed with respect to multiple tandem-connected DNA sequences. Thus, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well known to those skilled in the art.

[0090] As used herein, the term "(one or more) enhancer" may refer to a DNA sequence located adjacent to the DNA sequence encoding a recombinant product. Enhancer elements are typically located upstream of the promoter element, or downstream or within the encoding DNA sequence (e.g., the DNA sequence transcribed or translated into one or more recombinant products). Thus, enhancer elements may be located 100, 200, or 300 or more base pairs upstream or downstream of the DNA sequence encoding the recombinant product. Enhancer elements can increase the amount of recombinant product expressed from the DNA sequence beyond the high expression level achieved by the promoter element. Those skilled in the art will readily know that numerous enhancer elements are readily available.

[0091] In some embodiments, the AAV vector may further include a polyadenylation signal. For example, the poly(A) signal may typically be located at the 3' end of the cassette containing the polynucleotide encoding the polypeptide of interest. In one embodiment, the poly(A) signal is located downstream of the progranulin 3' untranslated region, i.e., between the 3'UTR sequence and one ITR adjacent to the cassette. In one embodiment, the poly(A) sequence includes or consists of a bovine growth hormone (bGH) poly(A) signal. Preferred polyadenylation signals (including bGH poly(A)) are known and are described, for example, in the literature of Choi et al., Molecular Brain 2014, 7:17; the literature of ECGoodwin, J Biol Chem. 1992;267:16330-16334 and U.S. Patent No. US5,122,458. In one embodiment, the bGH poly(A) signal includes the sequence of SEQ ID NO: 18, or its homolog or variant. [ka]

[0092] This disclosure provides pharmaceutical compositions or medicinal products comprising (a) the AAV vector described herein, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in humans.

[0093] "Pharmacopoeially acceptable" means a substance that is not biologically or otherwise undesirable, for example, a substance that can be administered to a subject without causing any undesirable biological effects. Accordingly, such pharmaceutical compositions can be used, for example, in the transfection of cells in vitro, or when viral particles or cells are directly administered to a subject.

[0094] Such excipients, carriers, diluents, and buffers include all medicinal agents that can be administered without excessive toxicity. Medicinal excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol.

[0095] As used herein, the term “one or more physiologically or pharmaceutically acceptable carriers, excipients, and / or diluents” is intended to include all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders, etc., that are suitable for administration to human or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are diluents, excipients, and / or carriers approved by a federal supervisory agency, a state government, or other supervisory agency, or listed in the United States Pharmacopeia and / or the European Pharmacopoeia or other generally accepted pharmacopoeias, for use in animals, including human and non-human mammals. The terms diluent, excipient, and / or “carrier” refer to the diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers may be sterile liquids such as water and oil, including petroleum-based, animal-based, plant-based, or synthetic-derived materials. Water, physiological saline, and aqueous glucose and glycerol solutions can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. These compositions may also contain small amounts of wetting agents, bulking agents, emulsifiers, or pH buffers, as needed. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. Examples of suitable pharmaceutical diluents, excipients, and / or carriers are described in E.W. Martin's literature "Remington's Pharmaceutical Sciences." Formulations must be adapted to the mode of administration. Appropriate diluents, excipients, and / or carriers will be obvious to those skilled in the art and will largely depend on the route of administration.

[0096] Medicinal salts may include, for example, mineral salts such as hydrochloride, hydrobromide, phosphate, and sulfate; and organic salts such as acetate, propionate, malonate, and benzoate. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may also be present in such vehicles. A wide variety of medicinal excipients are known in the art and do not need to be discussed in detail herein. Pharmaceutical excipients are well documented in various publications, including, for example, A. Gennaro's (2000) "Remington: The Science and Practice of Pharmacy," 20th edition; Lippincott, Williams, & Wilkins; "Pharmaceutical Dosage Forms and Drug Delivery Systems" (1999), edited by HCAnsel et al., 7th edition; Lippincott, Williams, & Wilkins; and "Handbook of Pharmaceutical Excipients" (2000), edited by AHKibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0097] Injectable preparations, such as sterile, injectable aqueous or oily suspensions, may be formulated according to known techniques using appropriate dispersants or wetting agents and suspending agents. Sterile, injectable preparations may also be sterile, injectable solutions, suspensions, or emulsions of non-toxic, parenterally acceptable diluents or solvents, such as a 1,3-butanediol solution. In acceptable vehicles and solvents, water, Ringer's solution, and isotonic sodium chloride solutions can be used. Furthermore, sterile non-volatile oils have conventionally been used as solvents or suspension media. For this purpose, any bland non-volatile oil, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid are used in injectable preparations. These injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0098] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated composition contains at least one inert and pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or (a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; (c) humectants such as glycerol; and (d) agar. (i) It is mixed with disintegrants such as calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) dissolution retarders such as paraffin; (f) absorption enhancers such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffering agents.

[0099] The medicinal products described herein may be provided as a kit comprising at least one container and a package insert. The container contains at least one dose of the medicinal product comprising the composition described herein. The package insert or label contains instructions for use in treating a patient with the medicinal product described herein. The kit may further include other materials that should be useful for administering the medicinal product, such as diluents, filters, intravenous (IV) bags and lines, injection needles and syringes.

[0100] The methods of the present invention provide means for delivering nucleic acid sequences into host tissues or cells. The vectors and other reagents, methods, and pharmaceutical formulations of the present invention are also useful in methods for administering proteins or peptides to subjects in need, for therapeutic or otherwise purposes. In this form, the proteins or peptides can be produced in vivo in the subject. The subject may require proteins or peptides for therapeutic or otherwise purposes due to a deficiency of the protein or peptide, which will be further described below.

[0101] As used herein, the terms “treatment,” “to treat,” etc., refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in the sense of completely or partially preventing or regressing a disease or its symptoms, and / or therapeutic in the sense of partially or completely curing a disease and / or adverse effects caused by it. As used herein, “treatment” encompasses the treatment of any disease of mammals, in particular humans, and includes (a) preventing the onset of the disease in subjects who have not yet been diagnosed with the disease but are susceptible to or at risk of developing the disease, (b) inhibiting the disease, i.e., stabilizing its progression, and (c) alleviating the disease, i.e., regressing it.

[0102] In general, the present invention can be used to deliver any foreign nucleic acid having a biological effect to treat or improve symptoms associated with any disorder related to gene expression in any organ, tissue, or cell, particularly symptoms associated with the brain, for example.

[0103] Gene transfer is quite likely to be useful in understanding disease states and providing therapies for them. For several hereditary diseases, the defective genes are publicly known and have been cloned. In some cases, the function of the cloned genes is known. These disease states generally fall into two categories: those that are typically enzyme deficiencies and are usually recessive, and those that are imbalanced and dominant, involving regulatory or structural proteins at least transiently. For deficiency diseases, gene transfer can be used to perform replacement therapy by introducing normal genes into affected tissue, and animal models of the disease can also be created using antisense mutations. For imbalanced disease states, gene transfer can also be used to create the disease state in a model system, and then this model disease can be used to attempt to address the disease state. Therefore, the methods of the present invention enable the treatment of hereditary diseases. The disease states used herein are treated by partially or completely improving the deficiency or imbalance that causes or exacerbates the disease. It is also possible to induce mutations or correct defects by employing site-specific integration of nuclear sequences.

[0104] One aspect of the present invention provides a method for delivering a gene product to a target tissue or cell (e.g., neuronal tissue or neuronal cells), comprising administering the AAV vector to the target. The gene product may be, for example, the polypeptide described above. The cells may be, for example, blood cells, stem cells, bone marrow cells (e.g., hematopoietic cells), hepatocytes, cancer cells, vascular cells, pancreatic cells, nerve cells, glial cells, epithelial or endothelial cells, dendritic cells, fibroblasts, lung cells, muscle cells, cardiac cells, intestinal cells, or kidney cells. Similarly, the tissue may be selected from, for example, blood, bone marrow, muscle tissue (e.g., skeletal muscle, cardiac muscle, or smooth muscle including vascular smooth muscle), central or peripheral nervous system tissue (e.g., brain, neuronal tissue, or retinal tissue), pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue, or cardiac tissue.

[0105] The delivery of gene products to nerve tissue or nerve cells may provide a treatment for neurological disorders. These gene products may be delivered to various cell types present in nerve tissue, such as neurons or glial cells (e.g., astrocytes, oligodendrocytes, and others).

[0106] This disclosure provides a method for treating a disease (e.g., a neurological disorder), comprising administering an effective amount of the AAV vector to an individual in need thereof. The AAV vector may be administered by intracranial injection, intracerebral injection, intraocular injection, intravenous injection, or any other convenient mode or route of administration.

[0107] Further exemplary modes of administration include oral administration, rectal administration, transmucosal administration, topical administration, transdermal administration, inhalation administration, parenteral administration (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intra-articular), as well as direct injection into tissues or organs, and separately, intrathecal injection, direct intramuscular injection, intraventricular injection, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection. The injectable preparation may be prepared in any conventional dosage form, such as a liquid solution or suspension, a solid dosage form suitable for liquid solution or suspension before injection, or an emulsion. Alternatively, the virus may be administered topically rather than systemically, for example, in a depot or sustained-release form.

[0108] The recombinant viral vector is preferably administered to the target cells (e.g., neuronal cells) in an amount sufficient to induce infection (or transduction) and expression of a heterologous nucleic acid sequence in those cells. Preferably, the target cells are nerve cells (including central and peripheral nervous system cells, particularly brain cells).

[0109] Preferably, the vector is administered in a therapeutically effective dose. As used herein, a “therapeutically effective” dose is sufficient to alleviate (e.g., reduce, decrease, or decrease) at least one symptom or etiology associated with a disease state. In other words, a “therapeutically effective” dose is sufficient to bring about some improvement in the symptom of interest. The “therapeutically effective dose” can be determined by experiment and / or clinical trials and covers a relatively wide range. For example, a therapeutically effective dose in in vivo infusion is approximately 10 6 ~about 10 15 AAV virus particles with a number of orders of magnitude, for example, about 10 8 ~10 12 This can be in the order of magnitude of AAV virus particles. In in vitro transduction, the effective amount of AAV virus particles delivered to cells is approximately 10 8 ~about 10 13 This would be an order of magnitude of AAV virus particles. Other effective doses will be readily established by those skilled in the art through routine trials to establish dose-response curves. As will be recognized by those skilled in the art, the effective dose of a composition or pharmaceutical product containing the AAV vector described herein may vary depending on factors such as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, and others. Additional factors that may be taken into consideration include the severity of the disease; the age, weight, and sex of the patient being treated; diet, timing, and frequency of administration; drug combinations; sensitivity to response; and tolerance / responsiveness to treatment.

[0110] In some embodiments, more than one dose (e.g., two, three, four or more doses) can be administered to achieve the desired level of gene expression over various intervals, such as once daily, once weekly, once monthly, once year, etc.

[0111] The present invention is useful in both veterinary and medical applications. Suitable subjects include both birds and mammals, but mammals are preferred. As used herein, the term "birds" includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. As used herein, the term "mammals" includes, but is not limited to, humans, cattle, sheep, goats, horses, cats, dogs, rabbits, etc. Human subjects are most preferred. Human subjects include fetuses, newborns, infants, adolescents, and adults.

[0112] Unless otherwise specified, all terms used in the disclosure of this invention, including technical and scientific terms, have the meanings generally understood by those skilled in the art to which this invention belongs. Further guidance may include definitions of terms to better understand the teachings of this invention.

[0113] It is understood that certain features of the Invention described in the context of separate embodiments for clarity may also be provided in combination within a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may also be provided separately or as any suitable subcombinations. All combinations of embodiments relating to the Invention are expressly encompassed by the Invention and are duly disclosed herein as each and all combinations are individually and expressly disclosed. Furthermore, all subcombinations of various embodiments and their elements are also expressly encompassed by the Invention and are duly disclosed herein as each and all subcombinations are individually and expressly disclosed herein. The following examples illustrate specific embodiments of the present invention. In no event is it intended to limit the present invention to the following. [Examples]

[0114] (Examples) (Example 1. Improving PGRN translation efficiency using a codon optimization algorithm) The rate of protein translation from RNA transcripts can be improved by modifying codons to use those that are optimal for a particular species and can increase gene expression within a specific cell type. Optimizing protein production from each transcript reduces the amount of viral vector used, lowering toxicity risk and cost. Optimization of wild-type PGRN transcripts was modeled using three open-source programs (IDT, Geneart, and Genscript: see Materials and Methods details). A rare codon analysis tool was used to validate the codon adaptation index (CAI; https: / / www.genscript.com / tools / rare-codon-analysis). A CAI of 1.0 is considered ideal, and a CAI >0.8 is ranked as good for expression in the target expression organism. A lower number indicates a higher likelihood of insufficient gene expression.

[0115] (1.1 Codon-Optimized Design) The code sequence of GRN(NM_002087.3) was redesigned using commercially available codon optimization algorithms (IDT®, https: / / eu.idtdna.com / CodonOpt), (GeneArt®, https: / / www.thermofisher.com / order / geneartgenes / projectmgmt), and (GenScript®, https: / / www.genscript.com / tools / rare-codon-analysis). The modified GRN sequences were ranked using the Codon Adaptation Index (CAI, GeneScript). CAI >0.8 was considered suitable for expression in the target organism, and sequences and CAIs created using various algorithms are shown in Figures 1a and 1b. Algorithms for humans and other species were applied to develop the best codon-optimized PGRN (Figure 1c). Fourteen novel constructs were designed and synthesized. The consensus Kozak sequence (GCCACC) was inserted before the ATG start codon, and the resulting sequences were created using IDT, GeneArt, and GenScript, respectively, and named PGRN-IDT, PGRN-GA, and PGRN-GS.

[0116] (1.1.1 Codon optimization for PGRN) To identify the optimal codon-optimized PGRN sequence, we used the GenScript algorithm service (https: / / www.genscript.com / codon_opt_pr.html) and applied dual codon optimization to ensure that the codon-optimized sequence was executable in animal models, human cell lines, and in humans. Codon optimization of PGRN for humans may not be applicable to other species such as mice or large animals (sheep or monkeys). Therefore, we designed 12 dual-codon-optimized human PGRN transgenes, which significantly improved CAI scores in both humans and other species (see Figure 1c). However, PGRN expression and secretion levels, as tested by Western blotting, did not dramatically improve PGRN protein expression except for those optimized for humans and mice (codop12, PGRN-GS, Figures 1a and 1b). Therefore, we applied additional algorithms for human PGRN to create PGRN-IDT and PGRN-GA. Next, all three constructs (PGRN-GS, PGRN-GA, and PGRN-IDT) were tested for optimal expression levels.

[0117] (1.1.2 Codon-optimized PGRN sequences designed using three different algorithms) (a) Human wild-type PGRN (PGRN-WT) DNA coding sequence (coding sequence (CDS) length: 1782 bp) [Sequence ID 1]: [ka] The codon adaptation index (CAI) of wild-type human PGRN is 0.83, and the GC content is 63.22%. The ideal percentage range for GC content is 30% to 70%.

[0118] (b) Candidate I (PGRN-IDT): DNA coding sequence of PGRN with artificially optimized codons (CDS length: 1782 bp) [SEQ ID NO: 2]: [ka] Codon-optimized candidate I (PGRN-IDT) has a CAI of 0.73 and a GC content of 59.33%. This codon-optimized sequence is 76.11% homologous to the wild type.

[0119] (c) Candidate II (PGRN-GA) Artificially codon-optimized PGRN DNA coding sequence (CDS length: 1782 bp) [SEQ ID NO: 3]: [ka] The CAI of codon-optimized candidate II (PGRN-GA) is 0.9, and the GC content is 56.23%. This codon-optimized sequence is 78.81% homologous to the wild type.

[0120] (d) Candidate III (PGRN-GS) DNA coding sequence of PGRN with artificially optimized codons (CDS length: 1782 bp) [SEQ ID NO: 4]: [ka] The CAI of codon-optimized candidate III (PGRN-GS) is 0.92 in humans and 0.94 in mice. The GC content of candidate 3 is 63.23%. This codon-optimized sequence is 81.34% homologous to the wild type.

[0121] Schematic diagrams of candidate hPGRNs I, II, and III, which have been codon-optimized using algorithms from different companies, are shown in Figure 2a). Candidate sequences I-III and the wild-type coding sequence (SEQ ID NOs: 1-4) each encode the amino acid sequence of progranulin (SEQ ID NO: 16). [ka]

[0122] (1.2. Preparation of PGRN fusion candidates) Since PGRNs are secreted proteins, their 5' guide sequence is crucial for secretory signaling. We hypothesized that this RNA sequence may play a vital role in guiding mRNA to the endoplasmic reticulum for translation. Therefore, we replaced the 5' region of the codon-optimized sequence with the wild-type 5' GRN sequence (392 bp) upstream of the codon-optimized protein-coding sequence using the Bamh1 and BstB1 restriction sites. This allowed us to create pAAV-CMV-PGRN-IDT, pAAV-CMV-PGRN-GA, and pAAV-CMV-PGRN-GS fusions.

[0123] Figure 2b) shows a schematic diagram of a fusion plasmid for three codon optimization candidates, in which the 5' sequence derived from the codon optimization sequence is replaced with the 5' sequence of a wild-type human PGRN.

[0124] (1.3. Complete evaluation of the characteristics of codon-optimized candidates and PGRN fusion candidates) The expression levels of codon-optimized PGRN were compared to wild-type human PGRN. The levels of secreted PGRN were directly measured in cell culture medium and cell lysates and standardized to cell number as a ratio to the housekeeping protein GAPDH. To directly compare the relative efficiency of PGRN production per PGRN cassette, the copy number of the vector genome per cell was quantified by quantitative qPCR (CMV / GAPDH) of CMV as a ratio to the same housekeeping gene GAPDH representing effective HEK-293 cell transduction. This was used as the denominator for standardization to accurately quantify the PGRN level per cassette.

[0125] Human kidney cell line (HEK-293) was maintained at 37°C in a humidified chamber containing CO2 (5%) and stored in Dulbecco's modified medium (DMEM, Thermo Scientific) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were passaged every 3 days.

[0126] HEK-293 cells were transfected at 80% culture density in 24-well Western blotting plates using Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol. A total of 250 ng of DNA was transfected or co-transfected into 40,000 cells. pAAV-CMV-EGFP or pAAV-Syn-EGFP was used as a control for transfection efficiency. To confirm PGRN secretion levels, the medium was changed the following day to serum-free medium supplemented with insulin transferrin selenium (ITS). After 48 hours, the medium was collected and immediately used for Western blotting analysis. The cells were lysed in mild cell lysis buffer (NP40) and then centrifuged at 10,000 × g for 10 minutes. The supernatant from the transfected wells was collected and used for PGRN ELISA, and the pellet was stored at -80°C for genomic DNA extraction.

[0127] Protein samples were loaded onto precast NuPage® Novex® 10% Bis-Tris Midi gels with MOPS SDS electrophoresis buffer (Thermo Fisher) and electrophoresed at a constant voltage of 100V. The gels were briefly immersed in NuPage transfer buffer (Thermo Fisher), and then the proteins were transferred using iBlot 2® (Thermo Fisher). The protein membranes were blocked for 1 hour using a blocking reagent (Roche, 10%) containing phosphate-buffered saline (PBS). The membranes were then incubated overnight at 4°C with primary antibodies diluted in 5% blocking buffer. After washing three times with Tris-buffered saline (TBS-T) containing Tween 20, the membranes were incubated with secondary antibodies for 1 hour. After three washes, the membranes were scanned using an Odyssey® CLx infrared imaging system (Li-Cor® Biosciences). The intensity of each protein band was measured using ImageJ and ImageStudio® light. The primary antibodies used for Western blotting detection were GRN (Abcam, ab191211) and GAPDH (Abcam, ab82485). The secondary antibodies used for Western blotting detection were goat anti-mouse IgG (H+L) DyLight® 680 / 800 conjugate (Thermo Fisher, 25518, SA535521, 1:5,000) and goat anti-rabbit (H+L) DyLight 680 / 800 conjugate (Thermo Fisher, 35568, SA535571, 1:5,000).

[0128] Western blotting of PGRN in culture medium showed significantly reduced expression in low-CAI candidate I (IDT) (see Figure 3a). However, PGRN expression and secretion in high-CAI candidates II (GA) and III (GS) were comparable to or higher than wild-type PGRN. Three biological replication studies were performed in four independent experiments. PGRN-GS significantly increased PGRN expression by 72% compared to wild-type (p=0.0327).

[0129] Several "fusion constructs" were also tested. These fusion constructs retained the signal sequence and adjacent regions of the wild-type genome until the BstB1 restriction site fused with the codon-optimized PGRN sequence (see Figure 2b, yellow area). Unfortunately, PGRN protein secretion from the PGRN fusion cassette was equivalent to or lower than that of the wild-type PGRN (see Figures 3a and 3b). This initial conclusion is that the 392 bp 5' generic adjacent DNA sequence of the wild-type PGRN did not significantly improve codon-optimized PGRN secretion with the CMV promoter alone. However, the results below show that the expression of the fusion constructs was enhanced when combined with a 5' intronic enhancement sequence (see below).

[0130] Western blot analysis of HEK cell lysates showed results similar to secreted PGRN, with codon-optimized PGRN-GA and PGRN-GS improving PGRN expression by 39.55% (not significant) and 127.7% (p=0.0014), respectively. However, PGRN-IDT and other "fusion" constructs failed to increase PGRN expression (see Figures 4a and 4b).

[0131] (Example 2. Development of an intronic enhancement element to increase PGRN expression) To further increase PGRN expression and secretion, we explored the possibility of enhancing gene expression by adding introns to the 5' sequence of PGRN. Introns can increase transcript levels by affecting transcription rate, nuclear export, transcript stability, or mRNA translation efficiency; this phenomenon is called intron-mediated enhancement (IME) (O. Shaul, Int J Biochem Cell Biol. 2017 Oct;91(Pt B):145-155). We searched for intron sequences less than 300 bp that could be used to test intron-mediated enhancement. Introns within the human growth hormone (hGH) gene were selected as suitable candidates (see Figure 5a).

[0132] The wild-type GRN sequence was synthesized and cloned into the pAAV plasmid (Addgene; 99280) using the BamH1 and Xho1 restriction sites. These constructs were used as a master vector (pAAV-CMV-PGRNwt) for subcloning the codon-optimized constructs described above. The human synapsin promoter sequence was replaced from the Addgene vector (58881) using the Pci1 and Bamh1 restriction sites of pAAV-Syn-PGRNwt.

[0133] The polynucleotide sequence of the synapsin promoter is shown in (SEQ ID NO: 15). [ka]

[0134] Intron sequences of human growth hormone (hGH1) were used to improve PGRN expression. hGH1 consisted of four introns synthesized by GenScript and cloned into the pAAV-CMV-PGRN plasmid using the BamH1 and Age1 restriction sites to create pAAV-CMV-hGHi1-PGRN, pAAV-CMV-hGHi2-PGRN, pAAV-CMV-hGHi3-PGRN, and pAAV-CMV-hGHi4-PGRN. Structural elements are important for intron-mediated enhancement (IME). Therefore, wild-type PGRN coding sequences were analyzed using the exonic splice enhancer (ESE) finder (ESE 3.0, http: / / krainer01.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi) and by the frequency of high ESEs predicted for GRN. To further enhance PGRN expression, the hGHi3 intron was introduced into a fusion construct that preserves the 5'ESE element of wild-type PGRN to create the pAAV-CMV-hGHi3-PGRN-GA fusion and the pAAV-CMV-hGHi3-PGRN-GS fusion. Using the Bamh1 and Age1 restriction sites, neuron-specific expression constructs, the pAAV-Syn-hGHi3-PGRN-GA fusion and the pAAV-Syn-hGHi3-PGRN-GS fusion, were created.

[0135] Sequence ID 5 is the intron 1 sequence (261 bp) of human growth hormone. [ka]

[0136] Sequence ID 6 is the intron 2 sequence (209 bp) of human growth hormone. [ka]

[0137] Sequence ID 7 is the intron 3 sequence (92 bp) of human growth hormone. [ka]

[0138] Sequence ID 8 is the intron 4 sequence (253 bp) of human growth hormone. [ka]

[0139] The regions adjacent to the restriction sites Bamh1 and BstB1 (1-392 bp) in pAAV-CMV-PGRNwt and pAAV-CMV-PGRN-GS were used for signal sequence substitution. The N-terminal region (1-51 bp) of the PGRN signal sequence was replaced with 78 bp of hGH1, and gene synthesis was performed. These were then subcloned into pAAV-CMV-PGRNwt and pAAV-CMV-PGRN-GS using the Bamh1 and BstB1 restriction sites to create pAAV-CMV-hGHs-PGRNwt and pAAV-CMV-hGHs-PGRN-GS.

[0140] Sequence ID 9 is a generic DNA signaling sequence for human growth hormone, and it replaces the PGRN signaling sequence. [ka]

[0141] Sequence ID 10 is the translated amino acid sequence for the generic signaling sequence of human growth hormone. [ka]

[0142] To test the effect of PGRN expression, the hGH intron 2, 3, and 4 sequences were cloned into the 5'UTR region of PGRN-WT (see Figure 5b). Furthermore, the wild-type signal sequence of PGRN was replaced with the hGH signal sequence (see Figure 5c).

[0143] (2.1 Addition of Intronic Enhancer Elements) The efficiency of each hGH intron in PGRN expression and secretion was evaluated by Western blotting (see above for method). The results showed that intron 3 significantly increased secreted PGRN (105%, p=0.0004), while intron 4 decreased secretion and intron 2 eliminated secretion (see Figures 6a and 6b). Furthermore, as shown in Figures 6a and 6b, replacing the wild-type PGRN signal peptide with the hGH signal peptide resulted in a significant increase in secreted PGRN (71%, p=0.0088).

[0144] Levels of PGRN expression measured in HEK-293 cell lysates confirmed that intron 3 significantly increased PGRN expression (148%, p=0.0009) (see Figures 7a and 7b), while intron 4 decreased PGRN expression by 40%, and intron 2 eliminated expression. As shown in Figures 7a and 7b, replacing the wild-type with an hGH signaling sequence increased PGRN expression by 23%, but this was not statistically significant. These results suggest that intron 3 enhances PGRN translation, which in turn increases PGRN secretion into the culture medium. Replacing the signal peptide with hGH slightly increased PGRN secretion, but not expression.

[0145] (Example 3. Combination of intronic enhancement element and exonic enhancement element) To test the intron-mediated enhancement of hGHi3 in codon-optimized PGRN-GS, the hGHi3 element was subcloned at the 5' position of PGRN-GS.

[0146] Surprisingly, hGHi3 had no enhancing effect on PGRN-GS expression (see Figures 8a and 8b), and insertion of intron 3 resulted in a slight decrease in PGRN. This suggests that exonic splicing elements (ESEs) within the wild-type PGRN sequence are necessary for the synergistic splicing effect of hGHi3 to occur. ESE elements within the wild-type PGRN and the codon-optimized PGRN-GS sequence were identified using the ESE Finder 3.0 software (see Figure 8c). The ESE distribution pattern within the codon-optimized PGRN-GS was significantly different compared to the wild-type PGRN. In other words, efforts to codon-optimize PGRN unintentionally removed ESEs necessary for enhanced delivery by hGHi3 insertion.

[0147] Based on the above results, the intronic enhancer hGHi3 was combined with a PGRN fusion construct containing the first 392 bp sequence of wild-type GRN fused to a PGRN-GA codon-optimized sequence (see Figure 9a). This was packaged in an AAV9 vector and delivered to cultured rat primary cortical neurons at an MOI of 1e6.

[0148] The 5'ESE adjacency sequences were as follows: [ka] .

[0149] The above 5'ESE adjacent sequences can be used in several embodiments of the present invention. However, these sequences were not further studied in the embodiments described below.

[0150] Each virus in a low subdivision (≤P30) is 10 × 145 cm. 2On the day of transfection, HEK-293T cells were used in plates at approximately 80% culture density. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 10% heat-inactivated FBS at 37°C and 5% CO2. For the transfection mixture, 800 μL of 1 mg / mL PEI was added to 15 mL of serum-free DMEM. In a separate tube, 240 μg of adeno helper plasmid (containing essential genes from the adenovirus genome that support the rescue and replication of the AAV genome), 80 μg of Rep2 / Cap9 plasmid, and 80 μg of plasmid containing a single-stranded transgene were added to serum-free DMEM to a final volume of 30 mL. This DNA-containing mixture was then filtered through a 0.45 μm polyethersulfone (PES, Sartorius, Epsom, UK) syringe filter to obtain the PEI-containing mixture. This solution was mixed and incubated at room temperature for 15 minutes. Next, these transfection solutions are applied to each 145 cm³. 2 It was added to the dish by dripping.

[0151] AAV virus particles were collected 72 hours after transfection. The soup and pellet were collected and subjected to a freeze-thaw cycle. All supernatants and pellets were treated with benzonase (50 units / mL, 37°C, 30 minutes), and then centrifuged at 2000×g for 30 minutes at 18°C. The supernatant was filtered through a 0.45 μM pore-size filter, and then AAV was purified using a pre-equilibriumized AKTA system AAVX POROS affinity column (Thermo Fisher). The purified AAV was kept at -80°C.

[0152] Neurons were isolated from cortical tissue on embryo day 18 (E18) (Sprague-Dawley rats). 100,000 cells were plated into PDL-coated 24-well plates. Cells were cultured in Neurobasal medium supplemented with penicillin / streptomycin (0.5%), glutamax (1%), and B27 (2%, Thermo Fisher). Cells were transduced in vitro on day 7 (DIV7) with AAV at an infection multiplicity (MOI) of 1e6. Neurons were harvested into DIV12 and analyzed by Western blotting (see above for method) and ELISA.

[0153] Genomic DNA was isolated 48 hours after transfection using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer's protocol. DNA concentration was measured using Nanodrop®.

[0154] The PGRN content of the samples was evaluated using the Adipogen Life Sciences Progranulin (Human) ELISA Kit (AG-45A-0018YEK-KI01). This sandwich ELISA kit captures human progranulin in the sample with polyclonal antibodies pre-coated on a microtiter plate, and detects the protein using biotinylated polyclonal secondary antibodies. The PGRN signal was detected by adding STREP-HRP solution to the wells, using TMB substrate for 10 minutes, adding acidic stop solution, and measuring the absorbance of each well at 450 nm. The PGRN concentration was calculated within the range of 0.063–4 ng / ml using recombinant human PGRN standards. All standard reagents and other reagents required for the assay were included in this kit.

[0155] As shown in Figures 9b and 9c, PGRN secretion by cortical neurons transduced by the hGHi3-PGRN-GA construct was greater than that by hGHi3 wild-type PGRN alone.

[0156] These results indicate that the increased expression and secretion induced by hGHi3 is due to hGHi3 functioning as an intronic splicing element (ISE). Preferably, hGHi3 functions in combination with an exonic splicing element (ESE) present in the first 392 bp sequence of the wild-type PGRN. Therefore, hGHi3 may contain a potential ISE sequence that appears to require an ESE present within the wild-type PGRN sequence to enhance splicing (McCarthy and Philips, (1998) Human Molecular Genetics. 7;1491-1496). While not limiting the scope of theory, the predicted mechanism is that this combination facilitates the binding of serine and arginine-rich (SR) splicing proteins, accelerating RNA processing and subsequent translation.

[0157] (Example 4. Restriction of expression using neuron-specific promoter synapsins) PGRNs are used by many cell types. However, in the brain, their expression and secretion are primarily determined by microglia and neurons, and both of these cell types are affected by PGRN deficiency. Because microglia are very difficult to transduce using viral vectors, the focus was on maximizing PGRN transduction and expression into postmittal neurons while simultaneously avoiding the risk of accelerating microglial and astrocyte cell division by potentially oncogenic PGRNs. Wild-type PGRN expression under the all-mammalian generic promoter CMV was compared with that under the human neuron-specific promoter synapsin. As shown in Figure 10, expression was restricted, and PGRN secretion under the synapsin promoter was significantly increased in rat cortical neuron cultures.

[0158] To test whether packaging the PGRN cassette within the AAV9 vector supports initial discovery from plasmid transfection, we created AAV9 holding either CMV-PGRNwt or Syn-PGRNwt. Primary rat cortical neurons were cultured for 7 days and transduced with the AAV9-GRN vector. After 5 days, cell culture medium was sampled and processed by Western blotting. The synapsin promoter increased GRN expression 5.4-fold compared to CMV, which was confirmed by PGRN ELISA (see Figure 11).

[0159] To validate these studies in vivo, AAV9-CMV-PGRNwt and AAV9-Syn-PGRN vectors were injected into 8-week-old C57BL\6J male mice via bilateral ventricles (ICV) (Figure 12a). Mice were deeply anesthetized with isoflurane and fixed to a stereotactic frame equipped with a digital stereotactic control panel. The vectors were injected bilaterally into the anterior ventricle or posterior thalamus of the mice using a Hamilton syringe. Each mouse was injected at a rate of 0.5 μL / min, delivering a total dose of 3 μL of 5e+10 VG, and its trochanter was removed after a 2-minute wait following injection. Mice were injected at 8 weeks of age and sacrificed at 12 weeks of age, 4 weeks after injection. Half of the brain, as well as the spinal cord, spleen, heart, liver, kidneys, lungs, testes, blood, and cerebrospinal fluid (CSF), were collected and immediately frozen in liquid nitrogen. The remaining half of the brain was post-fixed with paraformaldehyde (PFA) for histological and immunocytochemical (IHC) analysis.

[0160] For the collection of brain, plasma, and organs, mice were anesthetized with pentobarbital (100 mg / kg, Fatal Plus, Vortech Pharmaceuticals, Dearborn, Michigan), and blood was collected by cardiac puncture in a syringe containing EDTA (250 mM) for anticoagulation. The blood was kept on ice, and then the plasma was separated by centrifugation at 1000 × g for 10 minutes at 4°C. Next, the mice were perfused transcardially with PBS. The brain was removed and divided into two hemispheres. One hemisphere was microdissected into the prefrontal cortex, striatum, hippocampus, cerebellum, subcortical regions, and cortex, and rapidly frozen in liquid nitrogen for biochemical analysis. The other hemisphere was post-fixed in 4% paraformaldehyde for 24 hours for histological analysis. The spinal cord, spleen, heart, liver, kidneys, lungs, testes, blood, and cerebrospinal fluid were removed and immediately frozen in liquid nitrogen for ELISA and Western blot analysis.

[0161] The distribution and quantification of CMV-PGRN and Syn-PGRN transduction within each tissue were measured by amplifying human PGRN from genomic DNA using qPCR. Genomic DNA was diluted to 7.5 ng / µl with nuclease-free H2O. qPCR was performed using Powerup® SYBR® Green Master Mix according to the standard protocol. Standard curves were also performed for each reaction set using known DNA concentrations.

[0162] The immobilized hemispheres were cryoprotected in 30% sucrose and cut into 30 μm sections using a slide microtome (Leica Biosystems). The sections were then immunostained. For pathological analysis and qualitative evaluation of progranulin immunoreactivity, the sections were incubated overnight with primary antibodies (PGRN, neuronal marker (NeuN), or microglia marker (Iba1)), and the following day incubated with species-matched secondary antibodies: AlexaFluor®-488 conjugate antibody for PGRN, and AlexaFluor-647 conjugate antibodies for NeuN and Iba1, respectively.

[0163] Low-magnification, high-resolution images of progranulin immunostaining were acquired using an image analysis slide scanner (Olympus VS120).

[0164] Low levels of CMV-PGRN and Syn-PGRN were detected in the cerebral cortex, lungs, and spleen. However, relatively high copy numbers of the virus were detected in the liver (see Figure 12).

[0165] Using ELISA, we quantified PGRN expression levels from serum, CSF, and cortical tissue of transduced mice. Mice injected with AAV9-CMV-PGRN showed high levels of serum PGRN, which was almost undetectable in mice injected with AAV9-Syn-PGRN (see Figure 13). Conversely, PGRN levels in the cortex and CSF were significantly higher in AAV9-Syn-PGRN compared to mice injected with AAV9-CMV-PGRN (see Figures 13c and 13d), demonstrating the neuronal specificity of synapsin in vivo.

[0166] (Example 5. Broad PGRN expression in AAV9-Syn-PGRNwt after ICV injection) Mice injected with AAV9-Syn-PGRN-wt via ICV injection were disposed of after 4 weeks (Figure 15). Brains were sliced ​​after PFA fixation and used for IHC. Goat anti-human specific PGRN antibody (green) showed widespread GRN expression in the cortex (Figure 15c), hippocampus (Figure 15d), and thalamus (Figure 15e). Rabbit anti-neuron specific antibody NeuN was used for counterstaining. Human PGRN was not detected in the brain tissue of uninjected mice, while strong staining was detected in the hippocampus of mice injected with AAV-Syn-PGRNwt, and weaker staining was detected in the thalamus and cortex (Figure 15d). PGRN ELISA analysis of hippocampal tissue lysate demonstrated that the synapsin promoter resulted in a three-fold increase in PGRN expression compared to the CMV promoter (Figure 16b). Western blotting confirmed the presence of a strong PGRN band at approximately 68 kDA (Figures 16c and 16d).

[0167] (Example 6. Combination of intron sequence hGHi3 and 3'UTR element) Next, we tested whether PGRN expression could be enhanced and potentially regulated by using the 3'UTR as an exonality enhancing element (ESE). A 284 bp wild-type PGRN 3'UTR was synthesized and cloned into the 3' region in addition to the intronality enhancer, hGHi3, and our codon-optimized PGRN-GS to create Syn-hGHi3-PGRN-GS-UTR (Figure 17).

[0168] The 3'UTR sequence (284 bp) of the wild-type PGRN is shown in Figure 17b and below (Sequence ID 14). [ka]

[0169] This sequence, including 5'Pme1 and 3'XhoI, was synthesized using GenScript's gene synthesis service. This UTR sequence was cloned into AAV-Syn-hGHi3-PGRN-GS to create AAV-Syn-hGHi3-PGRN-GS-UTR. Figure 21 shows a vector map of the plasmid containing the cassette shown in Figure 17. The DNA sequence of the Syn-hGHi3-PGRN-GS-UTR vector cassette (from ITR to ITR) is shown below (SEQ ID NO: 17).

[0170] [ka] TIFF0007854715000018.tif154170

[0171] Sequence ID 17 contains the following array elements: AAV2 ITR residues 1–130 hSyn promoter residues 341-788 hGHi3 residues 801-892 PGRN-GS residues 905~2686 3'UTR residues 2702~2985 bGH poly(A) signaling residues 3015-3222 AAV2 ITR residues 3245~3385

[0172] (Example 7. Broad-spectrum PGRN expression in AAV9-Syn-PGRN-GS, AAV9-Syn-hGHi3-PGRN-GS, and AAV9-Syn-hGHi3-PGRN-GS-UTR after intrathalamic injection) The optimized dose of AAV particles containing the final PGRN cassette (Figures 2, 5, 9, 14, and 17) was injected into the mouse brain via intrathalamic injection, and vector efficiency was compared. The efficacy of AAV-PGRN virus transduction was tested by ELISA and IHC analysis of PGRN protein expression. Intrathalamic injection (IT) was chosen among the various routes because it improves the cortical expression of EGFP and PGRNwt. The addition of the hGHi3 intronic enhancement element consistently increased PGRN levels by approximately 27%, and the addition of the 3'UTR exonic enhancement element increased PGRN expression by approximately 37% (Figures 18b and 19). Cortical PGRN levels were quantified by ELISA and IHC analysis after IT injection of low-dose (6.2E+10), medium-dose (1.2E+11), and high-dose (2.5E+11) vectors to confirm the dose-rich effect in target tissue (Figure 18c).

[0173] Granular PGRN-positive foci are abundant in cortical neurons (Figures 20a and 20c). Because pathological changes are observed in microglia and neurons, we investigated whether PGRN secreted by neurons is taken up by non-neuronal cells, including microglia. Under the synapsin promoter, PGRN is expressed only in neurons, but it is detected in many NeuN-negative cells (Figure 20a), and small PGRN foci can be detected in IBA1-positive microglia (Figures 20b and 20d).

[0174] (Example 8. Analysis of GRN distribution, toxicology, and efficacy using an in vivo model) The optimized dose of AAV particles containing the PGRN cassette shown in Figures 2, 5, 9, 14, or 17 will be injected into non-transgenic (NTg) mice via intraparenchymal and intraventricular injection, and the efficiency of the different administration routes will be compared. The efficacy of AAV-PGRN transduction will be tested by qPCR and by protein expression analysis using ELISA and IHC.

[0175] The distribution and toxicology of AAV-PGRN will be tested in non-transgenic (NTg) mice and PGRN knockout (PGRN+ / - and - / -) mice (see above for stereotactic brain surgery procedures). Furthermore, the distribution will be investigated by injecting AAV-PGRN into wild-type sheep. Wild-type sheep have the same spinal cord length as humans, and their brains are twice the size of those of macaques, another common non-human primate model.

[0176] Efficacy studies will be conducted by injecting AAV-PGRN into PGRN+ / - and - / -, TDP-43 Q331K, and TDP-43 Q331KxWT transgenic mice. TDP-43 transgenic mice will develop either a slow disease progression (Q331K) or a faster disease progression (TDP-43 Q331K xWT) phenotype. Mouse behavior will be monitored using a rotarod test and grip strength test for motor function assessment, and an elevated cusp maze for short-term social working memory cognitive testing. Tissues will be collected and processed as described above. Animals will be injected at 8 weeks of age (IT or ICV) for PGRN and TDP-43 Q331K transgenic mice, and at 2 weeks of age for TDP-43 Q331KxWT transgenic mice due to their rapidly progressive phenotype. Animals will be housed for 4 weeks or 6 months, and their behavior will be monitored monthly using the tests described above. Tissues collected from both sheep and mice are processed for IHC, ELISA, ddPCR, and Western blotting to measure PGRN expression levels and PGRN protein levels, quantify PGRN mRNA levels, and determine vector genome levels. Therapeutic efficacy of TDP-43 transgenic animals is determined by quantification of insoluble TDP-43 levels and quantification of microglia and astrocyte activation by Western blotting and IHC analysis. Differences in disease progression rate and disease severity between PGRN and control vectors are statistically analyzed.

[0177] Efficacy studies of AAV-PGRN in PGRN+ / - and - / - mice are even more challenging because these mice exhibit only a very mild phenotype—a reduction in social dominance in in vitro studies—and do not show neuronal loss. - / - mice exhibit readily quantifiable lipofuscin accumulation and activated microglia. Target engagement can be measured by quantifying the decrease in lipofuscin and the activation levels of microglia and astrocytes. Furthermore, the localization of PGRN to lysosomes can be confirmed to establish correct cellular targeting. Differences in disease severity between PGRN and control vectors can be statistically analyzed.

[0178] (Example 9. GRN expression using different AAV capsid serotypes) The PGRN cassettes shown in Figures 2, 5, 9, 14, or 17 are packaged within AAV serotypes 9 and 5, and it is determined whether the enhanced PRGRN expression is specific to AAV9 or associated with other AAV serotypes. AKTA-purified AAVs are tested in vivo and in vitro.

[0179] Overall, the experiments described above demonstrate that codon-optimized AAV9-Syn-PGRN-GS, AAV9-Syn-hGHi3-PGRN-GS, and AAV-Syn-hGHi3-PGRN-GS-UTR expression cassettes significantly increase PGRN expression levels, PGRN transduction efficiency, and cell specificity of PGRN protein expression. These cassettes are expected to enable a reduction in the vector dose administered to FTD, NCL11, and ALS patients, thereby reducing toxicity risks and vector production costs.

[0180] This application claims priority to UK Patent Application No. 1913974.0, filed September 27, 2019, the contents of which are incorporated herein by reference. All documents cited in the above specification are incorporated herein by reference. Various modifications and variations of the embodiments of the invention described herein, without departing from the scope and spirit of the invention, will be apparent to those skilled in the art. Although the invention is described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these particular embodiments. In fact, various modifications of the style described for carrying out the invention, which will be apparent to those skilled in the art, are intended to fall within the scope of the claims below. This application provides the invention in the following embodiments. (Aspect 1) An adeno-associated virus (AAV) vector comprising a nucleic acid comprising (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synapsin promoter sequence, and / or (iii) a progranulin 3' untranslated region (UTR) sequence, operably ligated to a polynucleotide sequence encoding a polypeptide of interest. (Aspect 2) An adeno-associated virus (AAV) vector comprising a polynucleotide sequence encoding progranulin, wherein the polynucleotide sequence has at least 95% sequence identity with SEQ ID NO: 4. (Aspect 3) An AAV vector according to embodiment 1 or 2, comprising an hGHi3 sequence. (Aspect 4) The AAV vector according to embodiment 3, wherein the hGHi3 sequence includes the sequence of sequence number 7 or a variant or homolog thereof. (Aspect 5) The target polypeptide is progranulin (PGRN), preferably comprising the sequence of SEQ ID NO: 16 or a variant or homolog thereof, according to any one of embodiments 1 to 4, of the AAV vector. (Aspect 6) The AAV vector according to any one of embodiments 1 to 5, wherein the polynucleotide sequence is codon-optimized. (Aspect 7) The AAV vector according to embodiment 6, wherein the polynucleotide sequence is codon-optimized for expression in humans. (Pattern 8) The AAV vector according to any one of embodiments 1 to 7, wherein the nucleic acid further comprises an exonial splicing element (ESE). (Aspect 9) The AAV vector according to embodiment 8, wherein the ESE is located upstream of the polynucleotide coding sequence. (Aspect 10) The AAV vector according to embodiment 9, wherein the ESE is part of or inserted into a 5' adjacent sequence. (Aspect 11) The AAV vector according to any one of embodiments 8 to 10, wherein the ESE is part of a guide sequence, inserted into a guide sequence, or part of a guide sequence. (Aspect 12) The AAV vector according to any one of embodiments 8 to 11, in which the ESE is a part of the 5' adjacent sequence derived from a wild-type polynucleotide sequence, as described in embodiment 6. (Aspect 13) The AAV vector according to any one of embodiments 10 to 12, wherein the 5' adjacent sequence is a 5' guide sequence derived from a wild-type GRN. (Aspect 14) The AAV vector according to embodiment 13, wherein the 5' guide sequence of the wild-type GRN contains 350 to 450 base pairs. (Aspect 15) An AAV vector according to any one of embodiments 1 to 14, comprising a progranulin 3' untranslated region (UTR) sequence, preferably the 3'UTR sequence comprising the sequence of SEQ ID NO: 14 or a variant thereof. (Aspect 16) The AAV vector according to any one of embodiments 1 to 15, wherein the polynucleotide sequence includes a signal sequence derived from hGH. (Aspect 17) The AAV vector according to embodiment 16, wherein the hGH signal sequence includes the sequence of sequence number 9 or a variant or homolog thereof. (Aspect 18) The AAV vector according to embodiment 16 or 17, wherein the hGH signal sequence replaces base pairs 1 to 51 of the GRN. (Aspect 19) The codon-optimized sequence comprises the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a variant or homolog thereof, preferably SEQ ID NO: 4, according to any one of embodiments 6 to 18. (Aspect 20) The AAV vector according to any one of embodiments 1 to 19, comprising the nucleic acid and a neuron-specific promoter. (Aspect 21) The AAV vector according to embodiment 20, wherein the neuron-specific promoter includes a synapsin promoter. (Aspect 22) The AAV vector according to embodiment 21, wherein the synapsin promoter comprises the sequence of SEQ ID NO: 15 or a variant or homolog thereof. (Aspect 23) The AAV vector described above is an AAV vector of serotype AAV9, as described in any one of embodiments 1 to 22. (Aspect 24) The AAV vector according to any one of embodiments 1 to 23, wherein the AAV vector comprises a polynucleotide sequence having at least 85% sequence identity with sequence number 17. (Aspect 25) A nucleic acid comprising (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synapsin promoter sequence, and / or (iii) a progranulin 3' untranslated region (UTR) sequence, operably ligated to a polynucleotide sequence encoding progranulin (PGRN). (Aspect 26) A nucleic acid comprising a polynucleotide sequence having at least 81.34% sequence identity with sequence number 4. (Aspect 27) The nucleic acid according to embodiment 25 or 26, comprising a neuron-specific promoter. (Aspect 28) The nucleic acid according to any one of embodiments 25 to 27, further comprising one or more AAV inverted terminal repeats. (Aspect 29) An expression cassette containing nucleic acid as described in any one of embodiments 25 to 28, suitable for use within an AAV vector. (Aspect 30) A pharmaceutical composition or drug comprising an AAV vector as described in any one of embodiments 1 to 24, and one or more pharmaceutically or physiologically acceptable carriers, excipients and / or diluents. (Aspect 31) A pharmaceutical composition or drug according to embodiment 30 for use in the treatment of neurological disorders. (Aspect 32) The aforementioned neurological disorders include frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, Alzheimer's disease, and other neurological disorders, as described in Embodiment 31, a pharmaceutical composition or drug for use. (Aspect 33) A pharmaceutical composition or drug for use according to embodiment 31 or 32 for use in the treatment of (i) subjects with heterozygous, homozygous, or compound heterozygous GRN mutations, (ii) subjects suffering from sporadic neurological disorders, and / or (iii) subjects having PGRN levels lower than physiologically normal levels.

Claims

1. (i) A human growth hormone intron 3 (hGHi3) sequence, wherein the hGHi3 sequence includes the sequence of sequence number 7; (ii) Synapsin promoter sequence; (iii) a polynucleotide coding sequence encoding progranulin (PGRN) and comprising a 5' exon splicing element (ESE); and (iv) 3' untranslated region (UTR) sequence of PGRN, An adeno-associated virus (AAV) vector containing nucleic acids.

2. The AAV vector according to claim 1, wherein the polynucleotide coding sequence has at least 95% sequence identity with SEQ ID NO:

4.

3. The AAV vector according to claim 1 or 2, wherein the PGRN comprises the amino acid sequence of SEQ ID NO: 16 or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO:

16.

4. The AAV vector according to any one of claims 1 to 3, wherein the polynucleotide coding sequence is codon-optimized.

5. The AAV vector according to claim 4, wherein the polynucleotide coding sequence is codon-optimized for expression in humans.

6. The AAV vector according to claim 1, wherein the ESE is part of a 5' adjacent sequence upstream of the polynucleotide coding sequence or is inserted into the 5' adjacent sequence.

7. The AAV vector according to claim 6, wherein the ESE is a part of the 5' adjacent sequence, and the 5' adjacent sequence is derived from a wild-type polynucleotide sequence.

8. The AAV vector according to claim 6 or 7, wherein the 5' adjacent sequence is a 5' guide sequence derived from wild-type granulin (GRN).

9. The AAV vector according to claim 8, wherein the 5' guide sequence of the wild-type GRN contains 350 to 450 base pairs.

10. The AAV vector according to any one of claims 1 to 9, wherein the 3'UTR sequence of the PGRN includes the sequence of SEQ ID NO: 14, or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO:

14.

11. The AAV vector according to any one of claims 1 to 10, wherein the polynucleotide coding sequence includes a signal sequence derived from hGH.

12. The AAV vector according to claim 11, wherein the hGH signal sequence includes the sequence of SEQ ID NO: 9 or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO:

9.

13. The AAV vector according to claim 4 or 5, wherein the codon-optimized sequence includes the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

14. The AAV vector according to any one of claims 1 to 13, wherein the synapsin promoter comprises the sequence of SEQ ID NO: 15 or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO:

15.

15. The AAV vector according to any one of claims 1 to 14, wherein the AAV vector has the AAV9 serotype.

16. The AAV vector according to any one of claims 1 to 15, wherein the nucleic acid sequence of the AAV vector has at least 90% sequence identity with SEQ ID NO:

17.

17. A pharmaceutical composition comprising an AAV vector according to any one of claims 1 to 16, and one or more pharmaceutically or physiologically acceptable carriers, excipients and / or diluents.

18. The pharmaceutical composition according to claim 17, for use in treating neurological disorders in subjects requiring it.

19. The pharmaceutical composition for use according to claim 18, wherein the neurological disorder is frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease.

20. The pharmaceutical composition for use according to claim 18 or 19, wherein the subject is (i) a heterozygous, homozygous, or compound heterozygous GRN mutation, (ii) suffering from a sporadic neurological disorder, and / or (iii) having a PGRN level lower than physiologically normal.

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