Carrier

Abstract

The present invention relates to the field of vectors, in particular to recombinant viral vectors suitable for in vivo delivery of therapeutic genes. Described is an adeno-associated virus (AAV) vector comprising (i) a human growth hormone intron 3 (hGHi3) sequence, (ii) a synaptic promoter sequence and / or (iii) a granulin precursor 3'untranslated region (UTR) sequence operatively linked to a polynucleotide sequence encoding a polypeptide of interest. Particular uses of such vectors are to enhance the expression of a polypeptide of interest, such as a granulin precursor (PGRN), to treat subjects having gene mutations or intrinsic polypeptide levels below physiologically normal levels.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202080067598X, application date September 28, 2020, and invention name “Carrier”. Field of the Invention

[0002] The present invention relates to the field of recombinant viral vectors. Specifically, the present invention relates to a recombinant viral vector suitable for delivering therapeutic genes in vivo. Background of the Invention

[0004] Frontotemporal dementia (FTD) is the second most common form of dementia in people younger than 65 years who show symptoms. It is characterized by changes in behavior, personality, and language, and is very distressing for patients, their families, and friends. About 30% of FTD patients have a family history of dementia, and about 20% of them carry a loss of function (LoF) mutation in the GRN gene that encodes the protein progranulin. Children homozygous for GRN mutations develop the lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL11), which is characterized by neurodegeneration and visual impairment (Rohrer JD et al., (2015b) Lancet Neurol. 14:253–262). Adults heterozygous for LOF mutations develop adult-onset FTD with lysosomal storage lesions and TDP-43 inclusions in neurons of the frontal and temporal cortices. MRI scans of presymptomatic GRN mutation carriers show structural differences in the temporal and parietal lobes approximately 15 years before expected clinical onset (Rohrer et al., supra).

[0005] There are currently no effective treatments that significantly alter disease progression for any form of FTD, amyotrophic lateral sclerosis (ALS), or any form of neurodegenerative disease. The only medications prescribed for FTD are designed to mitigate the effects of depression (antidepressants) or problematic behaviors (major tranquilizers), and these often have significant deleterious short- and long-term side effects.

[0006] Progranulin (PGRN) is a secretory glycoprotein that has important nutritional effects on neurons, inhibits inflammation and plays a key role in autophagy (Chitramuthu BP et al., (2017) Brain.140(12):3081-3104). Progranulin is produced by microglia and neurons, but is taken up by all cell types. Once it binds to its receptor sortilin on the cell membrane, it is imported by the endosome and delivered to the lysosome, where it is degraded into multiple granules (Holler CJ et al., (2017) eNeuro.18:4). One of these granules acts as a chaperone for the protease cathepsin D to promote lysosomal acidification and efficient proteolysis. Lysosomal defects inhibit autophagy, and TDP-43 accumulates in nuclear and cytoplasmic inclusions.

[0007] GRN-\- mice show an imperceptible behavioral phenotype (reduced social dominance), and despite developing lipofuscinosis, reactive gliosis, and abnormal lysosomes, they do not develop TDP-43 proteinopathy or neuronal loss (Arrant AW et al., (2018) J. Neurosci. 38 (9): 2341-2358). Selective knockout of GRN from mouse neurons (Petkau TL et al., (2017) Neurobiol Dis. 106: 14-22) or partial knockout from microglia (Petkau TL et al., (2017) J. Neuroinflammation. 14 (1): 225) reduces PGRN levels by about 50%, but does not result in phenotypes or detectable pathology. This suggests that both cell types are able to secrete enough progranulin to deliver cross-correction of tissue breadth.

[0008] Although no good animal models exist, previous studies using viral vectors to deliver PGRN have shown promise. Intraparenchymal infusion of the mouse GRN gene delivered by adeno-associated virus 1 (AAV1) into 12-month-old GRN-\- mice reduced lipofuscinosis and inflammation, suggesting that supplementation with progranulin could reverse aspects of the pathology (Arrant AE et al., (2017) Brain. 140(5):1447-1465; Arrant AE et al., (2018) as above). However, another study using a human GRN delivered by AAV9 and AAV4 at a dose approximately 33 times higher reported significant hippocampal degeneration and T cell activation in the GRN after 3 months. - / - Infiltration in mice and non-transgenic mice (Amado DA et al., doi: https: / / doi.org / 10.1101 / 308692).

[0009] The remarkable success of a recent trial of AAV9-SMN in type 1 spinal muscular atrophy (SMA) has stimulated research into gene therapy for neurological disorders. SMA is caused by homozygous deletion of the survival motor neuron gene (SMN). These children are born floppy and gradually become weaker, so that they never sit, crawl, stand or talk, because 95% of the children are already on oxygen before the age of two. Of the 15 patients who were given a single injection of AAV9-SMN between the ages of 1 and 7 months, none required oxygen, and 14 / 15 had significantly increased muscle strength, so that they could stand and walk without help and all were able to speak (Mendell JR et al., (2017) N. Engl. J. Med. 377 (18): 1713-1722).

[0010] However, high doses of intravenous AAV9-SMN in non-human primates and minipigs have been reported to be toxic (Hinderer C et al., (2018) Hum. Gene Ther. 29: 285-298), so minimizing the vector dose in any clinical trial should be carefully considered. In addition, AAV vector production is an extremely expensive process, currently estimated to be as high as ~$1 million per patient.

[0011] Because GRN overexpression can double the survival of transgenic mice expressing TDP-43 mutant forms (which mimic amyotrophic lateral sclerosis (ALS) and FTD) (Beel S et al., (2018) Mol. Neurodegener. 13 (1): 55), AAV-GRN treatment can be a successful treatment for ALS and FTD due to TDP-43 accumulation unrelated to GRN mutations. GRN delivered by lentivirus to Alzheimer's disease transgenic mouse models also enhances the clearance of amyloid β deposits in the brain (Minami SS et al., (2014) Nat. Med. 20 (10): 1157-64), and progranulin gene delivery has been shown to have neuroprotective effects in the MTPT toxin model of Parkinson's disease (Van Kampen JM et al., (2014) PLoS One. 9 (5): e97032). Because GRN is able to enhance autophagy and reduce inflammation, AAV-GRN may also provide therapeutic benefits for many other neurodegenerative conditions in which misfolded proteins accumulate.

[0012] Many research groups have been investigating various approaches to find suitable treatments for FTD-GRN patients, including the use of AAV vectors to deliver progranulin. For example, Passage Bio has received significant funding to develop AAV-delivered treatments 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). WO 2017 / 151884 also describes the use of a vector comprising a nucleic acid encoding progranulin, wherein the vector transduces cells in contact with cerebrospinal fluid (CSF) to express progranulin. Specifically, the vector comprises an AAV capsid protein and a pregranulin nucleic acid inserted between a pair of AAV inverted terminal repeats.

[0013] However, given the known toxicity of high-dose AAV vectors, there is a real need to optimize the expression of progranulin proteins carried in such vectors so that administration of such vectors results in a return of brain progranulin levels to near physiological levels in subjects carrying loss-of-function (LoF) mutations in the GRN gene and in subjects with reduced, suppressed or low progranulin levels. Indeed, optimization of therapeutic protein expression can be applied and extended to many diseases where the cause is either a genetic defect or the inability of the physiological system to produce proteins at levels below physiological normal.

[0014] Therefore, there is a need for improved AAV vectors that show increased expression of heterologous genes, such as progranulin, in the brain. Summary of the invention

[0015] In one aspect, the present invention provides an adeno-associated virus (AAV) vector comprising a nucleic acid comprising a human growth hormone intron 3 (hGHi3) sequence operably linked to a polynucleotide sequence encoding a polypeptide of interest.

[0016] In another aspect, the present invention provides an adeno-associated virus (AAV) vector comprising a synapsin promoter sequence operably linked to a polynucleotide sequence encoding a polypeptide of interest. Preferably, the polypeptide of interest is progranulin.

[0017] In another aspect, the present invention provides an adeno-associated virus (AAV) vector comprising a progranulin 3' untranslated region (UTR) sequence operably linked to a polynucleotide sequence encoding a polypeptide of interest. Preferably, the polypeptide of interest is progranulin.

[0018] In another aspect, the present invention provides an adeno-associated virus (AAV) vector comprising a polynucleotide sequence encoding a granulin precursor, wherein the polynucleotide sequence has at least 95% sequence identity with SEQ ID NO:4.

[0019] In one example, the hGHi3 sequence may comprise the sequence of SEQ ID NO: 7 or a variant, fragment or homolog thereof. For example, a suitable variant may comprise a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 7, such as at least 30, 50, 70, 80, 90 residues or the full length of SEQ ID NO: 7.

[0020] Preferably, the polynucleotide sequence encodes a heterologous polypeptide or transgenic protein, such as a non-AAV protein. In some embodiments, the polynucleotide sequence can encode a mammalian polypeptide, such as a human polypeptide or protein. In one embodiment, the polynucleotide sequence can include the complete coding sequence of a polypeptide of interest or a portion of its coding sequence. For example, the coding sequence can be derived from GRN and encode a progranulin (PGRN), such as human progranulin. It is preferred that the polynucleotide sequence includes the complete coding sequence of PGRN.

[0021] Thus, the polynucleotide sequence preferably encodes an amino acid sequence as defined in SEQ ID NO: 16, or a fragment, homologue or variant thereof. In a preferred embodiment, the encoded amino acid sequence may have at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 16, such as at least 50, 100, 200, 300, 500 residues or the full length of SEQ ID NO: 16.

[0022] In one embodiment, the polynucleotide sequence may comprise the wild-type coding sequence of human progranulin (SEQ ID NO: 1), or a variant, fragment or homolog thereof.

[0023] In a preferred embodiment, the polynucleotide sequence can be codon optimized, for example, optimized for expression in humans. In a preferred embodiment, the codon optimized sequence has a codon adaptation index (CAI) of at least 0.8, more preferably at least 0.9 for humans. Most preferably, the codon optimized sequence has a CAI of at least 0.9 for humans and mice. Preferably, the codon optimized sequence has a GC content of 30 to 70%, most preferably 60 to 65%. In one embodiment, the codon optimized sequence has a sequence similarity or sequence identity of at least 50%, 60%, 70%, 75% or most preferably at least 80% to the wild-type progranulin sequence.

[0024] Codon optimization can introduce new or different regulatory elements that enhance protein production. Such regulatory elements can additionally limit expression to specific tissues and / or organs, such as brain. In one example, the codon-optimized sequence can include a sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, or a variant, fragment or homologue thereof. In a particularly preferred embodiment, the codon-optimized sequence can include a sequence of SEQ ID NO:4, or a variant, fragment or homologue thereof, i.e., based on candidate III (PGRN-GS) described in the examples herein.

[0025] Suitable variants may comprise a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, such as at least 100, 200, 300, 500, 1000 or 1500 residues or the full length of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In a particularly preferred embodiment, the polynucleotide sequence may comprise a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4, such as at least 100, 200, 300, 500, 1000 or 1500 residues or the full length of SEQ ID NO: 4.

[0026] Generally, suitable fragments may comprise at least 20, 30, 50, 100, 200, 300, 500, 1000 or 1500 residues of any one of the polynucleotide 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.

[0027] 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 in a 5' flanking sequence. It should be understood that the ESE may be a part of a flanking sequence or inserted into a 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, a 5' flanking sequence may be a 5' guide sequence derived from a wild-type GRN gene. In such an example, the wild-type GRN 5' guide sequence comprises about 300 to 500 base pairs, for example 350 to 450 or about 392 base pairs.

[0028] In a preferred embodiment, the AAV vector may further comprise a 3' untranslated region (UTR) from progranulin. The 3'UTR of PGRN may function as an exonic enhancing element to enhance and potentially regulate PGRN expression. The 3'UTR of PGRN may, for example, be located in a box or vector downstream of the polynucleotide coding sequence, i.e., in the 3' region. Preferably, the box or vector comprises the 3'UTR of human PGRN, or a variant, fragment or homolog thereof. For example, the box or vector may comprise the sequence of SEQ ID NO: 14, or a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 14, such as at least 100, 200, 250 residues or the full length of SEQ ID NO: 14.

[0029] In another embodiment, the polynucleotide sequence may comprise a signaling sequence (i.e., a sequence encoding a signal peptide) derived from human growth hormone (hGH). The hGH signal sequence may comprise 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 to 51 of GRN.

[0030] In another embodiment, the AAV vector may further comprise a sequence that confers tissue or cell type specificity to protein expression. For example, a neuron-specific promoter, such as a promoter derived from a synaptic protein, may be included in the expression cassette. For example, the promoter may comprise the sequence of SEQ ID NO: 15, or a variant, fragment, or homolog thereof. For example, the cassette or vector may comprise the sequence of SEQ ID NO: 15, or a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15, such as at least 50, 100, 200, 300, or 400 residues or the full length of SEQ ID NO: 15.

[0031] In another embodiment, the present invention provides an AAV vector comprising a human synapsin promoter, hGHi3 and a progranulin 3'UTR, wherein the progranulin 3'UTR is operably linked to a polynucleotide sequence encoding a polypeptide of interest. Preferably, the polynucleotide sequence can be codon optimized, for example, optimized for expression in humans. Even more preferably, the polypeptide of interest is progranulin. The AAV vector can, for example, comprise the sequence of SEQ ID NO: 17, or a variant, fragment or homolog thereof. For example, the AAV vector can comprise the sequence of SEQ ID NO: 17, or a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 17, such as at least 100, 200, 300, 500, 1000, 1500, 2000, 2500 or 3000 residues or the full length of SEQ ID NO: 17.

[0032] In another example, the sequence may additionally or alternatively restrict expression of the polypeptide of interest to dividing or non-dividing cells, such as non-dividing neurons. In this way, any carcinogenic risk may be reduced.

[0033] Thus, in one specific example, the present invention provides an AAV vector cassette for delivering the GRN gene to effectively express and secrete progranulin to replenish PGRN levels in the brain of subjects suffering from ALS, FTD, and NCL11 and related neurodegenerative disorders. Such subjects may have progranulin levels below normal physiological levels, have GRN mutations, or lack this protein partially or completely.

[0034] Although it should be understood that the present invention contemplates the use of any suitable AAV serotype, serotype AAV9 is a preferred example.

[0035] In a second aspect, the present invention consists in i) a pharmaceutical composition or medicament comprising an AAV vector as described herein and ii) one or more pharmaceutically or physiologically acceptable carriers, excipients and / or diluents.

[0036] In one embodiment, the pharmaceutical composition or medicament may be formulated for administration or delivery, directly or indirectly, to the brain or specific regions of the brain, such as the frontal, temporal, and / or parietal lobes.

[0037] In a third aspect, the pharmaceutical composition or medicament as described herein can be used to treat a disease, preferably a neurological disease or condition. Typically, the disease to be treated is a neurodegenerative disease. In a specific embodiment, the neurological condition includes frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease.

[0038] The pharmaceutical composition or drug can be used to treat any patient suffering from the disease or a subgroup of these patients. In some embodiments, the pharmaceutical composition or drug as described herein is used to treat (i) subjects with heterozygous, homozygous or compound heterozygous GRN mutations, (ii) subjects suffering from sporadic neurological diseases and / or (iii) subjects with PGRN levels below physiological normal levels. "Sporadic disease" generally refers to subjects who are not aware of other family members being affected or who do not have loss-of-function GRN mutations associated with the disease.

[0039] In other words, the use of a pharmaceutical composition or drug as described herein can be for the preparation of a drug for the treatment of a neurological disorder, such as a drug for the treatment of a patient with frontotemporal dementia (FTD-GRN) heterozygous for a GRN mutation or a patient with neuronal ceroid lipofuscinosis (NCL11) homozygous or compound heterozygous for a GRN mutation. The drug can also be used for other neurological disorders in a population whose PGRN level is not lower than a physiological normal level, such as: patients with frontotemporal dementia (FTD) without a GRN mutation, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease or Alzheimer's disease, and subjects with other sporadic neurological diseases.

[0040] The pharmaceutical compositions or medicaments of the present invention may also be used in therapeutic methods, wherein a therapeutically effective amount of a pharmaceutically or physiologically acceptable composition comprising an AAV vector as described herein may be administered to a subject in need thereof to treat a neurological disorder, such as frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease, such as in subjects with sporadic neurological diseases and / or subjects with PGRN levels below physiological normal levels. For example, the pharmaceutical compositions or medicaments of the present invention may be administered as gene therapy to recently symptomatic FTD and ALS patients to arrest the degenerative process. Such treatments may even rescue dysfunctional but still viable neurons. Even a modest improvement in dementia symptoms and prevention of progression would significantly reduce the care burden on their families and communities.

[0041] It will be appreciated that a composition or medicament may be administered prophylactically to prevent or limit the onset of a disease, particularly in subjects at greatest risk.

[0042] In another aspect, the invention provides a nucleic acid as described above, e.g., a nucleic acid (e.g., a polynucleotide) 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 heterologous polynucleotide sequence encoding a polypeptide of interest (e.g., a polypeptide other than human growth hormone). Preferably, the polypeptide of interest comprises progranulin. The coding sequence of the polypeptide may be codon optimized, e.g., as described herein.

[0043] In another aspect, the invention provides a nucleic acid sequence comprising a sequence having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:4, such as at least 100, 200, 300, 500, 1000 or 1500 residues or the full length of SEQ ID NO:4.

[0044] The nucleic acid sequence may further comprise one or more regulatory elements (e.g., promoters), or other elements as described above for AAV vectors comprising such nucleic acids. Thus, in one embodiment, the nucleic acid comprises a neuron-specific promoter (e.g., a synapsin promoter). The nucleic acid may, for example, comprise an expression cassette suitable for use in an AAV vector, and may therefore, for example, comprise one or more AAV inverted terminal repeats. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Screening of codon-optimized candidates for secreted PGRN expression. Figure 1 a: 12 codon-optimized PGRN sequences under CMV promoter were synthesized. HEK-293 cells were used for transfection (250 ng / 2E+10 cells). The culture medium was collected after 48 hours and analyzed by western blot. Figure 1 b: The PGRN expression intensity of each candidate was determined by ImageJ. Figure 1 c: CAI scores before and after double codon optimisation (human and other species).

[0047] Figure 2 : Cloning strategy for hPGRN fusions. Figure 2 a: Schematic diagram of hPGRN candidates I, II, and III codon-optimized by different algorithms. Figure 2 b: BamH1 and BstB1 were used to cleave the 5' sequence of wild-type human PGRN. The digested DNA fragment was replaced with the codon-optimized PGRN candidate to generate a fusion plasmid.

[0048] Figure 3: Quantification of PGRN secreted from codon-optimized constructs in cell culture medium. Figure 3 a: PGRN codon-optimized and fusion constructs were transfected into HEK-293 cells. After 48 hours, the cell culture medium was sampled and the level of PGRN was determined by Western blot. Figure 3 b: The secreted PGRN level of each candidate was determined by ImageJ, and the value was first normalized to the number of cells (protein GAPDH), and then normalized to the cassette copy number (qPCR of CMV / GAPDH). The results of three biological replicates and four independent experiments were plotted in a scatter plot and subjected to one-way ANOVA statistical analysis (*<0.05).

[0049] Figure 4 : Quantification of PGRN expressed from codon-optimized constructs in cell culture lysates. Figure 4 a: PGRN codon optimized and fusion constructs were transfected into HEK-293 cells. After 48 hours, cell cultures were lysed and the levels of PGRN were determined by Western blotting. Figure 4 b: The PGRN level expressed by each candidate was determined by ImageJ, and the value was first normalized to the number of cells (protein GAPDH), and then normalized to the cassette copy number (qPCR of CMV / GAPDH). The results of three biological replicates and four independent experiments were plotted in a scatter plot and subjected to one-way ANOVA statistical analysis (*<0.05).

[0050] Figure 5 : Detection of the intron-mediated enhancement of PGRN protein expression by human growth hormone. Figure 5 a: Schematic diagram of the human growth hormone intron sequence located in the genome sequence. Figure 5 b: hGH intron 1, 2 or 3 was cloned into the 5'UTR of human PGRN-wt. Figure 5 c: The human PGRN signal sequence (18 amino acid peptide) was replaced with the human growth hormone signal sequence to test whether it could enhance the secretion of PGRN.

[0051] Figure 6 : Enhancement of PGRN secretion by hGH intron 3 and hGH signal sequence. Figure 6 a: PGRN intron and signal peptide sequences were transfected into HEK-293 cells. After 48 hours, the cell culture medium was sampled and the level of PGRN was determined by Western blotting. Figure 6b: The secreted PGRN level of each candidate was determined by ImageJ, and the value was first normalized to the number of cells (protein GAPDH), and then normalized to the cassette copy number (qPCR of CMV / GAPDH). The results of three biological replicates and four independent experiments were plotted in a scatter plot and subjected to one-way ANOVA statistical analysis (*<0.05, ***<0.0005).

[0052] Figure 7 : PGRN expression is enhanced by hGH intron 3 but not by hGH signal in cell lysates. Figure 7 a: PGRN intron and signal peptide sequences were transfected into HEK-293 cells. After 48 hours, cell cultures were lysed and PGRN levels were determined by Western blotting. Figure 7 b: The PGRN level expressed by each candidate was measured by ImageJ, and the value was first normalized to the number of cells (protein GAPDH), and then normalized to the cassette copy number (qPCR of CMV / GAPDH). The results of three biological replicates and four independent experiments were plotted in a scatter plot and subjected to one-way ANOVA statistical analysis (***<0.0001).

[0053] Figure 8 : hGHi3 did not enhance GRN expression of codon-optimized PGRN-GS. Figure 8 a: PGRN-GS inserted with hGH intron 3 was transfected into HEK-293 cells. After 48 hours, the culture medium was collected and used for western blotting of GRN expression. Figure 8 b: Expression levels were quantified using image-J and image studio, and the values ​​were first normalized to the cell number (protein GAPDH) and then to the cassette copy number (qPCR of CMV / GAPDH). Figure 8 c and 8d: ESE elements of PGRN wild-type sequence and codon-optimized PGRN-GW were analyzed by ESE finder 2.0. SR protein binding scores were plotted as bar graphs. Each color represents a different type of SR splicing protein.

[0054] Fig. 9 : hGHi3-PGRN-fusion construct enhances GRN expression in primary neurons. Fig. 9 a: Schematic representation of hGHi3 cloning of hGHi3-PGRN-fusion construct. Fig. 9b: AAV carrying hGHi3-PGRN-GA was generated and transduced into 7-day-old rat primary cortical neurons. After five days of incubation, the culture medium was collected for Western blot analysis of PGRN expression levels. Fig. 9 c: The PGRN level expressed by each candidate was measured by ImageJ, and the value was first normalized to the number of cells (protein GAPDH), and then normalized to the cassette copy number (qPCR of CMV / GAPDH). The results of three biological replicates and four independent experiments were plotted in a scatter plot and subjected to one-way ANOVA statistical analysis.

[0055] Fig.10 : The synapsin promoter increases PGRN secretion in rat cortical neurons. Fig.10 a: Wild-type PGRN cloned under the CMV and synapsin promoters in an AAV shuttle vector. Plasmids were transfected into rat cortical neurons at day 7 for 5 days, and then the culture medium was sampled and processed for western blot analysis. Fig.10 b: The intensity of PGRN expression was measured by imageJ.

[0056] Fig.11 : AAV9 carrying Syn-PGRNwt enhances PGRN expression in cultured rat neurons. Fig.11 a: Viral particles were produced using neuron-specific AAV9 capsids. AAV particles were then used to transduce rat primary cortical neurons for 5 days using 1E+6 viral particles on day 7. The culture medium was used for Western blotting. Fig.11 b: PGRN protein levels were measured by ELISA assay (0.001).

[0057] Fig.12 : Biodistribution of AAV9-PGRN transduction in mouse organs. Fig.12 a: AAV9-CMV-PGRN-WT and AAV9-Syn-PGRN-WT were administered by bilateral ICV injection (1E+12 GC / Kg). Fig.12 b: qPCR of human PGRN was performed on genomic DNA to determine tissue biodistribution.

[0058] Fig.13 : AAV9 carrying Syn-PGRNwt enhances PGRN expression in mouse cortex. AAV9 vectors carrying CMV-PGRN-WT and Syn-PGRN-WT were delivered by bilateral ICV injection (1E+12 GC / Kg). Fig.13 a: Commercial ELISA kit (Adipogen) was used to quantify PGRN expression levels in serum, CSF, and cortex. Fig.13b: Sera were diluted 1:300 and used for ELISA (n=3). Fig.13 c: CSF was diluted at 1:200 and used for ELISA (N=1). Fig.13 d: RIPA lysates of cortex were diluted 1:300 and then used for ELISA (N=3).

[0059] Fig.14 : Examples of PGRN vectors suitable for gene therapy. Fig.14 a: Schematic representation of the PGRN codon-optimized construct containing hGH3 intron 3 in the 5'UTR driven by the CMV promoter. Fig.14 b: Schematic representation of the PGRN codon-optimized construct containing hGH3 intron 3 in the 5'UTR driven by the synapsin promoter.

[0060] Fig.15 : ICV delivery of AAV9-Syn-PGRNwt showed widespread distribution of PGRN in the mouse brain. Fig.15 a: 4-week-old mouse brain sections were used for IHC to detect human GRN expression. Images were taken by Nikon A1R confocal. To reconstruct the whole brain image, the scanned images were stitched with 20% overlap in the automatic setting of NIC software. Human GRN (green), NeuN (red). Fig.15 b: IHC performed on non-injected brain sections showed no detection of GRNs. Fig.15 c: leather, Fig.15 d: hippocampus, Fig.15 e: thalamus.

[0061] Fig.16 : AAV9 carrying Syn-PGRNwt enhances PGRN expression in the mouse hippocampus. AAV9 vectors carrying CMV-PGRN-WT and Syn-PGRN-WT were delivered by bilateral ICV injection (2.5E+10). Fig.16 a: Commercial ELISA kit (Adipogen) was used to quantify PGRN expression levels in serum, CSF, and cortex. Fig.16 b: PGRN ELISA of AAV9-Syn-EGFP, AAV9-Syn-PGRNwt, AAV9-CMV-EGFP and AAV9-CMV-PGRNwt (N=3). Fig.16 c: PGRN Western blot of AAV9-Syn-EGFP and AAV9-Syn-PGRNwt. PGRN of approximately 68 kDa was detected only in the hippocampal lysate injected with AAV9-Syn-PGRNwt. Fig.16d: PGRN western blot of AAV9-CMV-EGFP and AAV9-CMV-PGRNwt.

[0062] Fig.17 : Schematic diagram of Syn-hGHi3-PGRN-GS-UTR. Fig.17 a: Schematic diagram of the PGRN codon-optimized construct driven by the Syn promoter, which contains hGH3 intron 3 and 3'UTR. Fig.17 b: DNA sequence of human PGRN-UTR.

[0063] Fig.18 : AAV9 carrying Syn-PGRN-GS, Syn-hGHi3-PGRN-GS and Syn-hGHi3-PGRN-GS-UTR enhances PGRN expression in mouse cortex. AAV9 vectors carrying Syn-PGRN-GS, Syn-hGHi3-PGRN-GS and Syn-hGHi3-PGRN-GS-UTR were delivered by bilateral IT injection. Cell lysates of cortical tissue were subjected to PGRN ESLISA assay and Western blot. Fig.18 a: A commercial ELISA kit (Adipogen) was used to quantitatively analyze the PGRN expression level in the cortex. Fig.18 b: RIPA lysates of cortex were diluted 1:300 and then used for ELISA (N=3). Fig.18 c: RIPA lysates from low (6.2E+10), medium (1.2E+11) and high (2.5E+11) cortices were used for ELISA (N=3).

[0064] Fig.19 : Immunohistochemistry of AAV9-PGRN-GS and AAV9-hGHi3-PGRN-GS-UTR. Mice were harvested 4 weeks after IT injection. Free frozen tissues were sectioned at a thickness of 30 uM and immunostained overnight with human-specific goat anti-GRN antibody (green). DAPI was used as a counterstain (blue). Fig.19 a, Fig.19 a': AAV-EGFP, Fig.19 b, Fig.19 b'; AAV9-PGRN-GS and Fig.19 c, Fig.19 c') AAV9-PGRN-GS-UTR.

[0065] Fig. 20: High power and 3D-rendered images of PGRN in the mouse brain. The distribution of human PGRN in the mouse cortex was studied by immunofluorescence to determine whether PGRN is secreted and taken up by other cells. Fig. 20 a: Many cortical neurons (stained red with NeuN) are positive for human PGRN (green) in a 3D rendered image ( Fig. 20 Some NeuN-negative cells are also seen in c), indicating that PGRN secreted by neurons is taken up by glial cells. Fig. 20 b: Some microglial cells labeled with IBA1 ( Fig. 20 b) shows GRN-positive foci (green), and in the 3D rendered image ( Fig. 20 d), indicating that they are able to take up PGRN secreted by neurons. Brain slices were imaged using 2-photon microscopy ( Fig. 20 a, Fig. 20 b) or by A1R confocal continuous scanning. The stacked images were processed by 3D rendering using IMARIS 8.3 to show GRN particles. Fig. 20 c) and microglia ( Fig. 20 d). Scale bar = 2 μM.

[0066] Fig.21 : Plasmid map of AAV PGRN vector. Human growth hormone intron was inserted between synapsin promoter and hPGRN. Native sequence of hPGRN UTR was added to the 3' end of hPGRN. ITR to ITR was cloned into a newly synthesized kanamycin resistant backbone plasmid. Fig.21 A: Linear form. Fig.21 B: Circular plasmid format. AAV2 ITR = adeno-associated virus type 2 inverted terminal repeat; hSyn promoter = human synapsin promoter; hGHi3 = human growth hormone intron 2; Codon-optimized human progranulin; hPGRN-3'UTR=human progranulin 3' untranslated region; bGH poly(A) signal=bovine growth hormone polyadenylation signal; f1 ori=F1 origin of replication; KanR=kanamycin resistance gene.

[0067] Sequence Listing

[0068] SEQ ID NO: 1—human PGRN wild type (PGRN-WT) DNA coding sequence;

[0069] SEQ ID NO:2—Candidate 1 (PGRN-IDT) artificial codon-optimized PGRN DNA coding sequence;

[0070] SEQ ID NO:3—Candidate II (PGRN-GA) artificial codon-optimized PGRN DNA coding sequence;

[0071] SEQ ID NO:4—Candidate III (PGRN-GS) artificial codon-optimized PGRN DNA coding sequence;

[0072] SEQ ID NO:5—human growth hormone intron 1 sequence;

[0073] SEQ ID NO:6—human growth hormone intron 2 sequence;

[0074] SEQ ID NO:7—human growth hormone intron 3 sequence;

[0075] SEQ ID NO:8—human growth hormone intron 4 sequence;

[0076] SEQ ID NO:9—the conventional DNA signal sequence of human growth hormone;

[0077] SEQ ID NO: 10—Translated amino acid sequence of the conventional signal sequence of human growth hormone;

[0078] SEQ ID NO: 11 – 5’ ESE ​​flanking sequence RPL41;

[0079] SEQ ID NO: 12 – 5’ ESE ​​flanking sequence UCHL1;

[0080] SEQ ID NO: 13 - 5' ESE flanking sequence RPL38.

[0081] SEQ ID NO:14—Human PGRN 3'UTR sequence

[0082] SEQ ID NO:15—Synapsin promoter sequence

[0083] SEQ ID NO:16 - Human PGRN amino acid sequence

[0084] SEQ ID NO:17—Syn-hGHi3-PGRN-GS-UTR DNA cassette sequence (from ITR to ITR)

[0085] SEQ ID NO:18—Bovine Growth Hormone (bGH) poly(A) signal

[0086] SEQ ID NO:19—5' AAV2 UTR sequence

[0087] SEQ ID NO:20—3' AAV2 UTR sequence DETAILED DESCRIPTION OF THE INVENTION

[0089] 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, which is operably linked to a polynucleotide sequence encoding a polypeptide of interest.

[0090] The present invention encompasses a specific embodiment in which the AAV vector cassette contains a codon-optimized PGRN gene that significantly increases the production and secretion of progranulin protein. PGRN secretion can be further increased by placing the sequence under the 5' regulatory control of hGH intron 3. Using a neuron-specific promoter, synaptobrevin, progranulin expression is restricted to neurons in vitro and in vivo, thereby reducing the risk of peripheral organ toxicity and carcinogenesis. The 3'UTR from PGRN can also be included in the cassette to further enhance and regulate PGRN expression.

[0091] Since the late 1970s, it has been known that otherwise identical genes containing introns and intron-free versions can exhibit significantly different expression profiles. hGHi3 is an intronic splicing element (ISE). However, the specific role of hGHi3 in increasing transgene expression / secretion in AAV vectors was previously unknown. In addition, the inventors discovered that hGH introns 2 and 4 have opposite effects. Therefore, the increase in transgene expression / secretion from AAV vectors containing the hGHi3 sequence is a surprising and favorable result.

[0092] As used herein, the singular forms "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0093] As used herein, the term "comprising" is synonymous with "including" or "containing", and is inclusive or open-ended and does not exclude additional, unrecited members, elements, or method steps. The term also encompasses "consisting of" and "consisting essentially of."

[0094] Numerical ranges recited by endpoints include all numbers and fractions subsumed within the corresponding ranges, as well as the recited endpoints.

[0095] As used herein, the term "about", when referring to a measurable value such as a parameter, amount, duration, etc., is meant to encompass variations from the specified value, especially variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less from the specified value, as long as such variations are suitable for making in the disclosed invention. It should be understood that the value referred to by the modifier "about" itself is also specifically and preferably disclosed.

[0096] Although by way of further illustration, the term "one or more", e.g. one or more members of a group of members, is itself clear, the term specifically encompasses reference to any one of the members or any two or more of the members, e.g. any ≥3, ≥4, ≥5, ≥6 or ≥7 of the members, etc., and up to all of the members.

[0097] The term "nucleic acid" or "polynucleotide" refers to a (e.g., polymeric) form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses each of the double-stranded form and the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0098] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When discussed in the context of delivering a gene product to a mammalian subject, polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, and compositions thereof refer to the corresponding complete polypeptides or any fragments or genetically engineered derivatives thereof that retain the desired biochemical function of the complete protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for delivering gene products to mammalian subjects (which may be referred to as "transgenics" for delivery to recipient cells) include polynucleotides encoding complete polypeptides or any fragments or genetically engineered derivatives having the desired biochemical function.

[0099] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that, when aligned, this percentage of bases or amino acids are the same when comparing two sequences. Sequence similarity can be determined in a variety of different ways. To determine sequence identity, methods and computer programs (including BLAST) available on the world wide web at ncbi.nlm.nih.gov / BLAST / can be used to align sequences. Another alignment algorithm is FASTA, which is available from the Genetics Computing Group (GCG) package of Oxford Molecular Group, Inc., a wholly owned subsidiary of Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow for gaps in the sequence. Smith-Waterman is an alignment algorithm that allows for gaps in the sequence. See Meth. Mol. Biol. 70:173-187 (1997). In addition, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0100] Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to determine sequence identity. The gap creation penalty is usually in the range of 1 to 5, generally in the range of 2 to 4, and in many embodiments will be 3. The gap extension penalty is usually in the range of about 0.01 to 0.20, and in many cases will be 0.10. The program has default parameters determined by the sequence being input for comparison. Preferably, the default parameters determined by the program are used to determine sequence identity. The program is also available from the Genetic Computing Group (GCG) package in Madison, Wisconsin, USA.

[0101] Another program of interest 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. The percent sequence identity is calculated by FastDB based on the following parameters:

[0102] Mismatch penalty: 1.00;

[0103] Open field penalty: 1.00;

[0104] Gap size penalty: 0.33; and

[0105] Joining Penalty: 30.0.

[0106] The present 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, the term encompasses all subtypes and both naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes, for example, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10, including AAVrh10), AAV type 12 (AAV-12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine 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 bovine mammals, and so on.

[0107] The genomic sequences of various AAV serotypes, as well as the sequences of the natural terminal repeats (TR), Rep proteins and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers 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); the disclosures thereof are incorporated herein by reference. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303.

[0108] The AAV vectors described herein are generally recombinant AAV vectors (rAAV). As used herein, "rAAV vectors" refer to AAV vectors comprising polynucleotide sequences from non-AAV sources (i.e., polynucleotides heterologous to AAV), which are generally sequences for the purpose of genetic transformation of cells. In some embodiments, the flanks of the heterologous polynucleotides may be at least one, and sometimes two, AAV inverted terminal repeats (ITRs). Preferably, ITR is derived from AAV serotype 2, i.e., the rAAV vector comprises AAV2 ITR. In some embodiments, the vector comprises one or both of the following AAV2 ITR sequences or homologs or variants thereof:

[0109] SEQ ID NO:19:

[0110] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGT

[0111] CGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0112] AGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0113] SEQ ID NO:20:

[0114] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT

[0115] CGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG

[0116] CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0117] Other suitable AAV ITR sequences are discussed, for example, in Wilmott et al. (2019), Human Gene Therapy Methods Vol. 30, No. 6: 206-213 and can be obtained from publicly accessible databases.

[0118] The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. The rAAV vector can be either single-stranded (ssAAV) or self-complementary (scAAV). A suitable cloning vector comprising other sequence elements that can be used for the vector of the present invention is RS540-AAV-ErbB-RASER1C-OFPBidBH3, which is disclosed in GenBank Accession No. MK801287.1.

[0119] "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a virus particle comprising at least one AAV capsid protein (usually all capsid proteins of wild-type AAV) and a polynucleotide rAAV enclosed in the shell. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is usually referred to as a "rAAV vector particle" or simply "rAAV vector". Therefore, the production of rAAV particles necessarily includes the production of rAAV vectors, because such vectors are contained in rAAV particles.

[0120] As used herein, "recombinant" means that the vector, polynucleotide, polypeptide or cell is the product of various combinations of cloning, restriction or ligation steps (e.g., involving polynucleotides or polypeptides contained therein) and / or other processes that produce a construct different from that found in nature. A recombinant virus or vector is a viral particle that contains a recombinant polynucleotide. The terms include replicas of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0121] In an embodiment of the present invention, the AAV vector comprises a nucleic acid sequence encoding a gene product, such as a heterologous nucleotide sequence encoding a heterologous polypeptide. "Gene" refers to a polynucleotide containing at least one open reading frame, which is capable of encoding a specific protein after being transcribed and translated. "Gene product" is a molecule produced by the expression of a specific gene. Gene products include, for example, polypeptides, aptamers, interfering RNAs, mRNAs, etc.

[0122] "Heterologous" means derived from a genotypically different entity that is different from the rest of the entity to which it is compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally associated is a heterologous promoter. Thus, for example, a rAAV comprising a heterologous nucleic acid encoding a heterologous gene product is a rAAV comprising a nucleic acid that is not normally contained in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by a naturally occurring wild-type AAV.

[0123] In one embodiment, the gene product (polypeptide of interest) is a therapeutic protein. A "therapeutic" peptide or protein is a peptide or protein that can alleviate or reduce symptoms caused by a protein deficiency or defect in a cell or subject. Alternatively, a "therapeutic" peptide or protein is a peptide or protein that additionally confers a benefit to a subject, such as an anti-degenerative effect.

[0124] When the gene product is a polypeptide, the polypeptide is typically one that enhances the function of a cell, such as a cell present in neuronal tissue, such as a neuron, a glial cell, or a photoreceptor cell. Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptor; VEGF binding antibody; VEGF binding antibody fragment (e.g., single-chain anti-VEGF antibody); endostatin; tumstatin; angiostatin; soluble Fit polypeptide (Lai et al. (2005) Mol. Ther. 12:659); Fc fusion protein comprising soluble Fit polypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16:10); pigment epithelium-derived factor (PEDF); soluble Tie-2 receptor; etc.); tissue inhibitor of metalloproteinases-3 (TIMP-3); light-responsive opsin (light-responsive opsin, such as rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl); and the like. Suitable polypeptides include, but are not limited to, glial derived neurotrophic factor (GDNF); fibroblast growth factor 2; neurotrophin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain derived neurotrophic factor (BDNF); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and Sonic hedgehog. Suitable polypeptides are disclosed, for example, in WO 2012 / 145601. However, in a preferred embodiment, the encoded polypeptide comprises progranulin.

[0125] In embodiments of the invention, the polynucleotide sequence is operably linked to: (i) human growth hormone intron 3 (hGHi3) sequence, (ii) synapsin promoter sequence, and / or (iii) 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.

[0126] For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a neuron-specific regulatory element (e.g., a neuron-specific promoter), such as a regulatory element that confers selective expression of the operably linked gene in neurons. Suitable neuron-specific promoters include neuron-specific enolase (NSE) promoter, Andersen et al. Cell. Mol. Neurobiol., 13: 503-15 (1993; neurofilament light chain gene promoter, Piccioli et al., Proc. Natl. Acad. Sci. USA, 88: 561 1-5 (1991); and neuron-specific vgf gene promoter, Piccioli et al., Neuron, 15: 373-84 (1995)]; and others. However, the neuron-specific promoter is preferably a synapsin promoter.

[0127] "Control elements" or "control sequences" are nucleotide sequences that participate in molecular interactions that contribute to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiates transcription of a coding region that is usually located downstream (in the 3' direction) of the promoter.

[0128] "Operably linked" refers to a juxtaposition of genetic elements wherein the elements are in a relationship that permits them to operate in the intended manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region to maintain this functional relationship.

[0129] As used herein, the term "promoter" may refer to a DNA sequence adjacent to the DNA sequence position encoding a recombinant product. The promoter is preferably operably connected to an adjacent DNA sequence. The promoter generally increases the amount of the recombinant product expressed from the DNA sequence compared to the amount of the recombinant product expressed when the promoter is not present. A promoter from an organism can be used to enhance the expression of a recombinant product of a DNA sequence derived from another organism. For example, a vertebrate promoter can be used to express jellyfish GFP in vertebrates. In addition, a promoter element can increase the amount of the recombinant product expressed for multiple DNA sequences connected in series. Therefore, a promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well known to those of ordinary skill in the art.

[0130] As used herein, the term "enhancer" may refer to a DNA sequence adjacent to the DNA sequence position encoding a recombinant product. An enhancer element is usually located upstream of a promoter element or may be located downstream or within a coding DNA sequence (e.g., transcribed or translated into a DNA sequence of one or more recombinant products). Therefore, an enhancer element may be located 100 base pairs, 200 base pairs or 300 or more base pairs upstream or downstream of the DNA sequence encoding a recombinant product. An enhancer element may increase the amount of the recombinant product expressed from the DNA sequence to an expression level higher than that provided by the promoter element. A variety of enhancer elements are readily available to those of ordinary skill in the art.

[0131] In some embodiments, the AAV vector may further comprise a polyadenylation signal. For example, the poly (A) signal may generally be present at the 3' end of the box comprising a polynucleotide encoding a polypeptide of interest. In one embodiment, the poly (A) signal is present downstream of the 3' untranslated region of the progranulin, i.e., between the 3' UTR sequence and one of the ITRs flanking the box. In one embodiment, the poly (A) sequence comprises or consists of a bovine growth hormone (bGH) poly (A) signal. Suitable polyadenylation signals (including bGH poly (A)) are known and described in, for example, Choi et al. Molecular Brain 2014, 7: 17; Goodwin EC, J Biol Chem. 1992; 267: 16330–16334 and U.S. 5,122,458. In one embodiment, the bGH poly (A) signal comprises the sequence of SEQ ID NO: 18, or a homologue or variant thereof:

[0132] SEQ ID NO:18

[0133] GTCCTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT

[0134] GTC

[0135] The present disclosure provides a pharmaceutical composition or medicament comprising: a) an AAV vector as 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 human use.

[0136] "Pharmaceutically acceptable" means a substance that is not biologically or otherwise undesirable, e.g., the substance can be administered to a subject without causing any undesirable biological effect. Thus, such pharmaceutical compositions can be used, for example, to transfect cells ex vivo or administer viral particles or cells directly to a subject.

[0137] Such excipients, carriers, diluents and buffers include any agent that can be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol.

[0138] As used herein, the term "one or more physiologically or pharmaceutically acceptable carriers, excipients and / or diluents" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., which are compatible with administration to humans or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients and / or carriers are approved by regulatory agencies of federal, state governments or other regulatory agencies or listed in the U.S. and / or European Pharmacopoeia or other generally recognized pharmacopeias for animals (including humans and non-human mammals). The terms diluent, excipient and / or "carrier" refer to diluents, adjuvants, excipients or vehicles used together with the pharmaceutical composition. Such pharmaceutical diluents, excipients and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin. Water, saline solutions and dextrose and glycerol aqueous solutions can be used as liquid diluents, excipients and / or carriers, especially for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerine, propylene, glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent, swelling agent (bulking agent), emulsifier or pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, sustained release formulations, etc. Examples of suitable pharmaceutical diluents, excipients and / or carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin. The preparation should be suitable for the mode of administration. Suitable diluents, excipients and / or carriers will be apparent to those skilled in the art and will depend to a great extent on the route of administration.

[0139] Pharmaceutically acceptable salts may be included, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described fully in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AK Kibbe et al., eds., 3rd edition Amer. Pharmaceutical Assoc.

[0140] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injections. Injectable preparations can be sterilized, for example, by filtering through a bacteria-retaining filter, or by adding a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0141] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the encapsulated or unencapsulated composition is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants such as glycerol, (d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, (e) solution retardants (a) lubricants, such as paraffin, (b) absorption promoters, such as quaternary ammonium compounds, (c) wetting agents, such as cetyl alcohol and glyceryl monostearate, (d) absorbents, such as kaolin and bentonite, and (e) 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 contain a buffer.

[0142] The medicaments described herein can be provided as a kit comprising at least one container and a package insert. The container contains at least one dose of a medicament comprising a composition as described herein. The package insert or label includes instructions for using the medicament described herein to treat a patient. The kit may further comprise other materials useful for administering the medicament, such as diluents, filters, IV bags and lines, needles, and syringes.

[0143] Methods of the present invention provide methods for delivering nucleic acid sequences to host tissues or cells. Carriers and other reagents, methods and pharmaceutical preparations of the present invention can also be used for methods (as a therapeutic method or other methods) in which proteins or peptides are administered to a subject in need thereof. In this way, proteins or peptides can therefore be produced in the subject. Subjects may need proteins or peptides as a therapeutic method or other methods because the subject lacks proteins or peptides, as further explained below.

[0144] As used herein, the terms "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing or reversing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or an adverse reaction attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease or is at risk of developing the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing the disease to regress.

[0145] In general, the present invention can be used to deliver any foreign nucleic acid that has the biological effect of treating or ameliorating symptoms associated with any disorder associated with gene expression in any organ, tissue, or cell, especially those associated with, for example, the brain.

[0146] Gene transfer has great potential use in understanding disease states and providing treatment for them. There are many genetic diseases in which defective genes are known and have been cloned. In some cases, the functions of these cloned genes are known. Generally, the above disease states are divided into two categories: defective states, usually enzymes, which are usually inherited in a recessive manner; and unbalanced states, at least sometimes involving regulatory or structural proteins, which are inherited in a dominant manner. For diseases in defective states, gene transfer can be used to bring normal genes into affected tissues for replacement therapy, and to create animal models of the disease using antisense mutations. For diseases in unbalanced states, gene transfer can be used to create disease states in model systems, which can then be tried to combat disease states. Therefore, the method of the present invention allows the treatment of genetic diseases. As used herein, the disease state is treated by partially or completely remedying the defects or imbalances that cause the disease or make it more serious. It is also possible to use site-specific integration of nucleic acid sequences to produce mutations or correct defects.

[0147] In one aspect, the invention provides a method for delivering a gene product to a tissue or cell (e.g., neuronal tissue or cell) of a subject, the method comprising administering an AAV vector as described above to the subject. The gene product can be a polypeptide, such as described above. For example, the cell can be a blood cell, a stem cell, a bone marrow (e.g., hematopoietic) cell, a liver cell, a cancer cell, a vascular cell, a pancreatic cell, a nerve cell, a glial cell, an epithelial or endothelial cell, a dendritic cell, a fibroblast, a lung cell, a muscle cell, a cardiomyocyte, an intestinal cell or a kidney cell. Similarly, the tissue can be, for example, selected from blood, bone marrow, muscle tissue (e.g., skeletal muscle, myocardium 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 heart tissue.

[0148] Delivery of gene products to neuronal tissue or cells can provide treatment of neurological disorders.Gene products can be delivered to various cell types present in neuronal tissue, for example, neurons or glial cells (e.g., astrocytes, oligodendrocytes, etc.).

[0149] The present disclosure provides a method for treating a disease (e.g., a neurological disease), the method comprising administering an effective amount of an AAV vector as described above to an individual in need thereof. The subject AAV vector can be administered by intracranial injection, intracerebral injection, intraocular injection, intravenous injection, or by any other convenient mode or route of administration.

[0150] Other exemplary modes of administration include oral, rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, etc., and direct tissue or organ injection, or intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Injections can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquids prior to injection, or as emulsions. In addition, viruses can be administered in a local rather than systemic manner, such as in a depot or sustained release form.

[0151] Preferably, the recombinant viral vector is administered to the subject in an amount sufficient to cause infection (or transduction) and expression of the heterologous nucleic acid sequence in the subject's cells (e.g., neuronal cells). Preferably, the target cells are neural cells (including cells of the central and peripheral nervous systems, especially brain cells).

[0152] Preferably, the vector is administered in a therapeutically effective amount. As used herein, a "therapeutically effective" amount is an amount sufficient to alleviate (e.g., relieve, reduce, lower) at least one symptom or cause associated with a disease state. Alternatively, a "therapeutically effective" amount is an amount sufficient to provide some improvement in the subject's condition. A "therapeutically effective amount" will fall within a relatively broad range that can be determined experimentally and / or clinically. For example, for in vivo injection, a therapeutically effective dose may be about 10 6 to about 10 15 AAV virions, such as about 10 8 Up to 10 12 For in vitro transduction, an effective amount of AAV virions to be delivered to cells would be about 10 8 to about 10 13 The effective amount of a composition or drug comprising an AAV vector as described herein may vary according to factors such as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. Other factors that may be considered include the severity of the disease; the age, weight, and sex of the patient being treated; diet, time and frequency of administration; drug combination; reaction sensitivity; and tolerance / response to treatment.

[0153] In some embodiments, more than one administration (e.g., two, three, four, or more administrations) may be employed to achieve desired gene expression levels over various time intervals, such as daily, weekly, monthly, yearly, etc.

[0154] The present invention can be used for veterinary and medical applications. Suitable subjects include birds and mammals, preferably mammals. As used herein, the term "birds" includes, but is not limited to, chickens, ducks, geese, quail, turkeys and pheasants. As used herein, the term "mammal" includes, but is not limited to, humans, cattle, sheep, goats, horses, cats, dogs, rabbits, etc. Most preferably, human subjects. Human subjects include fetuses, newborns, infants, adolescents and adult subjects.

[0155] Unless otherwise specified, all terms, including technical and scientific terms, used to disclose the present invention have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs. By way of further guidance, term definitions can be included to better understand the teachings of the present invention.

[0156] It should be understood that, for the sake of clarity, certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the present invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to the present invention are specifically encompassed by the present invention and disclosed herein, just as if each and every combination were individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed by the present invention and disclosed herein, just as if each and every such sub-combination were individually and explicitly disclosed herein.

[0157] The following examples are provided to illustrate certain embodiments of the present invention. They are not intended to limit the present invention in any way. Example

[0158] Example 1: Improving the efficiency of PGRN translation by codon optimization algorithm

[0159] The rate at which proteins are translated from RNA transcripts can be increased by changing codons to use codons that are optimal for a particular species, and gene expression in a particular cell type can be increased. By optimizing protein production per transcript, fewer viral vectors are used, reducing toxicity risk and cost. Optimization of the wild-type PGRN transcript was modeled using three open-source programs (IDT, GeneArt, and Genscript: see Materials and Methods for details). The codon adaptation index (CAI; https: / / www.genscript.com / tools / rare-codon-analysis) was validated using the Rare Codon Analysis tool. A CAI of 1.0 is considered ideal, while a CAI>0.8 is rated as good expression in the target expression organism. The lower the number, the higher the likelihood that the gene is poorly expressed.

[0160] 1.1 Codon optimization design

[0161] Use commercially available codon optimization algorithms ( https: / / eu.idtdna.com / CodonOpt)、( https: / / www.thermofisher.com / order / geneartgenes / projectmgmt) and ( The codon adaptation index (CAI, GeneScript) was used to score the modified GRN sequences. CAI>0.8 was scored as suitable for expression in the target organism, and sequences generated using different algorithms and CAI were Figure 1 To develop the best codon-optimized PGRN, algorithms for humans and other species were used ( Figure 1 c). 14 new constructs were designed and synthesized. A consensus Kozak sequence (GCCACC) was inserted before the ATG start codon and prepared by IDT, GeneArt and GenScript and named PGRN-IDT, PGRN-GA and PGRN-GS, respectively.

[0162] 1.1.1PGRN codon optimization

[0163] To determine the best codon-optimized PGRN sequence, double codon optimization was applied using the GenScript algorithm (https: / / www.genscript.com / codon_opt_pr.html) service to ensure that the codon-optimized sequence was viable in animal models as well as human cell lines and humans: PGRN codon optimization for humans may not be applicable to other species, such as mice or large animals (sheep or monkeys). Therefore, twelve double codon-optimized human PGRN transgenes were designed that significantly improved the CAI scores of both humans and other species (see Figure 1 c). However, PGRN expression and secretion levels tested by Western blotting did not significantly improve PGRN protein expression, except for one transgenic optimized for humans and mice (codop12, PGRN-GS; Figure 1 a and 1b). Therefore, an additional algorithm for human PGRN was used to create PGRN-IDT and PGRN-GA. All three constructs (PGRN-GS, PGRN-GA and PGRN-IDT) were then tested for optimal expression levels.

[0164] 1.1.2 Sequences of codon-optimized PGRN designed using three different algorithms

[0165] a) Human PGRN wild type (PGRN-WT) DNA coding sequence (coding sequence (CDS) length: 1782 bp) [SEQ ID NO: 1]:

[0166]

[0167] The codon adaptation index (CAI) of wild-type human PGRN is 0.83 and the GC content is 63.22%. The ideal percentage range of GC content is between 30% and 70%.

[0168] b) Candidate I (PGRN-IDT) artificial codon-optimized PGRN DNA coding sequence (CDS length: 1782 bp) [SEQ ID NO: 2]:

[0169]

[0170] The codon optimized candidate I (PGRN-IDT) has a CAI of 0.73 and a GC content of 59.33%. The codon optimized sequence has 76.11% homology with the wild type.

[0171] c) Candidate II (PGRN-GA) artificial codon-optimized PGRN DNA coding sequence (CDS length: 1782 bp) [SEQ ID NO: 3]:

[0172]

[0173] The codon optimized candidate II (PGRN-GA) has a CAI of 0.9 and a GC content of 56.23%. The codon optimized sequence has 78.81% homology with the wild type.

[0174] d) Candidate III (PGRN-GS) artificial codon-optimized PGRN DNA coding sequence (CDS length: 1782 bp) [SEQ ID NO: 4]:

[0175]

[0176] The CAI of codon-optimized candidate III (PGRN-GS) is 0.92 in human and 0.94 in mouse. The GC content of candidate 3 is 63.23%. The codon-optimized sequence has 81.34% homology to the wild type.

[0177] Schematic diagram of codon-optimized hPGRN candidates I, II, and III using different company algorithms. Figure 2 As shown in a.

[0178] Each of candidates I to III and the wild-type coding sequences (SEQ ID NOs: 1 to 4) encodes the amino acid sequence of progranulin (SEQ ID NO: 16):

[0179] MWTLVSWVALTAGLVAGTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWG CCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTC CRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGC DQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL

[0180] 1.2. Generation of PGRN-fusion candidates

[0181] Since PGRN is a secretory protein, the 5' guide sequence is important for the secretion signal. It is speculated that the RNA sequence may play a key role in guiding mRNA to the endoplasmic reticulum for translation. Therefore, using Bamh1 and BstB1 restriction sites, the 5' region of the codon-optimized sequence was replaced with a wild-type 5' GRN sequence (392bp) upstream of the codon-optimized protein coding sequence. This produced pAAV-CMV-PGRN-IDT-fusion, pAAV-CMV-PGRN-GA-fusion and pAAV-CMV-PGRN-GS-fusion.

[0182] Figure 2 b) is a schematic diagram of the fusion plasmids of the three codon-optimized candidates, in which the 5' sequence of the codon-optimized sequence has been replaced with the 5' sequence of wild-type human PGRN.

[0183] 1.3. Complete Characterization of Codon-Optimized and PGRN Fusion Candidates

[0184] The expression level of codon-optimized PGRN is compared with wild-type human PGRN. The secreted PGRN level is directly measured in cell culture medium and cell lysate, and it is normalized relative to cell count as the ratio relative to housekeeping protein GAPDH. In order to directly compare the relative efficiency of the PGRN production of each PGRN box, the vector genome copy number of each cell is quantitatively measured by the quantitative qPCR of CMV (as the ratio (CMV / GAPDH) relative to the same housekeeping gene GAPDH, which represents effective HEK-293 cell transduction). This is used as the denominator (denominator) of the PGRN level of normalization and accurate quantification of each box.

[0185] Human kidney cell line (HEK-293) was maintained at 37°C in a humidified chamber with CO2 (5%) and maintained in Dulbecco's Modified Medium (DMEM, Thermo Scientific) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were passaged every three days.

[0186] According to the manufacturer's protocol, lipofectamine 2000 (Life Technologies) was used to transfect HEK-293 cells in 24-well plates at 80% confluence for western blotting. A total of 250ng DNA was transfected or co-transfected in 40,000 cells. pAAV-CMV-EGFP or pAAV-Syn-EGFP was used as a transfection efficiency control. In order to check the secretion level of PGRN, the culture medium was replaced with a serum-free culture medium, which was supplemented with insulin transferrin selenium (ITS) the next day. After 48 hours, the culture medium was collected and used for western blot analysis immediately. The cells were lysed with a gentle lysis buffer (NP40) and then centrifuged at 10,000xg for 10 minutes. The supernatant of the transfection well was collected for PGRN ELISA, and the precipitate was stored at -80C for genomic DNA extraction.

[0187] The protein samples were loaded into a pre-made HPLC column with MOPSSDS running buffer (Thermo Fisher). Novex TM 10% Bis-Tris Midi gel and run at a constant 100 V. The gel was briefly immersed in NuPage transfer buffer (Thermo Fisher) and then transferred using iBlot 2 TM (Thermo Fisher). Blocking reagent (Roche, 10%) containing phosphate buffered saline (PBS) was used to block the protein membrane for 1 hour. After that, the membrane was incubated in primary antibody diluted in 5% blocking buffer at 4°C overnight. After washing three times with Tris buffered saline containing Tween 20 (TBS-T), the membrane was incubated with secondary antibody for 1 hour. After washing three times, CLx Infrared Imaging System( Biosciences). Using ImageJ and ImageStudio TM -light was used to measure the intensity of each protein band. The primary antibodies used for western blot detection were GRN (Abcam, ab191211) and GAPDH (Abcam, ab82485). The secondary antibody used for western blot detection was goat anti-mouse IgG (H+L) 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).

[0188] Western blotting of PGRN in the culture medium showed that low CAI candidate I (IDT) showed significantly reduced expression (see Figure 3 a). However, the PGRN expression and secretion of high CAI candidates II (GA) and III (GS) were comparable or higher than that of wild-type PGRN. Three biological replicates were tested and performed in four independent experiments. PGRN-GS significantly increased PGRN expression by 72% compared to wild-type (p=0.0327).

[0189] Several "fusion constructs" were also tested. These fusion constructs retained the wild-type genomic signal sequence and flanking regions until the BstB1 restriction site was fused to the codon-optimized PGRN sequence (see Figure 2 b, yellow). Unfortunately, secretion of PGRN protein from the PGRN-fusion cassette was similar to or lower than that of wild-type PGRN (see Figure 3 a and 3b). The initial conclusion was that the 392 bp 5' conventional flanking DNA sequence of wild-type PGRN did not significantly improve codon-optimized PGRN secretion under the CMV promoter alone. However, subsequent results showed that expression of the fusion construct was enhanced when combined with the 5' intronic enhancing sequence (see below).

[0190] Western blots of HEK cell lysates showed similar results to secreted PGRN, with codon-optimized PGRN-GA and GS increasing PGRN expression by 39.55% (not significant) and 127.7% (p=0.0014), respectively. However, PGRN-IDT and other "fusion" constructs again failed to increase PGRN expression (see Figure 4 a and 4b).

[0191] Example 2: Development of intronic enhancing elements to increase PGRN expression.

[0192] To further increase the expression and secretion of PGRN, the addition of introns to the 5' sequence of PGRN was explored to see if gene expression could be enhanced. Introns can increase transcription levels by affecting transcription rate, nuclear export, transcript stability, or mRNA translation efficiency, a phenomenon known as intron-mediated enhancement (IME) (Shaul O. Int J Biochem Cell Biol. 2017 Oct; 91 (Pt B): 145-155). A search was conducted for intron sequences less than 300 bp that could be used to test intron-mediated enhancement. An intron within the human growth hormone (hGH) gene was selected as a suitable candidate (see Figure 5 a).

[0193] Wild-type GRN sequences were gene synthesized and cloned into pAAV plasmids (Addgene; 99280) using BamH1 and Xho1 restriction sites. These constructs were used as master vectors (pAAV-CMV-PGRNwt) to subclone previously codon-optimized constructs. For pAAV-Syn-PGRNwt, the human synapsin promoter sequence was replaced from the Addgene vector (58881) using Pci1 and Bamh1 restriction sites.

[0194] The polynucleotide sequence of the synapsin promoter is shown in (SEQ ID NO: 15):

[0195] AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCC TCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG

[0196] The intron sequence of human growth hormone (hGH1) was used to improve PGRN expression. hGH1 comprises four introns, which are synthesized by GenScript and cloned into pAAV-CMV-PGRN plasmid using BamH1 and Age1 restriction sites to generate 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, the wild-type PGRN coding sequence was analyzed by exon splicing enhancer (ESE)-finder (ESE 3.0, http: / / krainer01.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi) and the ESE high frequency predicted on GRN. To further enhance PGRN expression, the hGHi3 intron was introduced into a fusion construct retaining the 5' ESE element of wild-type PGRN to generate pAAV-CMV-hGHi3-PGRN-GA-fusion and pAAV-CMV-hGHi3-PGRN-GS-fusion. Bamh1 and Age1 restriction sites were used to generate the neuron-specific expression constructs pAAV-Syn-hGHi3-PGRN-GA-fusion and pAAV-Syn-hGHi3-PGRN-GS.

[0197] SEQ ID NO: 5 is the sequence of human growth hormone intron 1 (261 bp):

[0198] GTAAGCGCCCCTAAAATCCCTTTGGGCACAATGTGTCCTGAGGGGAGAGGCAGCGACCTGTAGATGGGACGGGGGCACTAACCCTCAGGTTTGGGGCTTCTGAATGTGAGTATCGCCATGTAAGCCCAGTAT TTGGCCAATCTCAGAAAGCTCCTGGTCCCTGGAGGGATGGAGAGAGAAAAACAAACAGCTCCTGGAGCAGGGAGAGTGCTGGCCTCTTGCTCTCCGGCTCCCTCTGTTGCCCTCTGGTTTCTCCCCAG(SEQ ID NO:5)

[0199] SEQ ID NO: 6 is the sequence of human growth hormone intron 2 (209 bp):

[0200] GTAAGCTCTTGGGGAATGGGTGCGCATCAGGGGTGGCAGGAAGGGGTGACTTTCCCCCGCTGGGAAATAAGAGGAGGAGACTAAGGAGCTCAGGGTTTTTCCCGAAGCGAAAATGCAGGCAGATGAGCACACGCTGAGTGAGGTTCCCAGAAAAGTAACAATGGGAGCTGGTCTCCAGCGTAGACCTTGGTGGGCGGTCCTTCTCCTAG(SEQ ID NO:6)

[0201] SEQ ID NO:7 is the sequence of human growth hormone intron 3 (92bp):

[0202] GTGAGTGGATGCCTTCTCCCCAGGCGGGGATGGGGGAGACCTGTAGTCAGAGCCCCCGGGCAGCACAGCCAATGCCCGTCCTTCCCCTGCAG(SEQ ID NO:7)

[0203] SEQ ID NO:8 is the sequence of human growth hormone intron 4 (253bp):

[0204] GTGAGGGTGGCGCCAGGGGTCCCCAATCCTGGAGCCCCACTGACTTTGAGAGCTGTGTTAGAGAAACACTGCTGCCCTCTTTTTAGCAGTCAGGCCCTGACCCAAGAGAACTCACCTTATTCTTCATTTCCCCTCGTGAATCCTCCAGGCCTTTCTCTACACCCTGAAGGGGAGGGAGGAAAATGAATGAATGAGAAAGGGAGGGAACAGTACCCAAGCGCTTGGCCTCTCCTTCTCTTCCTTCACTTTGCAG(SEQ ID NO:8)

[0205] The regions flanking the restriction sites Bamh1 and BstB1 in pAAV-CMV-PGRNwt and pAAV-CMV-PGRN-GS (1-392 bp) were used for signal sequence replacement. The N-terminal region of the PGRN signal sequence (1-51 bp) was replaced with 78 bp of hGH1 for gene synthesis. These were subcloned into pAAV-CMV-PGRNwt and pAAV-CMV-PGRN-GS using the Bamh1 and BstB1 restriction sites to generate pAAV-CMV-hGHs-PGRNwt and pAAV-CMV-hGHs-PGRN-GS.

[0206] SEQ ID NO:9 is the conventional DNA signal sequence of human growth hormone, which replaces the PGRN signal sequence:

[0207] ATG GCT ACA GGC TCC CGG ACG TCC CTG CTC CTG GCT TTT GGC CTG CTC TGCCTG CCC TGG CTT CAA GAG GGC AGT GCC (SEQ ID NO:9)

[0208] SEQ ID NO: 10 is the amino acid sequence of the conventional signal sequence translation of human growth hormone:

[0209] MATGSRTSLLLAFGLLCLPWLQEGSA(SEQ ID NO:10)

[0210] To test the effect on PGRN expression, hGH introns 2, 3, and 4 sequences were cloned into the 5'UTR region of PGRN-WT (see Figure 5 b). In addition, the wild-type signal sequence of PGRN was replaced with the hGH signal sequence (see Figure 5 c).

[0211] 2.1 Addition of intronic enhancer elements

[0212] The efficiency of each hGH intron on PGRN expression and secretion was assessed by Western blotting (methods, see above), which demonstrated that intron 3 greatly increased secreted PGRN (105%, p=0.0004), whereas intron 4 decreased secretion and intron 2 abolished secretion (see Figure 6 a and 6b). In addition, Figure 6 As shown in Figs. 6a and 6b, replacement of the wild-type PGRN signal peptide with the hGH signal peptide also showed a significant increase in secreted PGRN (71%, p=0.0088).

[0213] PGRN expression levels measured in HEK-293 cell lysates confirmed that intron 3 significantly increased PGRN expression (148%, p=0.0009) (see Figure 7 a and 7b), whereas intron 4 reduced PGRN expression by 40%, and intron 2 abolished expression. Figure 7 As shown in Figs. 7a and 7b, substitution of the wild-type hGH signal sequence increased PGRN expression by 23%, which was not significant.

[0214] These results indicate that intron 3 enhances PGRN translation, which in turn increases PGRN secretion into the culture medium. Substitution with the signal peptide of hGH slightly increased PGRN secretion but not expression.

[0215] Example 3: Combining intronic and exonic enhancer elements.

[0216] To test the intron-mediated enhancement of codon-optimized PGRN-GS by hGHi3, the hGHi3 element was subcloned into the 5' position of PGRN-GS.

[0217] Surprisingly, hGHi3 had no enhancing effect on PGRN-GS expression (see Figure 8 a and 8b), because the inclusion of intron 3 slightly reduced PGRN. This suggests that the action of hGHi3 may require the exonic splicing element (ESE) within the wild-type PGRN sequence to produce a synergistic effect on splicing. The ESE elements in the wild-type PGRN and codon-optimized PGRN-GS sequences were searched using the software ESE finder 3.0 (see Figure 8 c and 8d). The ESE distribution pattern in codon-optimized PGRN-GS was significantly altered compared to wild-type PGRN. Thus, attempts to codon-optimize PGRN inadvertently removed the ESEs required to provide enhancement due to the inclusion of hGHi3.

[0218] Therefore, the intronic enhancer hGHi3 was combined with a PGRN fusion construct carrying the original 392 bp sequence of wild-type GRN fused to the PGRN-GA codon-optimized sequence (see Fig. 9 a) It was packaged into AAV9 vector and delivered to rat primary cortical neurons in culture at an MOI of 1e6.

[0219] The 5'ESE flanking sequence is as follows:

[0220] RPL41:

[0221] CGACACCCGGCGCTCCATTAAATAGCCGTAGACGGAACTTCGCCTTTCTCTCGGCCTTAGCGCCATTTTTTTGGGTGAGTGTTTTTTGGTTCCTGCGTTGGGATTCCGTGTACAATCCATAGACATCTGACCTCGGCACTTAGCATCATCACAGCAAACTAACTGTAGCCTTTCTCTCTTTCCCTGTAGAAACCTCTGCGCC (SEQ ID NO: 11);

[0222] UCHL1:

[0223] TTTCCCCCTCGCTTGGTTCTGCCCCTGCTCCCCCTGCACAGGCCTCACAGTGCGTCTGGCCGGCGCTTTATAGCTGCAGCCTGGGCGGCTCCGCTAGCGTTTTTCGTCTTCCCTAGGCTATTTCTGCCGGGCGCTCCGCGAAGG (SEQ ID NO: 12);

[0224] RPL38:

[0225] ACTGCCCGGAAACGGAAGTCTCGTTCTTTTCGTCCTTTTCCCCGGTTGCTGCTTGCTGTGAGTGTCTCTAGGGTGATACGTGGGTGAGAAAG (SEQ ID NO: 13).

[0226] The above 5'ESE flanking sequences can be used in some embodiments of the present invention. However, in the following examples, these sequences were not further investigated.

[0227] For each virus, 10 × 145 cm of low-passage (≤P30) HEK-293T cells were collected. 2The plates were used at about 80% confluence on the day of transfection. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% heat-inactivated FBS at 37°C, 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 test 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-chain transgene were added to serum-free DMEM to make a final volume of 30 mL. The mixture containing DNA was then filtered into the mixture containing PEI through a 0.45 μm polyethersulfone (PES, Sartorius, Epsom, UK) syringe filter. The solution was mixed and incubated at room temperature for 15 minutes. The transfection solution was then added dropwise to each 145 cm 2 In a culture dish.

[0228] AAV viral particles were harvested 72 hours after transfection. Soup and pellet were collected and subjected to freeze-thaw cycles. All supernatants and pellets were treated with benzonase (50 Unit / mL, 37C, 30 minutes) and then centrifuged at 2000xg for 30 minutes at 18 ° C. The supernatant was filtered with a 0.45 μM pore size filter and then loaded onto a pre-equilibrated AAVX POROS affinity column (Thermo Fisher) on an AKTA system for AAV purification. The purified AAV was kept at -80 ° C.

[0229] Neuronal cells were isolated from cortical tissue at embryonic day 18 (E18) (Sprague-Dawley rats). 100,000 cells were plated on 24-well plates coated with PDL. Cells were grown in Neurobasal media supplemented with penicillin / streptomycin (0.5%), Glutamax (1%) and B27 (2%, Thermo Fisher). Cells were transduced with AAV at a multiplicity of infection (MOI) of 1e6 at day 7 in vitro (DIV). Neurons were harvested at DIV12 and subjected to Western blotting (methods see above) and ELISA.

[0230] Genomic DNA was isolated 48 h after transfection using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's protocol. TM Measure DNA concentration.

[0231] The PGRN content of the samples was assessed using the Adipogen Life Sciences Progranulin (Human) ELISA Kit (AG-45A-0018YEK-KI01). The sandwich ELISA kit uses a polyclonal antibody pre-coated on a microtiter plate to capture human progranulin in the sample and a biotinylated second polyclonal antibody to detect the protein. The STREP-HRP solution was added to the wells, and the PGRN signal was detected using TMB substrate for 10 minutes, an acidic stop solution was added, and the absorbance of each well at 450 nm was measured. The concentration of PGRN was calculated using a recombinant human PGRN standard in the range of 0.063-4ng / ml. The standards and all other reagents required for the assay are included in the kit.

[0232] like Fig. 9 As shown in b and 9c, PGRN secretion from cortical neurons transduced with the hGHi3-PGRN-GA construct was greater than that from hGHi3-wild-type PGRN alone.

[0233] These results prove that the expression and secretion increase produced by hGHi3 is because hGHi3 acts as an intron splicing element (ISE). Preferably, hGHi3 acts in combination with an exon splicing element (ESE) present in the initial 392bp sequence of wild-type PGRN. Therefore, hGHi3 may carry a cryptic ISE sequence, which may require an ESE to enhance splicing in the wild-type PGRN sequence (McCarthy and Philips (1998) Human Molecular Genetics.7; 1491–1496). Without wishing to be bound by theory, the predicted mechanism is that this combination promotes the combination of splicing proteins rich in serine and arginine (SR), which accelerates RNA processing and subsequent translation.

[0234] Example 4: Restricted expression of synapsin using the neuron-specific promoter.

[0235] PGRN is used by many cell types. However, in the brain, its expression and secretion are mainly determined by microglia and neurons, and both cell types are affected by PGRN deficiency. It is very difficult to transduce microglia using viral vectors, so the focus is on maximizing the transduction and expression of PGRN to post-mitotic neurons, which also avoids the risk of PGRN accelerating cell division of microglia and astrocytes (which may be carcinogenic). Wild-type PGRN expression under the pan-mammalian conventional promoter CMV was compared with the human neuron-specific promoter synapsin. As Fig.10As shown, in rat cortical neuron cultures with the synapsin promoter, expression was restricted and PGRN secretion was significantly increased.

[0236] To test whether packaging of the PGRN cassette in the AAV9 vector supports the initial findings from plasmid transfection, AAV9 carrying either CMV-PGRNwt or Syn-PGRNwt was generated. Rat primary cortical neurons were cultured for 7 days and transduced with the AAV9-GRN vector. Five days later, the cell culture medium was sampled and subjected to Western blotting. The synapsin promoter increased GRN expression by 5.4-fold compared to CMV, which was confirmed by PGRN ELISA (see Fig.11 ).

[0237] To validate these studies in vivo, AAV9-CMV-PGRNwt and AAV9-Syn-PGRN vectors were injected bilaterally intra-cerebroventricularly (ICV) into 8-week-old C57BL\6J male mice ( Fig.12 a). Mice were deeply anesthetized with isoflurane and fixed in a stereotaxic frame equipped with a digital stereotaxic control panel. Hamilton syringes were used to inject bilaterally into the anterior ventricle or posterior thalamus of mice. Each mouse received a total dose of 3 μL of 5e+10 VG, infused at a rate of 0.5 μL / min, with a waiting time of 2 minutes after infusion before removing the trochanter. Mice were injected at 8 weeks of age and killed at 12 weeks of age 4 weeks after injection. Half of the brain and spinal cord, spleen, heart, liver, kidney, lung, testis, blood and cerebrospinal fluid (CSF) were collected and freshly frozen in liquid nitrogen. The other half of the brain was post-fixed with polyformaldehyde (PFA) and processed for histology and immunocytochemistry (IHC).

[0238] For brain, blood plasma and organ collection, mice were anesthetized with pentobarbital (pentobarbital) (100mg / kg, Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI), and blood was collected in a syringe containing EDTA (250mM) by cardiac puncture to prevent blood coagulation. Blood was kept on ice, and then centrifuged at 4°C for 10 minutes with 1000xg to separate blood plasma. Then mice were perfused through the heart with PBS. The brain was taken out and divided into hemispheres, one of which was microdissected into prefrontal cortex, striatum, hippocampus, cerebellum, subcortical area and cortex, and was quickly frozen in liquid nitrogen for biochemical analysis, and one of which was post-fixed in 4% paraformaldehyde for 24 hours for histological analysis. Spinal cord, spleen, heart, liver, kidney, lung, testis, blood and cerebrospinal fluid were taken out and frozen in liquid nitrogen immediately, for ELISA and Western blot analysis.

[0239] The distribution and quantification of CMV-PGRN and Syn-PGRN transduction in each tissue was measured by amplifying human PGRN from genomic DNA using qPCR. Genomic DNA was diluted to 7.5 ng / ul in nuclease-free H2O. qPCR was performed using Powerup TM Green Master Mix was performed according to the standard protocol. For each set of reactions, a standard curve was also prepared using DNA of known concentration.

[0240] Fixed hemispheres were cryoprotected in 30% sucrose and cut into 30 μm sections on a sliding microtome (Leica Biosystems). The sections were then immunostained. For pathological analysis and qualitative assessment of progranulin immunoreactivity, sections were incubated overnight in primary antibodies (PGRN, a marker for neurons (NeuN) or microglia (Iba1)) and the next day incubated in a species-matched secondary antibody against PGRN. -488-conjugated antibodies were incubated with species-matched AlexaFluor-647-conjugated antibodies against NeuN and Iba1.

[0241] Low-magnification, high-resolution images of progranulin immunostaining were obtained using a slide scanner (Olympus VS120) for image analysis.

[0242] 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 Fig.12 ).

[0243] ELISA was used to quantify PGRN expression levels in serum, CSF, and cortical tissue of transduced mice. Mice injected with AAV9-CMV-PGRN showed high serum PGRN levels, which were barely detectable in mice injected with AAV9-Syn-PGRN (see Fig.13 ). In contrast, PGRN levels in the cortex and CSF were much higher in mice injected with AAV9-Syn-PGRN than in mice injected with AAV9-CMV-PGRN (see Fig.13 c and 13d), which confirms the neuronal specificity of synapsin in vivo.

[0244] Example 5: Ubiquitous PGRN expression by AAV9-Syn-PGRNwt after ICV injection

[0245] Mice injected with AAV9-Syn-PGRN-wt by ICV injection were harvested 4 weeks later ( Fig.15 ). The brain sections were fixed with PFA and used for IHC. Goat anti-human PGRN antibody (green) was expressed in the cortex ( Fig.15 c) Hippocampus ( Fig.15 d) and thalamus ( Fig.15 e) shows widespread GRN expression. Counterstaining was performed using the rabbit anti-neuron-specific antibody NeuN. Human PGRN was not detected in uninjected mouse brain tissue, but was strongly stained in the hippocampus and, to a lesser extent, in the thalamus and cortex of mice injected with AAV-Syn-PGRNwt ( Fig.15 d). PGRN ELISA of hippocampal tissue lysates confirmed that the synapsin promoter produced a 3-fold increase in PGRN expression compared with the CMV promoter ( Fig.16 b). Western blotting confirmed the detection of a strong PGRN band at approximately 68 kDa ( Fig.16 c, d).

[0246] Example 6: Combination of intron sequence hGHi3 and 3'UTR element.

[0247] We then tested whether the 3'UTR could function as an exonic enhancer element (ESE) to enhance and potentially regulate PGRN expression. We synthesized 284 bp of the wild-type PGRN 3'UTR and cloned it into the 3' region (in addition to the intronic enhancer hGHi3 and our codon-optimized PGRN-GS) to generate Syn-hGHi3-PGRN-GS-UTR ( Fig.17 ).

[0248] The 3'UTR sequence of wild-type PGRN (284 bp) is as follows Fig.17b and as shown below (SEQ ID NO: 14):

[0249] GGGACAGTACTGAAGACTCTGCAGCCCTCGGGACCCCACTCGGAGGGTGCCCTCTGCTCAGGCCTCCCTAGCACCTCCCCTAACCAAATTCTCCCTGGACCCCATTCTGAGCTCCCCATCACCATGGGAGGTGGGGCCTCA ATCTAAGGCCTTCCCTGTCAGAAGGGGGTTGTGGCAAAAGCCACATTACAAGCTGCCATCCCCTCCCCGTTTCAGTGGACCCTGTGGCCAGGTGCTTTTCCCTATCCACAGGGGTGTTTGTGTGTGTGCGCGTGTGCGTTTC

[0250] This sequence containing 5' Pme1 and 3' XhoI was synthesized using gene synthesis services from GenScript. The UTR sequence was cloned into AAV-Syn-hGHi3-PGRN-GS to form AAV-Syn-hGHi3-PGRN-GS-UTR. Fig.17 The vector map of the plasmid containing the cassette shown is as follows Fig.21 The DNA sequence of the Syn-hGHi3-PGRN-GS-UTR vector cassette (from ITR to ITR) is shown below (SEQ ID NO: 17).

[0251]

[0252] SEQ ID NO: 17 comprises the following sequence elements:

[0253] AAV2 ITR – residues 1-130

[0254] hSyn promoter – residues 341-788

[0255] hGHi3 – residues 801-892

[0256] PGRN-GS – residues 905-2686

[0257] 3'UTR – residues 2702-2985

[0258] bGH poly(A) signal – residues 3015-3222

[0259] AAV2 ITR – residues 3245-3385

[0260] Example 7: Broad PGRN expression of AAV9-Syn-PGRN-GS, AAV9-Syn-hGHi3-PGRN-GS, and AAV9-Syn-hGHi3-PGRN-GS-UTR after intrathalamic injection.

[0261] The optimized dose of the final PGRN cassette ( Figure 2 , 5 , 9, 14 and 17) were injected into the mouse brain to compare the efficiency of the vectors. The transduction efficiency of the AAV-PGRN virus was detected by PGRN protein expression using ELISA and IHC. We chose the intrathalamic (IT) administration route because this would provide better cortical expression of EGFP and PGRNwt. The addition of the hGHi3 intronic enhancer element consistently increased PGRN levels by approximately 27%, and the addition of the 3'UTR exonic enhancer element increased PGRN expression by approximately 37% ( Fig.18 b and 19). Quantification of cortical levels of PGRN by ELISA and IHC following IT injection of low (6.2E+10), medium (1.2E+11), and high (2.5E+11) doses of vector confirmed a dose-abundance effect in the target tissue ( Fig.18 c).

[0262] There are numerous granular PGRN-positive foci in cortical neurons ( Fig. 20a and c). Because pathological changes were seen in microglia and neurons, we sought to determine whether PGRN secreted by neurons was taken up by non-neuronal cells, including microglia. PGRN under the synapsin promoter was expressed only in neurons but was detected in many NeuN-negative cells ( Fig. 20 a), and small foci of PGRN can be detected in IBA1-positive microglia ( Fig. 20 b and d).

[0263] Example 8: GRN Biodistribution, Toxicology and Efficacy Using In Vivo Models

[0264] The optimized dose contains Figure 2 , 5 AAV particles containing the PGRN cassettes shown in , 9, 14 or 17 were injected into non-transgenic (NTg) mice by intraparenchymal and intracerebroventricular injections to compare the efficiency of different administration routes. The transduction efficacy of AAV-PGRN was tested by qPCR and protein expression determined by ELISA and IHC.

[0265] The biodistribution and toxicology of AAV-PGRN were examined in non-transgenic (NTg) and PGRN knockout (PGRN+ / - and - / -) mice (see above for stereotaxic surgical procedures). In addition, biodistribution was studied by injecting AAV-PGRN into wild-type sheep, which have spinal cords the same length as humans and brains twice the size of another common nonhuman primate model, the macaque.

[0266] Efficacy studies were performed by injecting AAV-PGRN into PGRN+ / - and - / -, TDP-43Q331K and TDP-43Q331KxWT transgenic mice. TDP-43 transgenic mice develop slow (Q331K) or faster disease progression (TDP-43Q331K xWT). Their behavior is monitored using a rotarod and grip strength test for assessing motor function and an elevated plus maze for short-term social working memory cognitive tests. Tissues are collected and processed as described above. For PGRN and TDP-43Q331K transgenic mice, animals are injected at 8 weeks of age (IT or ICV), or in the case of TDP-43Q331KxWT transgenic mice, animals are injected at 2 weeks of age due to their aggressive phenotype. Animals are raised for 4 weeks or 6 months, and the above-mentioned test is used to monitor behavior monthly. Tissues collected from both sheep and mice were processed by IHC, ELISA, ddPCR and Western blotting to measure the expression and protein levels of PGRN, to quantify PGRN mRNA levels and to determine vector genome levels. The therapeutic effect of TDP-43 transgenic animals was determined by quantifying insoluble TDP-43 levels and activation of microglia and astrocytes using Western blotting and IHC. The differences in disease progression rate and pathological severity between PGRN and control vectors were statistically analyzed.

[0267] The efficacy study of AAV-PGRN in PGRN+ / - and - / - mice is more difficult because mice show only a very mild phenotype of reduced social dominance in test tube tests and no neuronal loss. - / - mice do show accumulation of easily quantified lipofuscin and activated microglia. Target engagement will be measured by quantitative lipofuscin reduction and microglia and astrocyte activation levels. In addition, the localization of PGRN on lysosomes will be verified to establish correct cell targeting. The differences in pathological severity between PGRN and control vectors will be statistically analyzed.

[0268] Example 9: GRN expression using different AAV capsid serotypes

[0269] Will Figure 2 , 5 , 9, 14, or 17 were packaged into AAV serotypes 9 and 5 to determine whether the enhanced PRGRN expression was specific to AAV9 or was associated with other AAV serotypes. AKTA purified AAV was tested in vivo and in vitro.

[0270] Overall, the above experiments showed that the codon-optimized AAV9-Syn-PGRN-GS, AAV9-Syn-hGHi3-PGRN-GS, and AAV-Syn-hGHi3-PGRN-GS-UTR expression cassettes significantly improved the expression level, transduction efficiency, and cell specificity of PGRN protein expression. These cassettes are expected to reduce the vector dose administered to patients with FTD, NCL11, and ALS, thereby reducing the risk of toxicity and vector production costs.

[0271] This application claims priority to UK Patent Application No. 1913974.0 filed on September 27, 2019, the contents of which are incorporated herein by reference. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the embodiments of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to such specific embodiments. In fact, various modifications of the modes for implementing the present invention that are apparent to those skilled in the art are intended to fall within the scope of the following claims.

Claims

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 linked to a polynucleotide sequence encoding a polypeptide of interest.

2. An adeno-associated virus (AAV) vector comprising a polynucleotide sequence encoding a granulin precursor, wherein the polynucleotide sequence has at least 95% sequence identity with SEQ ID NO:

4.

3. The AAV vector of claim 1 or claim 2, comprising an hGHi3 sequence.

4. The AAV vector of claim 3, wherein the hGHi3 sequence comprises the sequence of SEQ ID NO: 7 or a variant or homolog thereof.

5. An AAV vector as claimed in any preceding claim, wherein the polypeptide of interest is progranulin (PGRN), preferably comprising the sequence of SEQ ID NO: 16 or a variant or homologue thereof.

6. The AAV vector of any one of claims 1 to 5, wherein the polynucleotide sequence is codon optimized.

7. The AAV vector of claim 6, wherein the polynucleotide sequence is codon-optimized for expression in humans.

8. The AAV vector of any one of claims 1 to 7, wherein the nucleic acid further comprises an exonic splicing element (ESE).

9. The AAV vector of claim 8, wherein the ESE is located upstream of the polynucleotide coding sequence.

10. The AAV vector of claim 9, wherein the ESE is part of or inserted into the 5' flanking sequence.

11. The AAV vector of any one of claims 8 to 10, wherein the ESE is part of a guide sequence or is inserted into a guide sequence.

12. An AAV vector as claimed in any one of claims 8 to 11 when dependent on claim 6, wherein the ESE is part of a 5' flanking sequence derived from a wild-type polynucleotide sequence.

13. The AAV vector of any one of claims 10 to 12, wherein the 5' flanking sequence is a 5' leader sequence derived from a wild-type GRN.

14. The AAV vector of claim 13, wherein the wild-type GRN 5' guide sequence comprises 350 to 450 base pairs.

15. The AAV vector of any preceding claim, comprising a progranulin 3' untranslated region (UTR) sequence, preferably wherein the 3'UTR sequence comprises the sequence of SEQ ID NO: 14 or a variant or homologue thereof.

16. The AAV vector of any one of claims 1-15, wherein the polynucleotide sequence comprises a signal sequence derived from hGH.

17. The AAV vector of claim 16, wherein the hGH signal sequence comprises the sequence of SEQ ID NO: 9 or a variant or homolog thereof.

18. The AAV vector of claim 16 or claim 17, wherein the hGH signal sequence replaces base pairs 1 to 51 of GRN.

19. The AAV vector of any one of claims 6 to 18, wherein 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.

20. The AAV vector of any one of claims 1 to 19, wherein the nucleic acid comprises a neuron-specific promoter.

21. The AAV vector of claim 20, wherein the neuron-specific promoter comprises a synapsin promoter.

22. The AAV vector of claim 21, wherein the synapsin promoter comprises the sequence of SEQ ID NO: 15 or a variant or homolog thereof.

23. The AAV vector of any one of claims 1 to 22, wherein the AAV vector has serotype AAV9.

24. The AAV vector of any one of claims 1 to 23, wherein the AAV vector comprises a polynucleotide sequence having at least 85% sequence identity to SEQ ID NO:

17.

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 linked to a polynucleotide sequence encoding progranulin (PGRN).

26. A nucleic acid comprising a polynucleotide sequence having at least 81.34% sequence identity to SEQ ID NO:

4.

27. A nucleic acid as claimed in claim 25 or claim 26 comprising a neuron-specific promoter.

28. The nucleic acid of any one of claims 25 to 27, further comprising one or more AAV inverted terminal repeats.

29. An expression cassette suitable for use in an AAV vector, comprising the nucleic acid of any one of claims 25 to 28.

30. A pharmaceutical composition or medicament comprising an AAV vector as claimed in any one of claims 1 to 24 and one or more pharmaceutically or physiologically acceptable carriers, excipients and / or diluents.

31. A pharmaceutical composition or medicament as defined in claim 30 for use in the treatment of a neurological disorder.

32. A pharmaceutical composition or medicament for use as claimed in claim 31, wherein the 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.

33. A pharmaceutical composition or medicament for use as defined in claim 31 or claim 32, for treating (i) a subject heterozygous, homozygous or compound heterozygous for a GRN mutation, (ii) a subject suffering from a sporadic neurological disease, and / or (iii) a subject whose PGRN levels are below physiological normal levels.

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