gene therapy

Abstract

The present invention relates to nucleic acid constructs comprising granulin (GRN) promoter sequences. The present invention further relates to vectors, viral particles, host cells, and pharmaceutical compositions comprising the nucleic acid constructs. The present invention also relates to the therapeutic use of the nucleic acid constructs, vectors, viral particles, and pharmaceutical compositions.

Description

Technical Field

[0001] The present invention relates to a nucleic acid construct comprising a granulin (GRN) promoter sequence. The present invention further relates to a vector, a viral particle, a host cell, and a pharmaceutical composition comprising the nucleic acid construct. The present invention also relates to the therapeutic use of the nucleic acid construct, vector, viral particle, and pharmaceutical composition.

Background Art

[0002] Adeno-associated virus (AAV) vectors / particles are commonly used vehicles for delivering molecular therapeutics to treat clinical diseases. Many AAV-based therapies are gene replacement therapies. However, for robust AAV production and transgene expression, the AAV construct containing the transgene of interest should be between 4.1 kb and 4.7 kb, which allows for optimal packaging of AAV. So-called "stuffing sequences" or inert DNA can be added to the transgene or vector backbone to increase the overall length of the construct. However, since the vector is sensitive to the stuffing sequence, it must be carefully selected so as not to adversely affect transgene expression, the patient's immune response, and AAV packaging efficiency. Another approach to increasing the length of the AAV construct is to modify the transgene sequence itself. However, this approach may not be appropriate when it is desirable to use the native (wild-type) transgene nucleotide sequence.

[0003] A further approach to increasing the overall length of the AAV construct is by including a manipulated promoter sequence. Such a promoter must be carefully selected to ensure appropriate in vivo transgene expression levels. Additionally, when site-specific transgene expression is required, such as for the treatment of neurological diseases, the selection of a promoter that provides target expression of the transgene of interest in the desired tissue or cell type is important.

[0004] Among the various neurological disorders that can be treated with AAV-based gene therapy is frontotemporal dementia (FTD). FTD is the second most common type of dementia after Alzheimer's disease (Olney et al., 2017). Mutations in one allele of the GRN gene, which encodes the protein progranulin (PGRN), are associated with the development of FTD (Baker et al., 2006). Homozygous mutations in GRN are associated with neuronal ceroid lipofuscinosis 11 (NCL11), which is characterized by cerebellar ataxia, seizures, retinitis pigmentosa, and cognitive impairment, and usually develops between the ages of 13 and 25 (Faber et al., 2020). Loss of PGRN function can be caused by various mutations. In PGRN-deficient mouse models, it has been shown that promoting neuronal expression of PGRN using an AAV gene therapy approach modifies behavioral disorders associated with FTD (Arrant et al., 2017). The association between PGRN deficiency and CNS disorders, including FTD and NCL11, is well established (Mole and Cotman, 2015; Chitramuthu et al., 2017; Huin et al., 2020).

[0005] Therefore, there is strong biological evidence to support therapeutic approaches that increase PGRN levels in central nervous system (CNS) tissues and cells to treat neurological disorders associated with PGRN deficiency.

[0006] To date, therapeutic approaches to increase PGRN levels in the central nervous system to treat PGRN-deficient neurological diseases, including FTD and NCL, have utilized nucleic acid constructs containing conventional promoters that promote potent and / or cell-specific expression, such as UBC, CMV, CAG, or chicken β-actin promoters. See, for example, U.S. Patent No. 10,689,625, or Zin et al. (2021), which describes the construction of a construct in which the CBA promoter is operably linked to a transgene encoding PGRN. However, long-term data on the performance of conventionally active promoters that promote POI or PGRN expression are not available, thus leading to the assumption that the use of endogenous or naturally occurring promoters that can maintain native transcriptional regulation may be advantageous.

[0007] Bhandari et al. (1996) described a structural and functional analysis of the human GRN promoter. This study identified specific sequences with basal promoter activity (i.e., cleavage-type promoter sequences), although the strongest activity appears to vary by cell type. Bhandari further identified regulatory elements capable of modulating promoter activity.

[0008] Sardiello et al. (2009) described the presence of numerous CLEAR elements (used to indicate coordinated lysosomal expression and regulatory elements) within the GRN promoter and the correlation between TFEB (as used herein, "TFEB" refers to transcription factor EB) binding to these sequences and the expression of lysosomal genes. She et al. (2017) further correlated TFEB occupancy in the GRN promoter with the upregulation of PGRN expression. Typically, the length of the nucleotide sequence encoding the PGRN coding sequence is about 1.8 kb, which is significantly shorter than the optimal length of 4.1–4.7 kb for packaging nucleic acid constructs into AAVs.

[0009] Therefore, there is still a need for promoter sequences that can be used to increase the length of viral vector constructs while simultaneously providing robust, CNS-targeted expression of target proteins such as PGRNs. [Overview of the project]

[0010] Promoters derived from the granulin (GRN) gene have not only been found to help provide transgenes with a length of 4.1kb–4.7kb, but have also been found to be highly effective in gene therapy for promoting the central nervous system (CNS) targeted expression of target proteins (POIs) such as PGRN.

[0011] Accordingly, the present invention provides a nucleic acid construct comprising a granulin (GRN) promoter operably ligated to a nucleotide sequence encoding a target protein (POI), such as a progranulin (PGRN) protein. The promoter is preferably a cleavage promoter.

[0012] The present invention further discloses a vector comprising the nucleic acid construct of the present invention. The vector may be a plasmid or a viral vector.

[0013] The present invention also relates to a viral particle comprising a capsid and a nucleic acid construct or vector of the present invention. The viral particle may be an adeno-associated virus (AAV) particle, or a viral particle comprising an AAV genome, or a derivative thereof. Alternatively, it may be a lentiviral particle, or a viral particle comprising a lentiviral genome or a derivative thereof.

[0014] The present invention further provides a host cell comprising a nucleic acid construct or vector of the present invention and / or producing a viral particle of the present invention, the host cell optionally being a HEK293 cell.

[0015] Further provided by the present invention are pharmaceutical compositions comprising the nucleic acid construct of the present invention, the vector of the present invention, and / or the viral particles of the present invention together with a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0016] The present invention also discloses nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention for use in methods of treating or preventing diseases characterized by POI deficiencies, such as progranulin (PGRN) deficiency, in patients in need.

[0017] The present invention further relates to a method for treating or preventing a disease characterized by a deficiency of a POI, such as progranulin (PGRN) deficiency, in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the nucleic acid construct of the present invention, the vector of the present invention, the viral particles of the present invention, and / or the pharmaceutical composition of the present invention.

[0018] The present invention also provides the use of nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention for manufacturing pharmaceuticals for treating or preventing diseases characterized by POI deficiencies, such as progranulin (PGRN) deficiency, in patients in need thereof. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows the evaluation of hPGRN expression in primary mouse neurons and astrocytes for the construct pPG-41. The bar graphs represent: A. Expression of mouse PGRN in untransduced and transduced primary neurons from WT mice and GRN KO mice. B. Expression level of human PGRN in transduced neurons. C. Percentage of neurons expressing human PGRN after transduction with pPG41. D. Percentage of astrocytes expressing human PGRN after transduction with pPG41. E. Expression level of human PGRN in WT and KO neurons transduced with pPG 41. [Figure 2] This is a schematic diagram showing the composition of component nucleic acid sequences in the AAV-06164, AAV-06262, and AAV-06263 constructs. The 1801-residue GRN1 promoter represents a cleaved form of the 1801-residue human granulin promoter. The 632-residue GRN2 promoter represents a cleaved form of the 632-residue human granulin promoter. The 489-residue GRN3 promoter represents a cleaved form of the 489-residue human granulin promoter. KOZAK indicates the presence of the KOZAK sequence. Human PGRN represents a polynucleotide sequence encoding human progranulin. SV40 PolyA represents a polynucleotide sequence containing multiple adenosine monophosphate residues. [Figure 3] This figure shows the evaluation of PGRN expression and secretion by Ad293 and N2A cells. A. Images of Western blot analysis of promoter activity in two different cell types. B. Bar graph showing the concentrations of expressed PGRN (cell lysate) and secreted PGRN (supernatant). hPGRN expression was analyzed for each of the AAV9-06164, AAV9-06262, and AAV9-06263 constructs. [Figure 4] This figure shows the evaluation of human PGRN expression and secretion in primary neurons of GRN- / - mice. The bar graphs show PGRN mRNA levels (A), PGRN protein expression (B), and PGRN secretion (C) in neuronal cultures transduced with AAV9-06164, AAV9-06262, and AAV9-06263. A control without transduction is also shown. [Figure 5]This figure shows that human PGRN expression corrects lysosomal deficiency in GRN- / - mouse primary neurons. A. Images of Western blot analysis performed to quantify the level of lysosomal protein cathepsin D in WT (GRN+ / +) and KO (GRN- / -) primary neurons transduced with a lentiviral vector containing the pPG41 construct. B-D. Bar graphs showing the levels of cathepsin D protein (immature, mature heavy chain, and mature light chain, respectively). The cathepsin D expression values ​​are normalized against actin and GADPH expression levels. [Figure 6] This figure shows the evaluation of human PGRN (hPGRN) expression in wild-type rats after striatal injection of AAVTT-06164. A. IHC staining of hPGRN was observed in the brains of wild-type rats administered striatically with AAVTT-06164. High levels of hPGRN were detected mainly throughout the forebrain of GRN- / -KO mice, particularly in the striatum, thalamus, hypothalamus, cerebral cortex and hippocampus, as well as in the substantia nigra of the midbrain. B. ELISA quantification of hPGRN in CSF of rats injected with AAVTT-06164 or a control. C. Western blotting was performed to analyze hPGRN expression levels and cathepsin D maturation levels. [Figure 7] Human PGRN expression affects lysosomal gene expression in GRN- / - neurons. The bar graph shows the measurement of lysosomal gene mRNA levels in GRN- / - neurons transduced using AAV9-06164, AAV9-06262, and AAV9-06263. Wild-type and untransduced GRN- / - neurons are shown as controls. [Modes for carrying out the invention]

[0020] As used herein and in the appended claims, the singular forms "a," "an," and "the" encompass multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to "a nucleic acid" includes "nucleic acids," etc.

[0021] The term "comprises" (including "comprise" and "comprising") has its ordinary meaning in the art, that is, the recited features or groups of features are included, but it is understood that this term does not exclude the presence of any other recited features or groups of features. For example, a promoter comprising a minimal promoter sequence may also include other components such as one or more introns. The term "consists of" (including "consist of" and "consisting of") also has its ordinary meaning in the art, that is, the recited features or groups of features are included and further features are excluded. For example, a promoter consisting of a minimal promoter sequence includes the minimal promoter sequence and no other components. For all embodiments in which "comprises" (or "comprising") is used, further embodiments in which "consists of" (or "consisting of") is used are contemplated. Therefore, all disclosures of "comprises" should be considered as also being disclosures of "consists of".

[0022] The terms "protein" and "polypeptide" are used interchangeably herein and in their broadest sense refer to compounds of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "protein" includes not only short peptide sequences but also longer polypeptides. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including both D- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0023] The terms "AAV prototype", "AAV TT", "AAV-TT" (AAV-TT”), or "AAV tt" are defined in International Publication No. 2015 / 121501 and Tordo J. et al., 2018 (both incorporated herein by reference) and relate to a capsid comprising or consisting of SEQ ID NO: 1. The "r" preceding these terms indicates recombinant. [[ID=​​​​​​​​​​​​​​The term “heterogeneic nucleic acid” refers to the nucleic acid sequence packaged within the rAAV capsid that forms the viral particle. Such nucleic acid sequences include the AAV reverse terminal repeat (ITR). In the examples herein, heterogeneic nucleic acid includes at least the AAV5'ITR from 5' to 3', a sequence encoding a transgene (i.e., a gene different from the gene encoding the viral protein), and the AAV3'ITR.

[0028] The terms "patient" and "subject" are used interchangeably in this specification. Typically, a patient is a human being.

[0029] As used herein, the term “nucleic acid construct” refers to an artificial (e.g., generated or synthesized by recombination) nucleic acid comprising at least one control sequence (e.g., a promoter that enables the expression of a target protein in vitro and / or in vivo) and at least one nucleotide sequence encoding a target protein (POI). Therefore, a nucleic acid construct in the context of the present invention can be considered an expression cassette. A nucleic acid construct may be isolated or substantially isolated. A nucleic acid construct of the present invention may include a suitable promoter, enhancer, initiator, and other elements, such as a polyadenylation (poly-A) signal and / or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence. A nucleic acid construct of the present invention may also include nucleotide sequences that facilitate genetic manipulation, such as restriction sites.

[0030] In this specification, the term “operatably linked” refers to the juxtaposition of two or more nucleotide sequences that enables each of the two or more sequences to perform its normal function. Typically, the term “operatably linked” is used to refer to the juxtaposition of a regulatory element (e.g., a promoter, enhancer, poly(A) signaling sequence, WPRE sequence, etc.) with a nucleotide sequence encoding a target protein (POI). For example, operatably linked between a promoter and a protein-encoding nucleotide sequence allows the promoter to function to promote the expression of POI in vitro and / or in vivo. The term “operatably linked” does not preclude the presence of further sequences between two or more operatably linked nucleotide sequences, as long as the two or more sequences can still perform their normal function.

[0031] Sequence homology can also be considered from the perspective of functional similarity (i.e., amino acid residues with similar chemical properties / functions), but in the context of this document, homology is preferably expressed from the perspective of sequence identity.

[0032] Sequence comparison can be performed visually, but more commonly, it can be done with the help of readily available sequence comparison programs. These publicly available and commercial computer programs can calculate the percentage of homology (or identity, etc.) between two or more sequences.

[0033] Identity percentages can be calculated over consecutive sequences. That is, one sequence is aligned with the other, and each amino acid in one sequence is directly compared to its corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are performed only for a relatively small number of residues (e.g., fewer than 50 consecutive amino acids). For comparisons of longer sequences, gap scoring is used to generate the best alignment that accurately reflects the identity level of related sequences with mutual insertions or deletions. Examples of software / algorithms that can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA, and the GENEWORKS suite of comparison tools.

[0034] Typically, sequence comparisons are performed over the length of a reference sequence. For example, if a user wants to determine whether a given sequence is 70% identical to sequence number 2, sequence number 2 would be the reference sequence. To evaluate whether a sequence is at least 90% identical to sequence number 2 (an example of a reference sequence), a person skilled in the art would perform an alignment over the length of sequence number 2 and identify how many positions in the test sequence were identical to the positions in sequence number 2. If at least 70% of these positions are identical, the test sequence is at least 70% identical to sequence number 2. If the sequence is shorter than sequence number 2, any gaps or missing positions should be considered non-identical positions.

[0035] As used herein, the term “fragment” refers to a contiguous portion of a reference sequence. For example, a fragment of sequence number 2 with a length of 50 nucleotides refers to the 50 consecutive nucleotides of sequence number 2.

[0036] In this specification, the term “functional variant” refers to a nucleic acid or amino acid sequence that is modified relative to a reference sequence but retains the biological function / characteristics, or at least one of the biological functions / characteristics, of the reference sequence. For example, a functional variant of the GRN promoter retains the ability to promote the expression of nucleotide sequences encoding POI in CNS cells such as neurons or astrocytes. Similarly, a functional variant of the PGRN protein retains the activity of the reference PGRN protein.

[0037] The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to polymeric forms of nucleotides of any length (either deoxyribonucleotides or ribonucleotides, or analogs thereof). Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, synthetic polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or substantially isolated forms. Substantially isolated means that the polypeptide is substantially isolated, though not completely, from any surrounding medium. Polynucleotides may be mixed with a carrier or diluent that does not interfere with their intended use and are still considered substantially isolated. The nucleic acid sequence that “encodes” a selected polypeptide is a nucleic acid molecule that, under the control of an appropriate regulatory sequence, for example, placed in an expression vector, is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA). The boundaries of a coding sequence are determined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxy) end.

[0038] As an example, as described by Sambrook et al (1989), polynucleotides can be synthesized according to methods well known in the art.

[0039] The term “GRN promoter” refers to a nucleotide sequence of a GRN gene (e.g., a mouse or human GRN gene) that is capable of functioning as a promoter, that is, capable of promoting the transcription of a nucleotide sequence to which the GRN promoter is operably linked, thereby promoting the expression of the protein encoded by the nucleotide sequence. Typically, GRN promoter sequences used in the context of the present invention are specific to a particular tissue or one or more cell types. Preferably, the GRN promoter used in the present invention will specifically promote the expression of a target protein (POI), such as PGRN, in neurons and / or astrocytes. Exemplary sequences of GRN promoters can be found as SEQ ID NO: 1 (mouse origin) or SEQ ID NO: 2 (human origin).

[0040] The term "intron" refers to a non-coding nucleotide sequence within a gene. Normally, introns are transcribed from DNA to messenger RNA (mRNA) during gene transcription, but are cut out from the mRNA transcript through splicing before translation.

[0041] The term "enhancer element" refers to a short region of DNA (50–1500 residues long) that can be bound by proteins (usually called transcription factors) to increase the likelihood of transcription of a particular gene. Enhancer elements may be added to promoters, for example (Liu, B. et al., 2004).

[0042] Since vectors can be single-stranded (ss) or double-stranded (ds), the length of the nucleic acid sequence is preferably expressed herein as a number of base lengths, such as "base," "b," "kilobase," "kb," or "residue length / length" (each applicable to both ss and ds), rather than "base pair" or "bp," which is applicable only to ds.

[0043] The terms "therapeutic dose" or "therapeutic effective dose" usually refer to the amount or dosage of a compound that, when administered to primates, exhibits a positive pharmacological and / or physiological effect on the disease and is therefore sufficient to treat the disease.

[0044] Terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in that it completely or partially prevents the onset or worsening of a disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects caused by the disease. Therefore, treatment encompasses all treatments for diseases in primates, especially humans, and includes (a) preventing the onset of the disease in subjects who may be predisposed to the disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., stopping its onset; and (c) alleviating the disease, i.e., causing its regression.

[0045] Progranulin (PGRN; also known as granulin-epicerin precursor, pro-epicerin, prostate cancer (PC) cell-derived growth factor, and acrogranin) is a secreted glycoprotein expressed by many cell types throughout the body. Encoded by a single gene (GRN; gene ID: 2896) on chromosome 17q21, PGRN is a 593-amino acid cysteine-rich protein with an estimated molecular weight of 68.5 kDa. It contains 7.5 granulin-like domains, each consisting of a highly conserved tandem repeat of a motif containing 12 cysteine ​​residues. Proteolytic cleavage of PGRN by extracellular proteases such as elastase yields small peptide fragments called granulins or epicerins (e.g., granulin A, granulin B, granulin C, etc.). These fragments range in size from 6 kDa to 25 kDa and are thought to be involved in various biological functions. An exemplary sequence of the PGRN protein is reported in Sequence ID No. 14. An exemplary sequence of the PGRN coding sequence is reported in Sequence ID No. 13.

[0046] The optimal length for packaging nucleic acid constructs in AAVs is typically 4.1–4.7 kilobases (kb). However, the nucleic acid sequence encoding the target protein is often too small to reach this optimal length, even considering the promoter length and the length of additional sequences (such as regulatory sequences). Surprisingly, we have found that a promoter derived from the granulin (GRN) gene can be used to increase / modulate the length of nucleic acid constructs while being highly effective in promoting CNS-targeted expression of target proteins (POIs) such as PGRNs in the context of gene therapy.

[0047] In a first embodiment, the present invention provides a nucleic acid construct comprising a granulin (GRN) promoter operably ligated to a nucleotide sequence encoding a target protein (POI). The promoter is preferably a cleavage-type GRN promoter. In a non-limited example, the POI is a progranulin (PGRN) protein.

[0048] In a second embodiment, a nucleic acid construct comprising a granulin (GRN) promoter operably ligated to a nucleotide sequence encoding a progranulin (PGRN) protein is described herein. The promoter is preferably a cleavage-type GRN promoter.

[0049] The GRN promoter to be used in accordance with the entirety of this invention specifically promotes the expression of a target protein (POI), such as PGRN, in neurons and / or astrocytes.

[0050] In the overall context of the present invention, the GRN promoter may be a full-length GRN promoter or a functional variant thereof. A functional variant of the GRN promoter is functional in the sense that it retains the characteristics of the corresponding non-mutant GRN promoter. Thus, a functional variant of the GRN promoter retains the ability to promote the transcription of a nucleotide sequence to which the functional variant is operably linked, thereby promoting the expression of the protein encoded by the nucleotide sequence. The functional variants of the GRN promoter described herein retain specificity for a particular tissue type. For example, a functional variant of the GRN promoter described herein would specifically promote the expression of a target protein (POI), such as PGRN, in neurons and / or astrocytes. Non-limiting examples of GRN promoter sequences that can be used in the nucleic acid constructs described herein include, or consist of, SEQ ID NO: 1 or SEQ ID NO: 2, or functional variants thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 1 or SEQ ID NO: 2. Alternatively, the GRN promoter of the present invention may be a cleavage-type GRN promoter, i.e., a full-length GRN promoter or a functional fragment of a functional variant thereof. The cleavage-type GRN promoter must be a functional cleavage-type GRN promoter (also called a functional GRN promoter fragment). It is functional in the sense that it retains the characteristics of the corresponding non-mutant or full-length GRN promoter. Thus, the cleavage-type GRN promoter according to the present invention retains the ability to drive the transcription of a nucleotide sequence to which the cleavage-type GRN promoter is operably linked, thereby promoting the expression of the protein encoded by the nucleotide sequence. The cleavage-type GRN promoter described herein retains specificity for a particular tissue type. For example, the cleavage-type GRN promoter described herein would specifically promote the expression of a target protein (POI), such as PGRN, in neurons and / or astrocytes.Such a cleavage promoter is a functional fragment of the promoter region of a sufficiently long GRN gene (or a functional variant thereof), which comprises or comprises at least the minimum elements necessary to function as a GRN promoter, namely, elements capable of promoting the transcription of the nucleotide sequence to which the GRN promoter is operably linked, thereby promoting the expression of the protein encoded by the nucleotide sequence. Non-limiting examples of such cleavage GRN promoters used in nucleic acid constructs of the present invention include or comprise GRN1 (SEQ ID NO: 3), GRN2 (SEQ ID NO: 4), or GRN3 (SEQ ID NO: 5). In the context of the present invention as a whole, the GRN promoter is preferably a cleavage promoter, and is preferably selected from the group consisting of: (a) A nucleotide sequence containing or consisting of Sequence ID No. 3; (b) A nucleotide sequence containing or consisting of Sequence ID No. 4; (c) A nucleotide sequence containing or consisting of Sequence ID No. 5; (d) Functional variants having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with any one of sequence numbers 3, 4, or 5; or (e) A functional fragment of any length of any one of (a), (b), (c), or (d) containing a minimal promoter sequence.

[0051] A cleaved promoter sequence that contains or consists of at least the minimum elements necessary to function as a GRN promoter is referred to herein as a “minimal promoter sequence.” When a cleaved promoter sequence or minimal promoter sequence is used, it is preferably about 300 to 1000 residues in length, for example about 350 to 900 residues, about 400 to 800 residues, about 450 to 700 residues, or even about 485 to 630 residues in length, for example, one of the following lengths: 485 bases, 486 bases, 487 bases, 488 bases, 489 bases, 490 bases, 491 bases, 492 bases, 493 bases, 494 bases, or 495 to 625 bases, 626 bases, 627 bases, 628 bases, 629 bases, 630 bases, 631 bases, 632 bases, 633 bases, 634 bases, or 635 bases.

[0052] A nucleic acid construct comprising a GRN promoter as described herein enhances the expression of a target protein (POI) it encodes, such as PGRN. The construct also provides enhanced transcriptional efficiency. In certain embodiments, the enhanced expression is due to transcriptional regulation by the GRN promoter. Thus, in certain embodiments, the expression of a POI, such as a PGRN protein, from a nucleic acid construct of the present invention comprising a GRN promoter can be increased compared to a construct having an alternative promoter, while the constructs are otherwise identical. In certain embodiments, a nucleic acid construct of the present invention comprising a GRN promoter provides increased transcriptional efficiency compared to a construct having an alternative promoter, while the constructs are otherwise identical.

[0053] In the overall context of the present invention, a GRN promoter may include one or more introns and / or one or more enhancer elements. A GRN promoter including a cleaved promoter sequence and introns and / or added enhancer elements is referred to herein as an "operated GRN promoter."

[0054] In the overall context of the present invention, the protein or interest (POI) encoded by the nucleic acid construct is selected from (but not limited to) the group consisting of: secreted proteins (including pre and / or pro forms of such secreted proteins), intracellular proteins, membrane-bound proteins, and transmembrane proteins. Non-limiting examples of such POIs include (but not limited to) progranulin (PGRN), neuronal ceroid lipofuscinosis proteins types 1-14 (e.g., NCL types 2, 3, 6, or 11), or heparin-α-glucosaminide N-acetyltransferase (known as EC 2.3.1.78, or HGSNAT). In certain non-limiting examples, the nucleotide sequence encoding the POI encodes a PGRN protein, such as human PGRN protein. In another non-limiting example, the nucleotide sequence encoding the POI is a wild-type PGRN protein, such as wild-type human PGRN protein. In yet another non-limiting example, the nucleotide sequence encoding the POI, such as PGRN, is not codon-optimized.

[0055] In the overall context of the present invention, preferred nucleotide sequences encoding a PGRN protein or a functional variant thereof include or consist of a functional variant of SEQ ID NO: 13 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 13. Functional fragments of the nucleotide sequences may also be used. Such fragments may have lengths of 1000 to 1781 bases, 1200 to 1750 bases, 1400 to 1700 bases, or 1500 to 1600 bases.

[0056] In the overall context of the present invention, preferred nucleotide sequences encoding a PGRN protein include or comprise a PGRN protein comprising SEQ ID NO: 14, or a functional variant thereof having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO: 14. Nucleotides encoding a functional fragment of the PGRN protein may also be used. Such a PGRN protein fragment may have a length of 300–592 amino acid residues, 350–490 amino acid residues, 400–480 amino acid residues, or 450–475 amino acid residues.

[0057] In any protein or polypeptide described herein, the amino acid sequence may be modified by addition, deletion, or substitution, provided that the polypeptide having the modified sequence exhibits similar / identical activity to the polypeptide having the unmodified sequence. “Similar” or “identical” means that the polypeptide with the modified sequence does not exhibit significantly reduced activity compared to the polypeptide with the unmodified sequence. Such a modified protein or the nucleotide sequence encoding such a modified protein may be considered a “functional variant.”

[0058] For example, PGRN has been observed to co-localize with the lysosomal marker protein LAMP-1 (lysosome-associated membrane protein 1) and play a role in regulating lysosomal function and biosynthesis through lysosome acidification (Tanaka et al., 2017). Therefore, in order to identify functional mutants and / or fragments of PGRN, those skilled in the art can evaluate the ability of such functional mutants and / or fragments of PGRN to co-localize with LAMP-1 or to regulate lysosomal acidification.

[0059] The co-localization of PGRN proteins and LAMP-1 can be evaluated and / or quantified using any suitable technique known in the art. For example, in cultured cells deficient in PGRN (e.g., GRN - / -Cells, or cells in which PGRN expression is downregulated by siRNA, may be transfected with a vector containing a nucleic acid construct comprising a functional variant or fragment of the nucleotide sequence encoding the PGRN protein. The cells may then be immunostained using a first fluorescently labeled (e.g., green) antibody specific to PGRN and a second fluorescently labeled (e.g., red) antibody specific to LAMP-1. Co-localization of red and green staining can then be assessed using a fluorescence microscope (see Tanaka et al., 2017). The co-localization of the variant fragment of the PGRN protein with LAMP-1 may be at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the co-localization between the corresponding non-variant or full-length PGRN protein under the same conditions. It may also be substantially the same as, or greater than, the co-localization between the corresponding non-variant or full-length PGRN protein under the same conditions.

[0060] The effect of PGRN on lysosome acidification can be evaluated using any appropriate technique in the art. For example, cultured cells deficient in PGRN (e.g., GRN - / -Cells, or cells in which PGRN expression (PGRN expression) has been downregulated by siRNA, may be transfected with a vector containing a nucleic acid construct that includes a functional variant or functional fragment of the nucleotide sequence encoding the PGRN protein. Lysosomal acidification in the transfected cells can then be evaluated using a cell-permeable dye such as LysoSensor DND-189 or acridine orange (see Tanaka et al., 2017). The fluorescence of LysoSensor DND-189 increases in a lysosomal acidity-dependent manner. The acridine orange monomer emits green fluorescence, but its dimers and oligomers are formed when it is protonated. Therefore, the red / green fluorescence ratio indicates the relative acidity of the lysosome. The regulation of lysosomal acidification by PGRN protein variants or fragments may be at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the regulation by the corresponding non-variant or full-length PGRN protein under the same conditions. The regulation of lysosomal acidification by PGRN protein variants or fragments may be substantially the same as, or greater than, the regulation by the corresponding non-variant or full-length PGRN protein under the same conditions.

[0061] Unless otherwise specified, modifications to polypeptide sequences are preferably conservative amino acid modifications (or substitutions). Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, chemical properties, and / or side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid they substitute for. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid modifications are well known in the art.

[0062] In addition to the GRN promoter (or a modified GRN promoter), the nucleic acid construct according to the present invention may include one or more further regulatory elements. One or more regulatory sequences that direct the expression of the transgene are selected from the group consisting of: a. One or more transcription start sequences (such as promoters), b. One or more translation initiation sequences, c. One or more mRNA stability sequences, d. One or more transcription termination sequences (such as polyadenylated sequences), e. One or more secretory sequences, f. One or more enhancer sequences, g. One or more introns, h. One or more TATA boxes, i. One or more microRNA target sequences, j. One or more polylinker sequences that facilitate the insertion of DNA fragments into the vector, k. One or more splicing signal sequences, l. One or more post-transcriptional regulatory elements, m. Any combination of one or more of groups a through l.

[0063] In the overall context of the present invention, preferred regulatory elements are those that function to stabilize mRNA transcribed from a nucleic acid construct / vector and / or to enhance the expression of a target protein (POI), such as PGRN, from the nucleic acid construct / vector. A non-limiting example of a regulatory element that may be used in the context of the present invention is the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). A WPRE is a DNA sequence that, when transcribed into mRNA, creates a tertiary structure in the mRNA transcript, thereby increasing the stability of the mRNA and also increasing the expression of the POI encoded by the nucleic acid construct. In the nucleic acid construct (or vector) of the present invention, the WPRE may be located at 3' relative to the nucleotide sequence encoding the POI or PGRN protein. WPRE may include the nucleotide sequence of SEQ ID NO: 20, or its functional variants or fragments having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the nucleotide sequence of SEQ ID NO: 20. Functional variants or fragments of WPRE retain the characteristics of the corresponding non-variant or full-length WPRE. Thus, variant or fragment WPRE may be capable of creating tertiary structures in mRNA transcripts and / or enhancing the stability of mRNA transcripts and / or enhancing the expression of POIs encoded by nucleic acid constructs. This enhancement is compared to mRNA without the variant or fragment WPRE. Another non-limiting example of a regulatory element that may be used in the context of the present invention is a polyadenylation (poly(A) or polyA) signal sequence. In eukaryotic cells, polyadenylation signal sequences within mRNA transcripts are recognized and processed to add a poly(A) tail consisting of multiple adenosine monophosphates to the 3' end of the mRNA transcript. The poly(A) tail functions to facilitate the transport of mRNA from the nucleus to the cytoplasm, preventing mRNA degradation and thereby enhancing the expression of POIs encoded by the nucleic acid construct. In the nucleic acid construct of the present invention, the polyadenylation signal sequence may be located 3' relative to the nucleotide sequence encoding a POI, such as a PGRN.The polyadenylation signal sequence may include the nucleotide sequence of SEQ ID NO: 21, or a functional variant or functional fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of SEQ ID NO: 21. The functional variant or functional fragment of the polyadenylation sequence retains the characteristics of the corresponding non-variant or full-length polyadenylation signal sequence. In further non-limiting examples, the nucleic acid construct according to the present invention includes two or more regulatory elements, e.g., the WPRE element described herein and the polyadenylation signal sequence. Thus, an example of the nucleic acid construct described herein may include, in the 5' to 3' direction, a GRN promoter (such as a cleaved GRN promoter or an engineered cleaved GRN promoter), a nucleotide sequence encoding a POI, e.g., a PGRN protein, a WPRE, and a polyadenylation signal sequence.

[0064] Another example of sequences that may be further incorporated into the nucleic acid constructs of the present invention is one or more restriction sites, such as BamH1, Xba1, or even the EcoR1 restriction site. Examples of such restriction site sequences can be found as SEQ ID NOs. 15-17.

[0065] In the overall context of the present invention, the nucleic acid construct may be approximately 2100 to approximately 4500 residues in length, for example, 2100 to 4400 residues, 2200 to 4000 residues, or 2400 to 3900 residues.

[0066] Examples of nucleic acid constructs according to the present invention include, in the 5' to 3' direction: (a) a GRN promoter sequence such as a cleavage-type GRN promoter (which may or may not be manipulated); (b) optionally a Kozak sequence; (c) a polynucleotide sequence encoding a POI such as a PGRN protein; and (d) optionally a post-transcriptional regulatory element, e.g., WPRE and / or a poly(A) sequence, e.g., an SV40 poly(A) sequence. In non-limiting examples, nucleic acid constructs or vectors comprising or comprising the following are provided herein: (a) Sequence ID 7, (b) Sequence ID 8, (c) Sequence ID 9, or (d) A functional variant or functional fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the nucleotide sequence of SEQ ID NO: 7, 8, or 9.9.

[0067] The nucleic acid constructs of the present invention may be provided in a vector such as a plasmid or a recombinant viral vector. Therefore, in a third embodiment, a vector containing the nucleic acid construct of the present invention is provided herein. A suitable vector may be any vector that contains a sufficient amount of genetic information and has the ability to enable POI expression in vivo. The vector containing the nucleic acid construct of the present invention may be administered directly to a patient in need, or it may be administered via particles such as viral particles. Such vectors are routinely constructed in the field of molecular biology. For an example therein, see Sambrook et al. (1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

[0068] The vectors described herein may be of any type. For example, the vector may be a plasmid vector, a minicircle DNA, or a viral vector. In the overall context of the present invention, the vector may be based on herpes simplex virus, adenovirus, or lentivirus. Alternatively, the vector may be an adeno-associated virus (AAV) vector or a derivative thereof. The vectors of the present invention may be single-stranded or double-stranded.

[0069] In the overall context of the present invention, vectors comprising the nucleic acid construct of the present invention, further comprising at least one ITR, preferably at least two ITRs (one at each end of the nucleic acid construct of the present invention, i.e., a 5' ITR and a 3' ITR), adjacent to the nucleic acid construct at 5' and / or 3', are also described herein. The ITR sequences function in cis to provide a functional origin for replication, enabling the incorporation and excision of the nucleic acid construct from the cell's genome. One or more ITRs may be obtained from viral genomes, such as AAV genomes having different serotypes, or may be chimeric ITRs or mutant ITRs. An example of a mutant ITR is one in which the terminal resolution site (trs) is deleted. This deletion allows for continued replication of the genome, generating a single-stranded genome containing both coding and complementary sequences, i.e., a self-complementary viral genome (e.g., a self-complementary AAV genome). This allows for bypassing DNA replication in target cells and thus enables acceleration of transgene expression. Preferred ITR sequences are derived from AAV genomes, e.g., AAV2, AAV9 and their variants. Non-limiting examples of such ITRs are disclosed as Sequence IDs 25-26.

[0070] Preferably, one or more ITRs are adjacent to the nucleic acid construct of the present invention, i.e., the nucleotide sequence containing the GRN promoter and the nucleotide sequence encoding a POI, such as a PGRN protein. Including one or more ITRs is preferable because it helps package the nucleic acid construct of the present invention (as is or incorporated into a vector) into a viral particle. In a preferred embodiment, the ITR element is the only sequence retained from the natural viral genome, e.g., the natural AAV genome. This is preferable for the reasons mentioned above and also preferable to reduce the likelihood of the nucleic acid construct of the present invention (as is or incorporated into a vector) being incorporated into the host cell genome. Furthermore, if the nucleic acid construct (as is or incorporated into a vector) contains the AAV genome, reducing the size of the AAV genome to only the ITRs makes it possible to increase the flexibility of incorporating other sequence elements (such as regulatory elements) into the nucleic acid construct in addition to the transgene.

[0071] In the overall context of the present invention, the vector preferably comprises (5' to 3' direction): (a) 5'ITR; (b) GRN promoter sequence such as a cleaved GRN promoter (may be manipulated or not); (c) optionally a Kozak sequence; (d) a polynucleotide sequence encoding a POI (e.g., a PGRN protein); (e) optionally a post-transcriptional regulatory element such as a WPRE; (f) optionally a poly(A) sequence such as an SV40 poly(A) sequence; and (g) a 3'ITR. In non-limiting examples, the vector of the present invention comprises (5' to 3' direction): (a) 5'ITR; (b) a cleaved GRN promoter sequence; (c) a Kozak sequence; (d) a polynucleotide sequence encoding a POI (e.g., a PGRN protein); (e) a WPRE and / or an SV40 poly(A) sequence; and (f) a 3'ITR. In further non-limiting examples, the vector may include: (a) a 5'ITR containing or comprising the nucleotide sequence of SEQ ID NO: 25, or a functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 25; (b) a cleaved GRN promoter sequence containing or comprising the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; (c) optionally a Kozak sequence containing or comprising the nucleotide sequence of SEQ ID NO: 27; (d) a polynucleotide sequence encoding a POI, wherein the POI contains or comprises the nucleotide sequence of SEQ ID NO: 13. (e) a PGRN protein comprising the same, or a functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 13; (e) a WPRE comprising or comprising the nucleotide sequence of SEQ ID NO: 20, or a functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 20, and / or an SV40 poly(A) sequence comprising or comprising the nucleotide sequence of SEQ ID NO: 21, or a functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 21; and (f) a 3'ITR comprising the nucleotide sequence of SEQ ID NO: 26, or a functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 26.

[0072] In a fourth embodiment, the present invention provides a viral particle comprising 1) a capsid (such as a viral capsid) and a nucleic acid construct of the present invention, or 2) a capsid (such as a viral capsid) and a vector of the present invention. In the overall context of the present invention, the viral particle may be based on herpes simplex virus, adenovirus, or lentivirus. Alternatively, the viral particle may be an adeno-associated virus (AAV) particle or a derivative thereof. The viral particle derivative may be a chimeric derivative, a shuffled derivative, or a capsid-modified derivative. The preferred viral particle is an AAV particle (also called a rAAV particle in the case of recombinant AAV particles).

[0073] Wild-type AAV, containing viral genes, inserts its genomic material into chromosome 19 of host cells (Kotin, et al., 1990). AAV serotypes determine the tissue specificity of AAV virus infection (or tropism). The AAV single-stranded DNA genome contains two reverse-ended repeats (ITRs) and two open reading frames containing structural (cap) genes and packaging (rep) genes (Hermonat et al., 1984). The AAV genome typically includes packaging genes such as rep genes and / or cap genes that encode the packaging function of AAV particles. Rep genes encode one or more proteins such as Rep78, Rep68, Rep52, and Rep40 or their variants. Cap genes encode one or more capsid proteins such as VP1, VP2, and VP3 or their variants. These proteins constitute the capsid of viral particles, such as AAV particles. Naturally occurring AAV viruses are replication-deficient and rely on the trans to provide helper functions (such as an adenovirus base) to complete the replication and packaging cycle. For therapeutic purposes, in addition to the gene encoding the POI, the only sequence required for cis is the ITR. Therefore, the vector (or viral vector) of the present invention (i.e., a vector comprising the nucleic acid construct and ITR sequence of the present invention) preferably does not contain the rep gene and cap gene.

[0074] Most gene therapy nucleic acid constructs (as well as vector constructs and viral particles) are based on AAV serotype 2 (AAV2). AAV2 binds to target cells via the heparin sulfate proteoglycan receptor (Summerford and Samulski, 1998). The AAV2 genome, like the genomes of all AAV serotypes, can be encapsulated in several different capsid proteins. AAV2 can be packaged in its native AAV2 capsid (AAV2 / 2) or can be pseudotyped with other capsids (e.g., AAV2 genome in an AAV1 capsid named AAV2 / 1; AAV2 genome in an AAV5 capsid named AAV2 / 5; or AAV2 genome in an AAV8 capsid named AAV2 / 8).

[0075] The AAV genome can be in a single-stranded form with a positive or negative sense strand, or in a double-stranded form. Using the double-stranded form allows for bypassing the DNA replication process in target cells, thus accelerating transgene expression. The AAV genome can originate from any naturally occurring serotype or AAV isolate or clade. As is known to those skilled in the art, naturally occurring AAV viruses can be classified according to various biological systems.

[0076] Generally, AAV viruses are referred to by their serotypes. Serotypes correspond to variant subspecies of AAV, each possessing a specific reactivity that can be used to distinguish it from other variant subspecies due to its expression profile of the capsid surface antigen. Typically, viruses with a particular AAV serotype do not efficiently cross-react with specific neutralizing antibodies against any other AAV serotype. AAV serotypes include AAV1 (accession number, e.g., NC_002077, AF063497), AAV2 (accession number, e.g., NC_001401), AAV3 (accession number, e.g., NC_001729, NC_001863), AAV4 (accession number, e.g., NC_001829), AAV5 (accession number, e.g., NC_006152), AAV6 (accession number, e.g., NC_001862), AAV7 (accession number, e.g., NC_006260), AAV8 (accession number, e.g., NC_006261), AAV9 (accession number, e.g., AY530579), and AAV10 (AAVrH10; accession number, e.g., AY243015), as well as recombinant serotypes such as Rec2 and Rec3 identified from primate brains.

[0077] AAV viruses can also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally occurring AAV viruses, usually tracing back to a common ancestor and encompassing all of its descendants. Furthermore, AAV viruses can be referred to in relation to specific isolates, i.e., genetic isolates of a particular AAV virus found in nature. The term genetic isolate describes a population of AAV viruses whose genetic mixing with other naturally occurring AAV viruses is limited, thereby defining a distinct population recognizable at the genetic level.

[0078] Those skilled in the art can, based on their common general knowledge, select an appropriate serotype, clade, clone, or isolate of AAV for use in the present invention.

[0079] The present invention encompasses viral particles containing capsids (or referred to as capsid proteins or viral capsids) derived from various serotypes, clades, clones, or isolates of AAV. The gene encoding the capsid is typically provided in trans, i.e., via a helper vector, in addition to the viral vector containing the ITR and the gene encoding the target protein. The present invention also encompasses viral particles in which the genome of one serotype is packaged (via the nucleic acid constructs of the present invention) into the capsid of another serotype, i.e., pseudotyping. Chimeric derivatives, shuffled derivatives, or capsid-modified derivatives may be selected to provide one or more desirable functionalities to the viral particle (p\article). Thus, these derivatives may exhibit improved gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism range, and / or improved targeting to specific cell types compared to AAV viral vectors containing naturally occurring AAV genomes such as AAV2. Improved gene delivery efficiency may be influenced by improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and nuclear transport, improved uncoating of viral particles, and improved conversion from single-stranded genome to double-stranded form. Improved efficiency may also be associated with changes in the tropism range or targeting of specific cell populations, so that the dose of viral particles is not diluted by administration to tissues where it is not needed.

[0080] Chimeric capsid proteins include those produced by recombination between two or more capsid-coding sequences of naturally occurring AAV serotypes. This can be done, for example, by marker rescue techniques, which involve co-transfecting a non-infectious capsid sequence of one serotype with a capsid sequence of a different serotype and using directed selection to select the capsid sequence with the desired properties. The capsid sequences of different serotypes can be modified by intracellular homologous recombination to produce novel chimeric capsid proteins. Chimeric capsid proteins also include those produced by manipulating capsid protein sequences to move specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes. Shuffled capsid proteins or chimeric capsid proteins can also be produced by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes can be constructed by randomly fragmenting the sequences of relevant AAV genes, such as genes encoding capsid proteins of multiple different serotypes, and then reconstructing the fragments using a self-priming polymerase reaction. This may result in crossovers in sequence homology regions. By shuffling several serotype capsid genes, libraries of hybrid AAV genes thus constructed can be screened to identify viral clones with desired functionality. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to create libraries of diverse mutants that can be selected for desired traits.

[0081] The capsid gene sequence may also be genetically modified to introduce deletions, substitutions, or insertions specific to the natural wild-type sequence. In particular, the capsid gene may be modified by inserting a sequence of an unrelated protein or peptide within the open reading frame of the capsid code sequence, or at the N-terminus and / or C-terminus of the capsid code sequence. The unrelated protein or peptide may, advantageously, act as a ligand for a particular cell type, thereby improving binding to target cells or improving the specificity of the vector's targeting to a particular cell population. The unrelated protein may also be a protein that assists in the purification of viral particles as part of the production process, i.e., an epitope or affinity tag. The insertion site is usually selected so as not to interfere with other functions of the viral particle, such as internalization or viral particle transport. Those skilled in the art can identify suitable insertion sites based on common general knowledge.

[0082] The viral particles according to the present invention include 1) a capsid identical to that of a wild-type serotype, AAV isolate, or clade, 2) a capsid derived from a wild-type serotype, AAV isolate, or clade, or 3) a fully engineered capsid. Similarly, the viral particles according to the present invention include a nucleic acid construct or vector containing an AAV genome or a portion thereof (such as at least one ITR contained in the nucleic acid construct or vector of the present invention) that 1) originates from a wild-type serotype, isolate, or clade of AAV, 2) is derived from a wild-type serotype, isolate, or clade of AAV, or 3) is fully engineered. The AAV genome (such as at least one ITR contained in the nucleic acid construct or vector of the present invention) and the capsid (such as the AAV capsid) may originate from the same serotype or different serotypes. Preferably, the viral particles according to the present invention (such as AAV viral particles) have the ability to transduce cells of the CNS, such as nerve cells, astrocytes, and / or oligodendrocytes. In the overall context of the present invention, the viral particle comprises an AAV genome and / or capsid that is identical to or derived from, for example, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or AAV serotype rh10 (AAVrh10). Preferably, the viral particle comprises an AAV genome and / or capsid that is identical to or derived from AAV2, AAV9, or AAVrH10. More preferably (but not limited to), the viral particle comprises an AAV genome and / or capsid that is identical to or derived from AAV2 or AAV9. In a non-limiting example, the AAV2 derivative is AAV-TT. AAV-TT is described in detail in Tordo et al., 2018 and International Publication No. 2015 / 121501, which are incorporated herein by reference in their entirety. For example, if the AAV genome (such as at least one ITR contained in the nucleic acid construct or vector of the present invention) and the capsid (such as the AAV capsid) are derived from the same serotype, the serotype may be AAV2 or a derivative of AAV2 such as AAV-TT.In another example, if the AAV genome (such as at least one ITR contained in the nucleic acid construct or vector of the present invention) and the capsid (such as the AAV capsid) originate from different serotypes, the serotype of the ITR may be AAV2 and the serotype of the capsid may be AAV-TT.

[0083] The nucleic acid constructs, vectors, and viral particles (such as AAV particles or rAAV particles) described herein can be prepared by standard means known in the art. Therefore, suitable vector preparations can be prepared using well-established production methods (including transfection, packaging, and purification methods).

[0084] Other non-limiting examples of nucleic acid constructs according to the present invention include or consist of: a) Sequence ID 10 (AAV-06164 construct); b) Sequence ID 11 (AAV-06262 construct); c) Sequence ID 12 (AAV-06263 construct); or d) A functional variant or functional fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with any one of (a) to (c).

[0085] Other non-limiting examples of vectors according to the present invention include or consist of: a) Sequence ID 22 (AAV-06164 plasmid); b) Sequence ID 23 (AAV-06262 plasmid); c) Sequence ID 24 (AAV-06263 plasmid); or d) A functional variant or functional fragment having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with any one of (a) to (c).

[0086] In a fifth embodiment, the present invention provides a host cell comprising the nucleic acid construct or vector of the present invention. The present invention also provides a host cell that produces viral particles of the present invention (such as AAV particles or rAAV particles). Any suitable host cell may comprise the nucleic acid construct or vector of the present invention. Furthermore, any suitable host cell may be used to produce the viral particles of the present invention. In the overall context of the present invention, the host cell is preferably an insect cell or a mammalian cell. Non-limiting examples of such cells are sf9, HEK293 (including, e.g., HEK293F, HEK293S, or HEK293T), BHK, or CHO cells. Non-limiting examples, the host cell may comprise the nucleic acid construct or vector of the present invention, and further comprise a further nucleic acid construct or vector that provides the minimum additional genomic sequences necessary for packaging the nucleic acid construct into viral particles (in the form of an AAV helper plasmid providing the AAV rep gene and cap gene). In a further non-limiting example, a host cell may include a nucleic acid construct or vector of the present invention, further including two additional nucleic acid constructs or vectors that provide the minimum additional genomic sequences necessary for packaging the nucleic acid construct within a viral particle (e.g., one nucleic acid construct or vector providing a minimum additional genomic sequence based on AAV (e.g., an AAV helper plasmid) and one nucleic acid construct or vector providing a minimum additional genomic sequence based on adenovirus (AdV) (e.g., an AdV helper plasmid)).

[0087] The nucleic acid constructs, vectors, and / or viral particles (such as AAV particles or rAAV particles) described herein can be formulated into pharmaceutical compositions. Accordingly, in the sixth embodiment, a pharmaceutical composition is provided herein comprising the nucleic acid construct, vector, and / or viral particles (such as AAV particles or rAAV particles) of the present invention together with a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0088] The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, and / or other materials well known to those skilled in the art. Such materials must be nontoxic and must not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other materials can be determined by those skilled in the art according to the route of administration.

[0089] Pharmaceutical compositions may be provided in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water or physiological saline. When injected to the affected area, the active ingredient is in the form of an aqueous solution that does not contain pyrogens and has appropriate pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using an isotonic solvent such as sodium chloride injection, Ringer's injection, Ringer's lactate injection, or Hartmann's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0090] The dosage and administration plan can be determined within the scope of the physician's normal skill level when administering the composition.

[0091] The nucleic acid constructs, vectors, and viral particles of the present invention have the ability to rescue the loss of function of the original POI (e.g., PGRN) that may occur due to mutations in one or both alleles of a patient's coding gene (e.g., the GRN gene). "Rescue" generally means any improvement or delay of the progression of the phenotype associated with the deficiency in a given POI (e.g., PGRN deficiency), such as the restoration of the presence of the POI (e.g., PGRN protein) in the brain and / or the reduction of neurological lesions.

[0092] The properties of the nucleic acid constructs and vectors of the present invention can be tested using techniques known to those skilled in the art. For example, if the POI is PGRN, the nucleic acid construct of the present invention can be incorporated into the vector of the present invention, delivered to PGRN-deficient test animals such as mice or primates, and its effect can be observed and compared with a control.

[0093] In a seventh embodiment, the present invention also encompasses the use of nucleic acid constructs, vectors, viral particles and / or pharmaceutical compositions described herein for treating or preventing a patient's disease or condition.

[0094] This specification provides nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention for use in methods of treating or preventing a disease or condition in a patient in need. The present invention further provides methods of treating or preventing a disease or condition in a patient in need, the methods comprising administering a therapeutically effective amount of the nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention to the patient. The present invention also provides the use of the nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention for producing pharmaceuticals for treating or preventing a disease or condition in a patient in need.

[0095] A disease or condition may be characterized by a deficiency of a specific target protein (POI), such as PGRN deficiency. Such deficiency (e.g., PGRN deficiency) may result from the loss of a loss-of-function mutation in one or both alleles of the corresponding gene (e.g., the GRN gene) in the patient being treated.

[0096] In certain embodiments, where the POI is PGRN, the present invention provides nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention for use in a method of treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient in need. The present invention further provides a method of treating or preventing a disease characterized by progranulin (PGRN) deficiency in a patient in need, the method comprising administering a therapeutically effective amount of the nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention to the patient. The present invention also provides the use of the nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention for producing pharmaceuticals for treating or preventing a disease characterized by progranulin (PGRN) deficiency.

[0097] Diseases characterized by PGRN deficiency to be treated in a patient with the nucleic acid constructs, vectors, viral particles and / or pharmaceutical compositions of the present invention may be (i) diseases of the central nervous system (CNS), (ii) diseases characterized by PGRN deficiency in the patient's neurons and / or astrocytes, (iii) diseases characterized by loss-of-function mutations in at least one allele of the patient's GRN gene, and / or (iv) diseases characterized by loss-of-function mutations in both alleles of the patient's GRN gene.

[0098] Diseases characterized by PGRN deficiency to be treated with the nucleic acid constructs, vectors, viral particles and / or pharmaceutical compositions of the present invention may be frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis type 11 (NCL11).

[0099] Diseases characterized by PGRN deficiency to be treated with the nucleic acid constructs, vectors, viral particles and / or pharmaceutical compositions of the present invention may further be characterized by lysosomal dysfunction, such as dysregulation of lysosomal acidification. The lysosomal dysfunction may be characterized by increased expression levels and / or activity of cathepsin D, preferably mature heavy chain and / or light chain cathepsin D.

[0100] Patients requiring treatment with the nucleic acid constructs, vectors, viral plasmids, and / or pharmaceutical compositions of the present invention may be male or female. Such patients may have been previously identified as being at risk of or having a disease characterized by PGRN deficiency. Such patients may have been previously identified as being at risk of or having FTD or NCL11.

[0101] The dosage of the nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention may be determined according to various parameters, particularly the age, weight, and condition of the patient being treated; the route of administration; and the required regimen. A physician can determine the necessary route of administration and dosage for any particular patient.

[0102] The nucleic acid constructs, vectors, viral particles, and / or pharmaceutical compositions of the present invention may be administered to the brain and / or cerebrospinal fluid (CSF) of a patient. Delivery to the brain may be selected from intracerebral delivery, intraparenchymal delivery, intraputaminal delivery, and combinations thereof. Further target regions within the brain include the thalamus, cerebellum, subthalamic nucleus, and combinations thereof. Delivery to the CSF may be selected from intracisional delivery, intrathecal delivery, intraventricular (ICV) delivery, and combinations thereof.

[0103] Delivery to the patient's brain and / or cerebrospinal fluid (CSF) may be by injection. Injections to the brain may be selected from intracerebral injection, intraparenchymal injection, intraputaminal injection, and combinations thereof. Delivery to the CSF may be selected from intracisional injection, intrathecal injection, intraventricular (ICV) injection, and combinations thereof.

[0104] Injections into the brain and / or cerebrospinal fluid may include convection-enhanced delivery (CED). The CED procedure involves placing a small-diameter catheter directly into a target area of ​​the brain after minimally invasive surgical exposure of the brain. CED is described, for example, by Debinski et al. (2009).

[0105] The doses of the nucleic acid constructs, vectors, virus particles, and / or pharmaceutical compositions of the present invention may be provided as single doses, but may be administered repeatedly if the vector does not target the correct region. Treatment is preferably a single injection, but repeated injections over several years and / or with different AAV serotypes may be considered.

[0106] The present invention further provides a kit comprising nucleic acid constructs, vectors, viral vectors, and / or pharmaceutical compositions of the present invention.

[0107] The present invention will be further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and the following examples, individually or in any combination thereof, can serve as materials for realizing the present invention in its various forms.

[0108] example Example 1 - Materials and Methods Lentivirus production: All lentiviral vectors used in this study were second-generation, produced using standard virus production methods. Briefly, 5.7 million HEK293T cells were seeded per 10 cm dish. The following day, the cells were transfected with lipofectamine 2000, 10 μg of transfer vector, 3 μg of pMD2G, and 8 μg of psPAX2. The culture medium was changed 12–14 hours after transfection. The viral supernatant was collected 24 and 48 hours after this medium change to obtain a total of 20 mL of virus, which was filtered through a 0.45 μm filter. The viral supernatant was concentrated 20-fold in PBS using a Lenti-X® concentrator (CloneTech) before flash freezing.

[0109] Titration of lentiviruses: All lentiviruses were titrated using the Lenti-X qRT-PCR titration kit (Takara).

[0110] AAV production: HEK293T cells (obtained from the American Tissue Collection Center, ATCC) were cultured in 150 mm tissue culture dishes at 37°C in DMEM containing 10% FBS and 1% S / P antibiotics. Once the cells reached 80% confluence, they were transfected (triple cotransfection, i.e., via one plasmid containing the nucleic acid / vector of the present invention, one rep / cap plasmid, and one helper plasmid). The cells were harvested and lysated according to a standard protocol. Subsequently, AAV particles were purified by iodixanol gradient ultracentrifugation according to a standard protocol.

[0111] Co-culture of neurons and astrocytes and transduction of lentiviruses or AAV9: Co-cultures of primary neurons and astrocytes were prepared from embryonic day 17 C57BL / 6J mice (Janvier Labs). Freshly dissected cortical tissue was first dissociated using papain solution. Cells were diluted in neuronal attachment medium and seeded into 96-well plates pre-coated with poly-D-lysine (Corning) (10,000 cells / well). The neuronal attachment medium consisted of Neurobasal Plus medium supplemented with 2.5% heat-inactivated FBS, 1 mM sodium pyruvate, 2 mM Glutamax-100X, B27 Plus Supplement, and 50 units / ml penicillin / streptomycin (all additives from ThermoFisher Scientific). Cells were maintained by weekly replenishment with fresh serum-free neurobasal medium. Lentivirus-mediated transduction was performed on day 3. Lentivirus or AAV9 stock was diluted in culture medium and spread onto cells at a given MOI as shown in the figure caption. Ten days after transduction, the cells were fixed and immunocytochemistry was performed. A MOI of 20 was used for lentiviruses, and a MOI of 10,000 was used for AAV9.

[0112] Immunolabeling and imaging:Immunocytochemistry was performed after transduction into primary neurons and astrocytes. Cells were washed three times (1×PBS) and then fixed with 4% PFA (ThermoFischer Scientific) at room temperature for 10 minutes. Next, cells were permeabilized with 0.25% Triton-X / 3% BSA / 1X PBS solution for 10 minutes. After permeabilization, cells were blocked with 3% BSA / 1X PBS solution for 30 minutes. Next, cells were labeled with primary antibody (60 minutes) and then with fluorescently conjugated secondary antibody (45 minutes) (see Table B for a list of antibodies). Imaging was performed using Zeiss LSM 880 (SH-SY5Y cells) and Perkin Elmer OperaPhenix (neurons / astrococytes) instruments. Threshold setting and quantification were performed using Image J or Perkin Elmer Harmony software. [Table 1]

[0113] ELISA: The levels of PGRN secreted after transduction of mouse neuronal-astroglial cell co-cultures were quantified using human and mouse progranulin ELISA kits (both Adipogen). The same reagents were used for in vivo CSF ​​hPGRN measurement. Cell culture media were collected 10 days after transduction. Samples were diluted 1:100 to 1:1000 and ELISA measurements were performed according to the supplier's instructions. For CSF, the sample was diluted 1:100. Colorimetric reactions were measured using a standard plate reader (Flex Station 3, Molecular Devices).

[0114] Quantitative real-time polymerase chain reaction (qRT-PCR):Total neuronal RNA was isolated using the RNeasy 96 kit (Qiagen) according to the manufacturer's protocol. Briefly, cDNA was synthesized from 1 μg of RNA using a high-volume cDNA reverse transcription kit (40 μl). Quantitative real-time PCR experiments were performed using specific Taqman gene expression probes for Hexb (Mm00599880_m1), Ctsd (Mm00515586_m1), Gpnmb (Mm01328586_g1), Lgals3 (Mm00802901_m1), β-actin (Mm02619580_g1), and Pgk1 (Mm00435617_m1). Reactions were performed on a ViiA 7 RT-PCR system (Applied Biosystems) using 5 ng of cDNA sample, recommended concentrations of specific probes, and the qPCR master mix Luminaris. The PCR reaction was performed in two sequences, and the compound change in mRNA levels was calculated using the 2-ΔΔCt method (Schmittgen and Livak, 2008), and normalized to the mRNA levels of β-actin and Pgk1.

[0115] Western blot analysis: Cells were transfected with the target construct using (flowing)X-treme GENE® 360 Transfection Reagent (Sigma) according to the manufacturer's instructions. 48 hours after transfection, the cell pellet was lysed in cell lysis buffer (Cell Signaling) supplemented with PMSF (Sigma-Aldrich), and protein concentrations were measured using BCA protein assay reagent (ThermoFisher). The culture medium was collected simultaneously and subsequently centrifuged at 12000 g at 4°C for 10 minutes. The lysates and medium were mixed with 4X loading buffer (Li-Cor), and an equal volume of protein was electrophoresed on Mini-PROTEAN TGX 4-15% precast polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane using the Trans-Blot Turbo System (Bio-Rad).

[0116] Nonspecific antibody binding was blocked at room temperature for 1 hour using Intercept TBS blocking buffer (Li-Cor). The membranes were incubated with the following primary antibodies: anti-PGRN (1:1000 dilution, Abcam) in Intercept T20 TBS (Li-Cor) overnight at 4°C; anti-GAPDH (1:5000 dilution, Sigma-Aldrich) in Intercept T20 TBS (Li-Cor) overnight at 4°C. The membranes were washed three times with TBST for 5 minutes each, incubated with donkey anti-mouse 680RD (Li-Cor, 1:5000) antibody and donkey anti-rabbit 800CW (Li-Cor, 1:5000) antibody in Intercept T20 TBS for 1 hour, and then washed three times with TBST for 5 minutes each. The membranes were visualized using Odyssey CLx (Li-Cor). For the culture medium samples, the blot was stained with Revert® Total Protein Stains to obtain the BSA signal.

[0117] Cell line culture: Ad293 cells were obtained from Agilent, and Neuro-2A cells were obtained from Sigma. Both cell lines were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin.

[0118] Brain section preparation, immunohistochemical staining, and acquisition: Brain sectioning was performed at Neuroscience Associates. First, the brain was treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent freeze artifacts, and then embedded in a gelatin matrix using MultiBrain® Technology. After curing, the block was rapidly frozen by immersion in isopentane cooled to -70°C with crushed dry ice and mounted on the freezing stage of an AO860 slide microtome. The MultiBrain® block was sectioned coronally into 40 μm sections. All sections were sequentially collected in 24 containers per block and filled with antigen preservation solution (49% PBS pH 7.0, 50% ethylene glycol, 1% polyvinylpyrrolidone). Sections that were not immediately stained were stored at -20°C.

[0119] Suspension sections were stained immunochemically with an antibody against human progranulin (R&D) diluted 1:15,000. All incubation solutions from blocking serum onward were Tris-buffered saline (TBS) containing Triton X-100 as the solvent. All rinsing was performed with TBS. Endogenous peroxidase activity was blocked by 0.9% hydrogen peroxide treatment, and nonspecific binding was blocked with 1.26% whole normal serum. After rinsing, sections were stained overnight at room temperature with primary antibody. The solvent solution contained 0.3% Triton X-100 for permeabilization. After rinsing, sections were incubated with avidin-biotin-HRP complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) at room temperature for 1 hour. After rinsing, the sections were treated with diaminobenzidine tetrahydrochloride (DAB) and 0.0015% hydrogen peroxide to produce visible reaction products. These were then placed on gelatinized (primed) glass slides, air-dried, lightly stained with thionine, dehydrated with alcohol, cleared with xylene, and mounted in covers using Permount mounting medium. Digital images of the stained sections were obtained using an AxioScan Z1 slide scanner with a 20x objective lens (Zeiss).

[0120] AAV vectors / AAV particlesCorresponding plasmid sequences are provided in SEQ ID NO: 22 (AAV-06164 plasmid; containing the AAV-06164 construct of SEQ ID NO: 10), SEQ ID NO: 23 (AAV-06262 plasmid; containing the AAV-06262 construct of SEQ ID NO: 11), and SEQ ID NO: 24 (AAV-06263 plasmid; containing the AAV-06263 construct of SEQ ID NO: 12). Viral (AAV) particles were generated using a triple plasmid transfection method involving a helper plasmid, a Rep / Cap encoding plasmid, and the plasmids reported above, respectively, using HEK 293T cells or HEK293 cells, as previously reported by Grieger et al (2016). Figure 2 provides schematic diagrams showing the constituent nucleotide sequences of SEQ ID NOs: 10, 11, and 12.

[0121] Example 2 - Generation of GRN promoter constructs The lentiviral vector construct pPG41 was generated as described above.

[0122] The construct pPG41 (SEQ ID NO: 6) contains the mouse GRN promoter region (SEQ ID NO: 1). The mouse GRN promoter sequence has a length of 2508 residues.

[0123] Construct 06164 (SEQ ID NO: 7) contains a cleaved human GRN1 promoter region (SEQ ID NO: 3). The cleaved GRN1 promoter sequence is 1801 residues long. The corresponding AAV-06164 construct corresponds to SEQ ID NO: 10.

[0124] Construct 06262 (SEQ ID NO: 8) contains a cleaved human GRN2 promoter region (SEQ ID NO: 4). The length of the cleaved GRN2 promoter sequence is 632 residues. The corresponding AAV-06262 construct corresponds to SEQ ID NO: 11.

[0125] Construct 06263 (SEQ ID NO: 9) contains a cleaved human GRN3 promoter region (SEQ ID NO: 5). The length of the cleaved GRN3 promoter sequence is 489 residues. The corresponding AAV-06263 construct corresponds to SEQ ID NO: 12.

[0126] Example 3 - Evaluation of transgene expression induced by mouse GRN promoter in primary neurons and astrocytes ELISA and immunocytochemistry experiments demonstrated the functionality of the mouse GRN promoter in WT and KO mouse primary neurons. As shown in Figure 1, mouse primary neurons were transduced to express the hPGRN (human progranulin) protein under the control of the mouse GRN promoter (pPG41). Ten days after transduction, the culture medium was collected and ELISA was performed to specifically detect mouse (A) and human (B) PGRN proteins. The mPGRN (mouse progranulin) protein was detected only in WT (both transduced and untransduced) cells and not in KO neuron cultures (A). Similarly, the hPGRN protein was detected only in transduced neuron cultures (both WT and KO). Immunocytochemistry and confocal imaging were performed on cells to quantify the percentage of transduced neurons (C), the percentage of transduced astrocytes (D), and the hPGRN expression level (E) in WT and KO neurons transduced to pPG41. In both WT and KO neurons, nearly 60% of neurons were transduced at a MOI of 20 using the lentiviral construct. hPGRN expression in astrocytes was minimal.

[0127] Example 4 - Evaluation of PGRN expression using a cleavage-type GRN promoter To evaluate the strength of various granulin-cleaving promoters, three constructs containing PGRN promoters of different sizes (see Example 2) were investigated for human progranulin expression in Ad293 and Neuro2A cells. Progranulin expression under the three different promoters was analyzed using Western blotting. As shown in Figure 3, all three promoters successfully promoted progranulin expression. Among them, the GRN3 promoter was found to result in the highest levels of transgene expression in both cell lines. Since progranulin is a secreted glycoprotein, progranulin secretion was examined by simultaneously collecting the culture medium. It was shown that the level of secreted progranulin was more than three times higher when induced by the GRN3 promoter than when induced by the GRN1 promoter, which is consistent with previous results in cell lysates.

[0128] hPGRN expression using three cleavage promoters was also investigated in GRN- / - mouse primary neurons (Figure 4). Mouse primary neurons were transduced using the AAV9 viral vector to express the hPGRN protein under the control of various cleavage human GRN promoters (AAV-06164, AAV-06162, AAV-06163). Ten days after transduction, the culture medium was collected and ELISA was performed to quantify the secreted hPGRN, or the cells collected for mRNA and protein extraction. Compared to the GRN1 promoter, the smaller promoters GRN2 and GRN3 showed enhanced levels of mRNA (A) and protein (B). Furthermore, in the case of the smaller promoters, higher levels of secreted hPGRN were detected in the culture medium compared to GRN1 (C).

[0129] Example 5 - CNS expression of human PGRN (hPGRN) in WT rats after striatal injection of AAVTT-06164 Adult (2-3 months old) Sprague Dolly rats were given an AAV-TT viral vector containing a construct of the GRN1 promoter (1801 residues) + human PGRN transgene (SEQ ID NO: 22) or a solvent (PBS) in a total dose of 2 10 The rats were injected bilaterally with a vector genome (vg). After 4 weeks, the animals were sacrificial, and CSF, plasma, and brain tissue were collected and analyzed (Figure 6). Transcardiac perfusion with 1×PBS was performed before dissection. Half of the brain was fixed for immunohistochemical (IHC) analysis, and the other half was used for biochemical analysis (Western blotting). IHC using hPGRN-specific antibodies revealed hPGRN protein expression not only in the striatum (injection site) but also in brain regions distant from the injection site, namely the thalamus, midbrain, substantia nigra, cortex, and hippocampus (Figure 6A). No hPGRN staining was observed in PBS-injected rats (control). hPGRN staining resulted from both cellular expression (strong expression in the cell body near the injection site - striatum, parts of the cortex, parts of the hippocampus, thalamus, midbrain) and secretory / uptake (distal to the injection site, diffuse staining). Diffused staining in the distal region suggests that secreted hPGRN is widely distributed by ISF / CSF flow within the rat's CNS. The concentration of hPGRN in rat CSF was also quantified using a human-specific ELISA assay (Figure 6B). CSF samples from animals injected with AAVTT-06164 and PBS were collected at 2 and 4 weeks post-surgery. hPGRN expression was detected only in the CSF of rats injected with AAVTT-06164 and not in rats injected with PBS. In particular, a slight increase in hPGRN concentration was detected between 2 and 4 weeks post-surgery. Therefore, AAVTT-06164 has the ability to translate hPGRN in the rat CNS via GT delivery under the control of the endogenous human GRN promoter.

[0130] Example 6 - Effect of human PGRN (hPGRN) expression on lysosomal gene expression Quantitative PCR analysis of lysosomal genes, cathepsin D (CTSD), glycoprotein NMB (GPNMB), hexosaminidase B (HEXB), and galectin 3 (LGALS3) was performed using primary neuron cultures from WT and Grn- / -(KO) mice. As shown in Figure 7, increased expression of these genes was observed in KO neurons compared to WT neurons, indicating lysosomal stress. hPGRN expression in KO neurons transduced with AAV9-06164, AAV9-06262, or AAV9-06263 reduced the levels of these lysosomal markers compared to control KO neurons. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17

[0131] References Olney et al., Neurol. Clin., 2017 May;35(2):339-374 Baker et al.,Nature,2006 Aug 24;442(7105):916-919 Faber et al.,Brain,2020;143(1):303-31 Arrant et al.,Brain,2017;140.5:1447-1465 Mole and Cotman,Biochimica et Biophysica Acta,2015;1852:2237-2241 Chitramuthu et al.,Brain,2017;140:3081-3104 Huin et al.,Brain,2020;143:303-319 US10,689,625 Zin et al.,Mol.Ther.Methods Clin.Dev.,2021 May 29;22:40-5 Bhandari et al.,Biochem.J.,1996;319:441-447 Sardiello et al.,Science,2009;325(5939):473-477 She et al.,Cell Chemical Biology,2017;24(7):892-906.e5 Sambrook et al.,1989,Molecular Cloning-a laboratory manual;Cold Spring Harbor Press. Liu B et al.,Gene Ther.,2004;11:52-60. Tanaka et al.,Human Molecular Genetics,2017;26(5):969-988 Kotin,et al.PNAS USA 1990.87:2211-2215 Hermonat et al.,J.Virol 1984.51:329-339 Summerford and Samulski J.Virol,1998,72:1438-1445 Tordo et al.,Brain,2018;141(7):2014-2031 WO2015121501 Debinski et al.,Expert Rev.Neurother.,2009;9(10):1519-27 Schmittgen and Livak,Nature Protocols,2008;3:1101 Grieger et al.,Molecular Therapy,2016;24(2):287-297

Claims

1. A nucleic acid construct comprising a granulin (GRN) promoter operably ligated to a nucleotide sequence encoding a target protein (POI), However, the GRN promoter is either a full-length GRN promoter or a cleavage-type GRN promoter.

2. The nucleic acid construct according to claim 1, wherein POI is a progranulin (PGRN) protein.

3. The nucleic acid construct according to any one of claims 1 to 2, wherein the GRN promoter comprises or consists of the following: (a) the nucleotide sequence of SEQ ID NO: 1 or 2, or a functional variant and / or functional fragment thereof having at least 90% identity with the nucleotide sequence of SEQ ID NO: 1 or 2; (b) the nucleotide sequence of SEQ ID NO: 3, or a functional variant and / or functional fragment thereof having at least 90% identity with the nucleotide sequence of SEQ ID NO: 3; (c) the nucleotide sequence of SEQ ID NO: 4, or a functional variant and / or functional fragment thereof having at least 90% identity with the nucleotide sequence of SEQ ID NO: 4; (d) The nucleotide sequence of SEQ ID NO: 5, or a functional variant and / or functional fragment thereof having at least 90% identity with the nucleotide sequence of SEQ ID NO:

5.

4. The nucleic acid construct according to any one of claims 1 to 3, wherein the nucleic acid construct comprises or consists of the following in the 5' to 3' direction: (a) GRN promoter sequences such as cleavage-type GRN promoters; (b) A Kossack array of any choice; (c) Polynucleotide sequences encoding POI, e.g., PGRN protein; and, (d) Optional post-transfer regulatory elements, such as poly(A) sequences including WPRE and / or SV40 poly(A) sequences.

5. A vector comprising a nucleic acid construct according to any one of claims 1 to 4, further comprising at least one reverse terminal repeat sequence (ITR), preferably a 5'ITR and a 3'ITR, adjacent to the 5' and / or 3' of the nucleic acid construct.

6. 1) A capsid and a nucleic acid construct according to any one of claims 1 to 4 packaged therein, or 2) comprising a capsid and the vector described in claim 5, Virus particles.

7. The virus particle according to claim 6, selected from the following: (a) Viral particles comprising adeno-associated virus (AAV) particles or AAV genome or derivatives thereof, wherein the derivative is optionally a chimeric derivative, a shuffled derivative, or a capsid-modified derivative; or (b) Lentiviral particles or viral particles containing lentiviral genomes or derivatives thereof.

8. The virus particle according to claim 7, comprising a capsid identical to the following or a capsid derived from the following capsid: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or AAV serotype rh10 (AAVrh10) Preferably, the AAV particles contain capsids identical to or derived from AAV2, AAV9, or AAVrH10.

9. The virus particle according to claim 8, wherein the particle comprises a capsid derived from AAV2, and preferably the AAV2 derivative is AAV-TT.

10. A host cell that produces a nucleic acid construct according to any one of claims 1 to 4 and / or a vector according to claim 5, and / or a viral particle according to any one of claims 6 to 9, wherein the host cell is optionally a HEK293 cell or a HEK293T cell.

11. A pharmaceutical composition comprising a nucleic acid construct according to any one of claims 1 to 4, a vector according to claim 5, and / or a viral particle according to any one of claims 6 to 9, together with a pharmaceutically acceptable carrier, excipient, and / or diluent.

12. A nucleic acid construct according to any one of claims 1 to 4, a vector according to claim 5, a viral particle according to any one of claims 6 to 9, and / or a pharmaceutical composition according to claim 11, for use in a method of treating or preventing a disease characterized by a deficiency of a target protein (POI), such as a deficiency of progranulin (PGRN), in a patient who requires treatment or prevention.

13. A method for treating or preventing a disease characterized by a deficiency of a target protein (POI), such as progranulin (PGRN), in patients requiring treatment or prevention, including the following: Administer to the patient a therapeutically effective amount of the nucleic acid construct according to any one of claims 1 to 4, the vector according to claim 5, the viral particle according to any one of claims 6 to 9, and / or the pharmaceutical composition according to claim 11.

14. The use of a nucleic acid construct according to any one of claims 1 to 4, a vector according to claim 5, a virus particle according to any one of claims 6 to 9, and / or a pharmaceutical composition according to claim 11, For use in the manufacture of pharmaceuticals for the treatment or prevention of diseases characterized by a deficiency of a target protein (POI), such as progranulin (PGRN), in patients requiring treatment or prevention.

15. A nucleic acid construct, vector, viral particle, or pharmaceutical composition for use according to claim 12, the method according to claim 13, or the use according to claim 14, wherein POI is progranulin. (i) Diseases characterized by PGRN deficiency are diseases of the central nervous system; (ii) The disease characterized by PGRN deficiency is characterized by a deficiency of PGRN in the patient's neurons and / or astrocytes; (iii) The patient has a loss-of-function mutation in at least one allele of the GRN gene; and / or (iv) The patient has loss-of-function mutations in both alleles of the GRN gene.

16. The nucleic acid construct, vector, viral particle, or pharmaceutical composition according to claim 15, the method according to claim 15, or the use according to claim 15, wherein the disease characterized by PGRN deficiency is frontotemporal dementia (FTD) or neuronal ceroid lipofuscinosis type 11 (NCL11).

17. A nucleic acid construct according to any one of claims 1 to 4, comprising the following nucleotide sequence: (a) SEQ ID NO: 7 ((a)(b)SEQ ID NO: 7), or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 7; (b) SEQ ID NO: 8, or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 8; (c) A functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 9; (d) SEQ ID NO: 10, or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 10; (e) SEQ ID NO: 11, or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 11; or (f) A functional variant or functional fragment thereof having at least 70% identity with SEQ ID NO: 12, or the nucleotide sequence of SEQ ID NO:

12.

18. The vector according to claim 5, comprising the following nucleotide sequence: (a) SEQ ID NO: 22, or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 22; (b) SEQ ID NO: 23, or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 23; or (c) SEQ ID NO: 24, or a functional variant or functional fragment thereof having at least 70% identity with the nucleotide sequence of SEQ ID NO: 24.

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