Treatment for central nervous system disorders

Abstract

The present application discloses a method for treating a protein deficiency in the central nervous system of a subject in need thereof, the method comprising systemically administering to the subject a therapeutically effective amount of a fusion polypeptide comprising a first protein, the fusion polypeptide comprising (a) the first protein, (b) a second protein that provides an extended circulatory residence time in vivo, and (c) a blood-brain barrier crossing-promoting peptide, wherein the fusion polypeptide crosses the blood-brain barrier (BBB).

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates generally to a dual-action therapeutic system for treating central nervous system disorders such as Alzheimer's disease. [Background technology]

[0002] General background and state of the art Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide, characterized by the progressive loss of neurons, typically resulting in severe impairment of cognitive functions, including memory and learning. Pathological hallmarks of AD include the presence of extracellular amyloid plaques and the formation of neurofibrillary tangles (NFTs) in the brain, which lead to neuronal dysfunction and cell death. One of the greatest challenges in developing AD therapeutics is achieving sufficient blood-brain barrier (BBB) ​​penetration (Banks, 2016). The BBB is a protective contact site that controls the transport of molecules, ions, and cells between the blood and the brain (Patabendige, 2013). Other molecules, such as biomolecules like antibodies, are too large to efficiently cross the BBB. Clinically, attempts to enhance the penetration of therapeutic proteins across the BBB in sufficient quantities to treat major brain disorders have so far been unsuccessful (Gabathuler, 2010). This makes it difficult to use biomolecules for central nervous system disorders such as AD. In recent years, various therapeutic BBB carriers have been developed for central nervous system diseases (Malakoutikhah, 2011), including chemical delivery systems (Brewster, 1997; Carelli, 1996), carrier-mediated transport (Manfredini, 2002; Gynther, 2008; Gynther, 2009), and molecular Trojan horses (Boado, 2007; Boado, 2008; Boado, 2009; Boado, 2013). Cell-penetrating peptide (CPP)-mediated drug delivery is one method to enhance brain delivery. In vivo studies in rodents have revealed that linking biomolecules to the HIV-1 trans-acting transcriptase (TAT), the most well-known CPP, results in constructs that can facilitate the delivery of biomacromolecules across the blood-brain barrier (Cao, 2002; Kilic, 2002; Banks, 2005).

[0003] However, the therapeutic efficacy of these delivery systems is unsatisfactory due to their limited penetration and retention capacity. Drug concentrations in the lesion area are significantly lower than the therapeutic threshold. To address these issues, receptor-mediated BBB delivery systems are considered a promising strategy to improve BBB penetration efficacy. Many researchers have focused on exploring specific receptors overexpressed on the cell membrane of the blood-brain barrier to gain access to the brain at therapeutic concentrations (Wang, 2017).

[0004] Recently, novel biomolecules have been shown in preclinical and clinical studies to enter the brain and reduce amyloid-beta plaques (Sevigny, 2016; Reiman, 2016; Chang, 2017). Because biomolecules offer promising therapeutic options for Alzheimer's disease, novel strategies to enhance their brain delivery are needed. Utilizing endogenous transport systems remains an underutilized strategy for drug delivery to the brain (Banks, 2012).

[0005] Alzheimer's disease (AD) is characterized by the extracellular deposition of misfolded amyloid beta (Aβ) peptides and the intracellular formation of neurofibrillary tangles (NFTs, also known as phosphorylated tau or p-tau), yet no strategy has yet been identified to reduce these pathological events. Development of therapeutics for AD is limited by the presence of the blood-brain barrier (BBB), which prevents efficient uptake of most therapeutics from the blood into the brain. We developed a human serum albumin (HSA) fusion platform containing a BBB-permeable cell-penetrating peptide (CPP, dTAT) for the reduction of Aβ and NFTs (p-tau).

[0006] The microtubule-associated tau protein is thought to be involved in the formation and stabilization of microtubules (Spillantini, 2013; Lee, 2001). Tau exists as a phosphoprotein, and even in healthy adult brains, tau is phosphorylated, at least to a minimal extent (Seubert, 1995). However, what characterizes Alzheimer's disease is the consistent phosphorylation of 19 of the 441 specific amino acids along the tau protein, resulting in a specific phosphorylation pattern and a greater burden of phosphorylated tau in Alzheimer's disease brains (Augustinack, 2002; Neddens, 2018; Medina, 2015). The magnitude of the difference in phosphorylated tau burden between Alzheimer's disease and healthy adult brains has been reported to be a 3-4-fold increase in moles of phosphate per mole of tau (Ksiezak, 1992). Neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau assembled into paired helical filaments (PHFs), are a valid pathological feature in the Braak staging system for Alzheimer's disease (Braak and Braak, 1995; Grundke-Iqbal, 1986; Kosik, 1986; Lee, 1991; Braak, 1995). Tangles are the only pathological finding in Alzheimer's disease that has been shown to correlate topographically and quantitatively with clinical symptomatology (Arriagada, 1992; Braak, 1991; Goedert, 1993; Ballatore, 2007).

[0007] Tau expression is high in unmyelinated axons in the cortex, particularly in brain regions involved in memory consolidation, such as the limbic cortex, including the hippocampus (Trojanowski, 1989). Hyperphosphorylation of tau causes the protein to detach from microtubules, thereby destabilizing them and disrupting axonal transport (Bramblett, 1993; Ishihara, 1999). Tau phosphorylation and dephosphorylation are regulated by the balance between the activity of protein kinases (e.g., GSK3β, cdK5, Akt / PKB, PKA, ERK1 / 2, and AMPK) and phosphatases (e.g., PP1, PP2A, and PP5) (Ballatore, 2007; Chung, 2009; Wang, 2007).

[0008] AMP-activated protein kinase (AMPK) is a key intracellular energy sensor and metabolic regulator. AMPK is involved in cellular energy homeostasis through the regulation of glycolytic flux and mitochondrial biogenesis (Hardie, 2011). Mammalian AMPK is a heterotrimeric complex consisting of a catalytic α subunit (isoforms α1 and α2) and regulatory β (β1 and β2) and γ (γ1, γ2, and γ3) subunits. AMPK is activated by the cellular metabolic state and its upstream kinases and inhibited by one of several phosphatases. Increased cytosolic AMP and calcium concentrations are key activators of AMPK signaling in neurons (Nakamura, 2001; Salminen, 2011; Steinberg, 2009). Metabolic dysfunction and AMPK activity have been reported as regulators of both tau phosphorylation and amyloid formation in the pathogenesis of Alzheimer's disease (Thornton, 2011; Vingtdeux, 2011). In primary mouse neurons, endogenous AMPK activation was shown to induce increased tau phosphorylation at multiple sites, and AMPK inhibition caused a rapid decrease in tau phosphorylation (Domise, 2016).

[0009] PP2A is the most important phosphatase in the tau phosphorylation process; under normal conditions, PP2A inhibition is accompanied by tau hyperphosphorylation. In vivo PP2A activity is downregulated by the endogenous inhibitory protein inhibitor 2 (I2PP2A; also known as SET) (Li, 1996). In Alzheimer's disease brains, lysosomal aspartic endopeptidase (AEP) cleaves the I2PP2A protein (full-length, approximately 39 kDa) into active fragments of approximately 20 kDa (Rosenmann, 2014; Basurto-Islas, 2013). PP2A activity is inhibited by interaction of the activated I2PP2A fragment with the catalytic subunit of PP2A (Arnaud, 2011). Fragments of I2PP2A have also been reported to be responsible for inhibiting PP2A activity and inducing increased abnormal hyperphosphorylation of tau (Basurto-Islas, 2013). Summary of the Invention

[0010] These and other objects of the present invention will be more fully understood from the following description of the invention, the referenced drawings herein, and the appended claims.

[0011] In one aspect, the present invention is directed to a method for treating a protein deficiency in the central nervous system of a subject in need thereof, the method comprising systemically administering to the subject a therapeutically effective amount of a fusion polypeptide, wherein the fusion polypeptide comprises: (a) a first protein, the loss of which in the brain correlates with Alzheimer's disease; (b) a second protein, the loss of which in vivo results in an extended circulatory residence time; and (c) a blood-brain barrier crossing-promoting peptide, wherein the fusion polypeptide crosses the blood-brain barrier (BBB). The amino acid sequence of the first protein may be covalently linked to the amino acid sequence of the second protein. The amino acid sequence of the second protein may be cleavably covalently linked to the blood-brain barrier crossing-promoting peptide. The covalent bond may be cleavable by a change in pH, or the introduced cleavage site may be glycyl phenylalanyl leucyl glycine (GFLG). In one aspect, the therapeutically effective amount may be at least about 1-10 mg / Kg of body weight.

[0012] In another aspect, the first protein may be a protein that reduces amyloid beta (Abeta) deposition in the brain and / or a protein that reduces the level of hyperphosphorylated tau in the brain. Alternatively, the first protein may have the dual function of reducing amyloid beta (Abeta) deposition in the brain and reducing the level of hyperphosphorylated tau in the brain.

[0013] In one aspect according to the present invention, the first protein may be cystatin C, low-density lipoprotein receptor-related protein 1 cluster IV (LRP1-C4), soluble receptor for advanced glycation end products (sRAGE), RAGE-v, or myelin basic protein (MBP). In particular, the first protein may be cystatin C or RAGE-v. Furthermore, particularly, the first protein may be cystatin C.

[0014] In another aspect, the second protein can be human serum albumin.

[0015] In another aspect, the blood-brain barrier crossing-facilitating peptide may be dTAT.

[0016] In another aspect, in the above method, the first protein may be a protein that reduces amyloid beta (Abeta) deposition in the entorhinal cortex or hippocampus and / or reduces the level of hyperphosphorylated tau in the entorhinal cortex or hippocampus.

[0017] In particular, a fusion protein containing a protein that reduces amyloid beta (Abeta) deposition in the brain as a first protein is co-administered or sequentially administered with a fusion protein containing a protein that reduces the level of hyperphosphorylated tau in the brain as a first protein.

[0018] In yet another aspect, the present invention is directed to a method for treating a central nervous system degenerative disease in a subject in need thereof, the method comprising systemically administering to the subject a therapeutically effective amount of a fusion polypeptide, wherein the fusion polypeptide comprises a first protein that reduces amyloid beta (Abeta) deposition in the entorhinal cortex or hippocampus and a second protein that reduces hyperphosphorylated tau levels in the entorhinal cortex or hippocampus, and the fusion polypeptide comprises a protein that confers extended in vivo circulatory residence time and a blood-brain barrier crossing-promoting peptide; wherein the fusion polypeptide crosses the blood-brain barrier (BBB). In the above method, the amino acid sequence of the protein that confers extended in vivo circulatory residence time is cleavably covalently linked to the blood-brain barrier crossing-promoting peptide. The covalent bond may be cleavable by a change in pH, or the introduced cleavage site may be glycyl phenylalanyl leucyl glycine (GFLG). In one aspect, according to the above method, the therapeutically effective amount may be at least about 1 to 10 mg / kg of human body weight. The disease may be Alzheimer's disease. In particular, the first protein may be cystatin C, low-density lipoprotein receptor-related protein 1 cluster IV (LRP1-C4), soluble receptor for advanced glycation end products (sRAGE), RAGE-v, or myelin basic protein (MBP). In another aspect, according to the above present invention, the second protein may be cystatin C, Pten-long, PDZ domain-deleted Pten-long, TFEB, or SIRT1. The protein that prolongs in vivo circulatory residence time may be human serum albumin. The blood-brain barrier-crossing-promoting peptide may be dTAT. The first protein may be, in particular, cystatin C or RAGE-v.

[0019] The present invention will be more fully understood from the detailed description set forth herein below and the accompanying drawings, which are provided by way of illustration and therefore not by way of limitation. [Brief explanation of the drawings]

[0020] [Figure 1] The construction of the pOptivec / HSA fusion plasmid for expressing human serum albumin fusion protein version 1 or 2 is shown. The bicistronic pOptivec / HSA fusion plasmid contains HSA fusion protein version 1 (or 2) and the selectable marker DHFR. Restriction enzyme recognition sites used in the cloning strategy are shown in italics. [Figure 2] A schematic diagram of the HSA fusion protein (version 1) is shown. The diagram includes the following: a mechanism of action (MOA) protein for reducing beta-amyloid plaques or tau tangles; a GS linker (GGSAS (SEQ ID NO: 1) or GGGSGGGS (SEQ ID NO: 2)); HSA (human serum albumin); a cleavable linker (GFLG); and a cell-penetrating peptide (CPP) in the hatched region. The diagram of AL04 (80 kDa) contains cystatin C, HSA, and a modified TAT peptide as the MOA. The numbers above the boxes represent the amino acid numbers from the N-terminus of the fusion protein. [Figure 3] Schematic diagrams of HSA fusion proteins (version 2, AL06–AL10) are shown. These proteins are MOA proteins (MOA1 or MOA2) for reducing β-amyloid plaques or tau tangles; GS linkers (GGSAS or GGGSGGGS); human serum albumin (HSA); cleavable linkers (GFLG (SEQ ID NO: 3) or GFLGGGGSAS (SEQ ID NO: 4)); and cell-penetrating peptides (CPPs). Schematic diagrams of AL06 (145 kDa), AL07 (135 kDa), AL08 (129 kDa), AL09 (144.5 kDa), and AL10 (156 kDa) contain RAGE-V or RAGE-V-C1 as MOA1; Pten-long, PDZ domain-deleted Pten-long, or TFEB or SIRT1 as MOA2; HSA as carrier; and a modified TAT peptide as CPP. The numbers above the boxes indicate the amino acid numbers from the N-terminus in AL04. [Figure 4]A schematic diagram of the HSA fusion protein (version 2.1, AL12) is shown. The construct contains a mechanism of action (MOA) protein for reducing beta-amyloid plaques (MOA1) or tau tangles (MOA2); a GS linker (GGGSGGGS); human serum albumin (HSA); a cleavable linker (GFLGGGGSAS); and a cell-penetrating peptide (CPP). A schematic diagram of AL12 (88 kDa) contains RAGE-V as MOA1; HSA as the carrier; SIRT1-exon 4 as MOA2; and a modified TAT peptide as the CPP. The numbers above the boxes represent the amino acid numbers from the N-terminus of each HSA fusion protein. [Figure 5] Characterization of purified AL04 (80 kDa), AL07 (135 kDa), AL08 (129 kDa), and AL12 (88 kDa) by SDS-PAGE (a) and Western blot (b) is shown. Purified samples were run on a 4-12% gradient gel under reducing conditions; (a) the gel was stained with Coomassie blue; (b) the gradient gel containing each protein was transferred to a PVDF membrane for Western blotting using purified anti-human serum albumin antibody. [Figure 6] Figures 6A and 6B show that AL04 attenuates Aβ1-42-induced cell death in undifferentiated PC12 cells. (A) Nonspecific cytotoxicity of AL04 in undifferentiated PC12 cells; (B) PC12 cells were exposed to 10 μM Aβ1-42 for 72 hours in the presence or absence of AL04 (CysC-HSA-TAT) (0.01 μM). Cell viability was assessed by WST-8 (water-soluble tetrazolium salt) reduction assay. [Figure 7] Figure 1 shows that AL04 attenuates Aβ1-42-induced cell death in differentiated PC12 cells. Cells were treated with either soluble or aggregated (aged) Aβ1-42 for 3 days in the presence or absence of AL04. Cell viability was assessed by WST-8 (water-soluble tetrazolium salt) reduction assay. *Soluble Aβ1-42, Aβ1-42 peptide in DPBS buffer; **Aggregated Aβ1-42, Aβ1-42 solution aged at 37°C for 3 days. [Figure 8]The BBB permeability of AL04 was evaluated using a human BBB model. (A) Dose-dependent permeability was evaluated at 120 minutes using 1 μM and 10 μM AL04 and recombinant human serum albumin (rHSA). (B) The permeability of AL04 was compared at three time points (60 minutes, 120 minutes, and 240 minutes) over the time course. [Figure 9] Figures 9A and 9B show that the HSA fusion protein penetrates cells in a dose-dependent manner. PC12 cells were differentiated with 100 ng / ml NGF in DMEM medium containing 1% horse serum. After 4 days, NGF-deprived PC12 cells were treated with various concentrations (1–5 μg / ml) of AL04 (A) or AL12 (B) for 24 hours. Cells were rinsed twice with ice-cold DPBS (pH 7.5) and lysed. Cell lysates were subjected to SDS-PAGE followed by Western blotting for cystatin C (AL04) or anti-human serum albumin (AL12). [Figure 10] Selection of carrier and cell-penetrating proteins for the constructs is indicated. * indicates transient expression and quality integrity of the fusion protein as determined by Western blot in CHO-S cells. ** indicates stable expression of the fusion protein as determined by SDS-PAGE from MTX (2000 nM) amplification in CHO-DG44 cells. The Fc fusion protein formed a dimer via the Fc region. [Figure 11] A summary of dual-acting therapeutics (versions 1, 2, and 2.1) for potential therapeutic development is presented. [Figure 12] Figures 12A and 12B show the reduction of hyperphosphorylated tau levels in NGF-deprived PC12 cells by treatment with HSA fusion proteins. (A) Representative blots of phosphorylated tau (Ser202 / Thr205), phosphorylated tau (Thr231), and total tau in NGF-deprived PC12 cells treated with AL04, AL07, or AL08 for 24 hours. GAPDH was used as a loading control. (B) Quantitative analysis of the ratio of tau phosphorylation levels at Ser202 / Thr205 and Thr231, normalized to total tau. [Figure 13]We show that AL04 treatment reduces phosphorylated tau levels during NGF deprivation through downregulation of I2PP2A (an inhibitor of the phosphatase PP2A). PC12 cells were differentiated with 100 ng / ml NGF in DMEM medium containing 1% horse serum. After 4 days, NGF-deprived PC12 cells were treated with various concentrations of AL04 (1-10 μg / ml) for 24 hours. Cell lysates were subjected to SDS-PAGE and Western blotting for cystatin C (AL04), P-tau (Ser202 / Thr205), and I2PP2A. GAPDH was used to confirm that the amounts of protein loaded in different lanes were identical. [Figure 14] Figures 14A and 14B show that AL04 treatment increases the interaction between tau and tubulin in NGF-depleted PC12 cells. (A) PC12 cells were differentiated with 100 ng / ml NGF in DMEM medium containing 1% horse serum. Four days later, NGF-depleted PC12 cells were treated with or without 1 μg / ml AL04 for 24 hours. Cells were then lysed and subjected to immunoprecipitation using an anti-β-tubulin antibody. The immunoprecipitates were probed with an anti-tau antibody. The lysate loaded per lane was 1% of the total lysate, indicating uniform loading of proteins. Cell lysates were subjected to Western blotting for total tau, β-tubulin, and cystatin C (with respect to AL04). Relative quantification of tau bound to β-tubulin is shown in (B). Values ​​were normalized to the total amount of input tau and are expressed as the mean + / - standard error of triplicate determinations. P<001, Student's t-test. [Figure 15] AL04 dose-dependently reduces hyperphosphorylated tau by modulating AMPK. Representative blots of cystatin C (AL04), phosphorylated tau (Ser202 / Thr205), phosphorylated AMPα (Thr172), phosphorylated AMPKβ (Ser182), and phosphorylated ULK1 (Ser555) are shown after NGF-deprived PC12 cells were treated with various concentrations of AL04 (1-5 μg / ml) for 24 hours. GAPDH was used as a loading control. [Figure 16]1 shows that treatment with AL04 reduces amyloid beta (Abeta) deposition in the hippocampus of Tg2576 mice. [Figure 17] 1 shows that treatment with AL04 reduces amyloid beta (Abeta) deposition in the entorhinal cortex of Tg2576 mice. [Figure 18] 1 shows that treatment with AL04 reduces the levels of hyperphosphorylated tau in the hippocampus and entorhinal cortex of JNPL3 mice. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present application discloses the creation of a fusion polypeptide that crosses the blood-brain barrier for the treatment of central nervous system diseases, particularly neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, or Huntington's disease, more specifically Alzheimer's disease. In some embodiments, at least about 100 μg or at least about 1 mg / kg body weight, at least about 2 mg / kg or 3 mg / kg, 4 mg / kg, or 5 mg / kg or more of the fusion polypeptide is intraperitoneally delivered to the human body, and about 0.01% of the administered amount is expected to be localized in the brain. In some embodiments, a therapeutically effective amount of the fusion polypeptide comprises at least about 0.5 mg / kg body weight. In some embodiments, systemic administration is parenteral, intravenous, subcutaneous, intramuscular, intranasal, intraarterial, transdermal, or respiratory administration.

[0022] As used herein, "treatment" or "treating" includes achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to the eradication or amelioration of the underlying disease or condition being treated. For example, in an individual suffering from Alzheimer's disease, a therapeutic benefit includes partially or completely halting the progression of the disease or partially or completely reversing the disease. A therapeutic benefit is also achieved by the eradication or amelioration of one or more physiological or psychological symptoms associated with the underlying condition, thereby resulting in an improvement in the patient, even if the patient is still affected by the condition. A prophylactic benefit of treatment includes preventing a condition, slowing the progression of a condition (e.g., slowing the progression of Alzheimer's disease), or reducing the likelihood of developing a condition. As used herein, "treating" or "treatment" includes prevention.

[0023] As used herein, "therapeutic protein" refers to the therapeutically active protein component of the fusion polypeptide.

[0024] As used herein, a "fusion polypeptide" is a polypeptide construct in which multiple protein components are fused to create a polypeptide that is capable of crossing the BBB and providing long-lasting activity to a therapeutic protein.

[0025] As used herein, the term "effective amount" may refer to an amount sufficient to achieve a beneficial or desired result in the central nervous system when administered systemically, such as a beneficial or desired clinical result, or improved cognition, memory, mood, or other desired result in the central nervous system. An effective amount also refers to an amount that produces a prophylactic effect, e.g., an amount that delays, reduces, or prevents the onset of a pathological or undesirable condition. Such conditions include, but are not limited to, neurodegeneration. An effective amount may be administered in a single dose or multiple doses. In terms of treatment, an "effective amount" of a composition of the invention is an amount sufficient to slow, alleviate, halt, reverse, or delay the progression of a disease (e.g., a neurological disorder). An "effective amount" is any composition of the invention used alone or in combination with one or more drugs used to treat a disease or disorder. An "effective amount" of a therapeutic agent within the meaning of the present invention is determined by a patient's physician. Such an amount can be readily determined by one of ordinary skill in the art and, when administered in accordance with the present invention, will produce a therapeutic effect. Factors that influence a therapeutically effective amount include the Alzheimer's disease-specific activity of the administered fusion polypeptide, its absorption characteristics (e.g., brain uptake rate), time since onset of the disease, and the age, physical condition, presence of other pathologies, and nutritional status of the individual being treated. Additionally, other medications the patient is receiving will affect the determination of the therapeutically effective amount of the therapeutic agent to administer.

[0026] As used herein, a "subject" or "individual" is an animal, e.g., a mammal. In some embodiments, a "subject" or "individual" is a human. In some embodiments, the subject is suffering from Alzheimer's disease.

[0027] In some embodiments, a pharmaceutical composition comprising a fusion polypeptide is "administered peripherally" or "peripherally administered." As used herein, these terms refer to any mode of administration of an agent (e.g., a therapeutic agent) to an individual, where the administration is not directly into the central nervous system (i.e., where the agent contacts the non-brain side of the blood-brain barrier). As used herein, "peripheral administration" includes intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, transdermal, inhalation, buccal, intranasal, rectal, oral, parenteral, sublingual, or nasal.

[0028] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Such carriers are well known to those skilled in the art. A detailed description of pharmaceutically acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences (Gennaro, AR, ed., 20th ed., 2000: Williams and Wilkins PA, USA). Examples of pharmaceutically acceptable carriers include salts, such as mineral acid salts (hydrochloride, hydrobromide, phosphate, sulfate, etc.) and organic acid salts (acetate, propionate, malonate, benzoate, etc.). For example, the compositions of the present invention may be provided in liquid form and formulated in saline-based aqueous solutions of different pHs (5-8), with or without surfactants such as 0.01%-1% polysorbate 80, or carbohydrate additives such as mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citrate.

[0029] A "recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion (e.g., direct uptake, transduction, F-mating, or other methods known in the art for creating recombinant host cells). The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host's genome.

[0030] The terms "polypeptide," "peptide," and "protein" are used synonymously herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide can equally be used to describe a peptide or a protein, and vice versa. These terms apply to naturally occurring polymers of amino acids as well as to polymers of amino acids in which one or more amino acid residues are unnatural amino acids, e.g., amino acid analogs. As used herein, these terms include amino acid chains of any length (including full-length proteins) in which the amino acid residues are linked by covalent peptide bonds.

[0031] The term "amino acid" refers to natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium) that have the same basic chemical structure as natural amino acids (i.e., an α-carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids.

[0032] Amino acids are referred to herein by either their commonly known three letter amino acid symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are likewise referred to by their commonly accepted single-letter codes for nucleotides.

[0033] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof, in either single- or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0034] Regarding the "linker" connecting the various polypeptide components of the fusion polypeptide, in some embodiments, the linker comprises glycine, serine, and / or alanine residues in any combination or order. In some cases, the combined percentage of glycine, serine, and alanine residues in the linker is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the total number of residues in the linker. In some preferred embodiments, the combined percentage of glycine, serine, and alanine residues in the linker is at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the total number of residues in the linker. In some embodiments, a combination of any number of amino acids (including natural or synthetic amino acids) can be used in the linker. In some embodiments, a three-amino acid linker is used. In some embodiments, the linker has a Ser-Ser-Ser sequence. In some embodiments, the two-amino acid linker comprises glycine, serine, and / or alanine residues in any combination or order (e.g., Gly-Gly, Ser-Gly, Gly-Ser, Ser-Ser, Ala-Ala, Ser-Ala, or Ala-Ser linkers). In some embodiments, the two-amino acid linker consists of one glycine, serine, and / or alanine residue and one other amino acid (e.g., Ser-X, where X is any known amino acid). In yet other embodiments, the two-amino acid linker consists of any two amino acids except gly, ser, or ala (e.g., XX).

[0035] In some embodiments, linkers longer than two amino acids in length may be used. Such linkers may also contain glycine, serine, and / or alanine residues in any combination or order, as further described herein. In some embodiments, the linker consists of one glycine, serine, and / or alanine residue and another amino acid (e.g., Ser-nX, where X is any known amino acid and n is the number of amino acids). In yet other embodiments, the linker consists of any two amino acids (e.g., XX). In some embodiments, the any two amino acids are Gly, Ser, or Ala, in any combination or order, separated by a variable number of amino acids. In one embodiment, the linker consists of at least one Gly. In one embodiment, the linker consists of at least one Ser. In one embodiment, the linker consists of at least one Ala. In some embodiments, the linker consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Gly, Ser, and / or Ala residues. In preferred embodiments, the linker includes Gly and Ser in a repeat sequence in any combination or order (such as (Gly4Ser)3 or other variations).

[0036] Linkers for use in the present invention can be designed using any method known in the art. For example, there are several publicly available programs for determining optimal amino acid linkers in engineering fusion proteins. Publicly available computer programs (e.g., the LINKER program) that automatically generate optimal linker amino acid sequences based on user-entered protein sequences and desired linker lengths may be used in the methods and compositions of the present invention. In many cases, such programs utilize trends observed in natural linkers connecting protein subdomains to predict optimal protein linkers for use in protein engineering. In some cases, such programs use other methods for predicting optimal linkers.

[0037] The peptide linker sequence may include a cleavage site for a protease.

[0038] As used herein, "activity" includes physiological activity (eg, ability to cross the BBB and / or therapeutic activity) or enzymatic activity of the protein of interest transported by the fusion polypeptide.

[0039] The compositions of the present invention are particularly suitable for injection (e.g., as pharmaceutical compositions for intravenous, subcutaneous, intramuscular, or intraperitoneal administration). Aqueous compositions of the present invention comprise an effective amount of the compositions of the present invention, which may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refers to a molecular entity or composition that does not produce adverse, allergic, or other untoward reactions, as appropriate, when administered to an animal, e.g., a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be added to the composition.

[0040] Examples of pharmaceutically acceptable carriers for injectable compositions include salts, such as mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.) and organic acid salts (e.g., acetate, propionate, malonate, benzoate, etc.). For example, the compositions of the present invention may be provided in liquid form or formulated in saline-based aqueous solutions of different pHs (5-8), with or without surfactants such as 0.01%-1% polysorbate 80, or carbohydrate additives such as mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citric acid. Under ordinary conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms. The action of microorganisms can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol; phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents (e.g., sugars or sodium chloride). Prolonged absorption of the injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0041] For human administration, the preparations meet sterility, pyrogenicity, general safety, and purity standards as required by FDA and other regulatory agency standards. The active compound is usually formulated for parenteral administration (e.g., formulated for injection by intravenous, intramuscular, subcutaneous, intralesional, or intraperitoneal routes). The preparation of an aqueous composition containing an active element or component will be understood by those skilled in the art based on the present disclosure. Typically, such compositions can be prepared as injectable preparations (either solutions or suspensions). Solid forms suitable for use in preparing solutions or suspensions by adding liquid prior to injection can also be prepared, and the preparations may be emulsified.

[0042] Sterile injectable solutions are prepared by incorporating the required amount of the active compound in the appropriate solvent with various other ingredients as enumerated above, and then, if necessary, filter sterilization is carried out. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient and any desired additional ingredients from a previously filter-sterilized solution thereof.

[0043] Upon formulation, solutions will be systemically administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically effective based on the criteria described herein. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug-release capsules and the like may also be used.

[0044] The appropriate amount of pharmaceutical composition to be administered, frequency of treatment, and unit dose will vary depending on the central nervous system uptake characteristics of the fusion polypeptides described herein and the subject of treatment (the subject's condition and desired effect). In any event, the person responsible for administration will determine the appropriate dose for each subject.

[0045] Oral formulations contain commonly used excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. In certain embodiments, oral pharmaceutical compositions contain an inert diluent or an assimilable edible carrier, or may be enclosed in hard or soft gelatin capsules, compressed into tablets, or directly combined with dietary food. For oral therapeutic administration, the active compound may be combined with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations may contain at least 0.1% of the active ingredient. The percentage of the compositions and preparations may, of course, be varied and may conveniently range from about 2% to about 75%, or from about 25% to 60% by weight of a unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0046] Tablets, troches, pills, capsules, and the like may further contain: binders such as tragacanth gum, acacia gum, cornstarch, or gelatin; excipients such as dibasic calcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint, oil of wintergreen, or cherry flavoring. When the unit-dose form is a capsule, it may contain a liquid carrier in addition to the above-mentioned materials. Various other materials may be present as coatings or to modify the physical form of the unit-dose form. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup of elixir may contain the active compound, sucrose as a sweetener, methylene and propylparabens as preservatives, a coloring agent such as cherry or orange flavor, and a flavoring agent. In some embodiments, oral pharmaceutical compositions may be enterically coated to protect the active ingredient from the stomach environment; enteric coating methods and formulations are well known in the art.

[0047] Dual acting therapy (DAT) The present invention is directed to methods for treating central nervous system disorders, particularly Alzheimer's disease, by administering to a patient a dual-acting construct as described herein. Such dual-acting treatment involves administering a construct that expresses (i) a molecule that reduces amyloid beta (Abeta) deposition and / or (ii) a molecule that reduces levels of hyperphosphorylated tau in the brain. Both components may be expressed from a single construct.

[0048] Alternatively, the present invention is also directed to a method of treating central nervous system diseases, particularly Alzheimer's disease, by administering multiple constructs, wherein each construct comprises a molecule that either (i) reduces amyloid beta (Abeta) deposition and / or (ii) reduces the level of hyperphosphorylated tau in the brain. Such constructs may be co-administered or sequentially administered, thereby providing a dual-action effect on the patient. Such a dual-action treatment comprises providing a molecule that (i) reduces amyloid beta (Abeta) deposition and / or (ii) reduces the level of hyperphosphorylated tau in the brain, as described herein.

[0049] AL04 activity Accumulation of amyloid beta has often been implicated in neuronal dysfunction and neuronal loss during the development of Alzheimer's disease (Hensley, 1994). In this study, we first investigated whether a human serum albumin fusion protein (AL04, CysC-HSA-dTAT) could protect PC12 cells from Aβ1-42-induced toxicity. Our results showed that 10 μM Aβ1-42 significantly reduced cell viability.

[0050] In a second series of experiments, we subjected PC12 cells to NGF and, after differentiation, treated them with Aβ1-42 for 72 hours in the presence or absence of AL04. As observed in undifferentiated cells, AL04 exerted a significant protective effect in the presence of Aβ1-42. However, the percentage of protection was lower than that in undifferentiated cells because differentiated PC12 cells were less sensitive to Aβ1-42.

[0051] From clinical use to preclinical trials, several drugs used to treat Alzheimer's disease are known to utilize transport systems. Donepezil, one of only two groups of drugs approved for the treatment of Alzheimer's disease, and possibly other cholinesterase inhibitors, are transported across the BBB by organic cation transporters, most likely choline transporters (Kang, 2005; Kim, 2010). As proof of concept, whether BBB penetration is a critical factor for therapeutic biomolecules for the treatment of Alzheimer's disease, we report here for the first time an HSA-based therapeutic biomolecule containing tandem repeats of the HIV-1 trans-acting transcriptase (dTAT) peptide and its efficacy in an in vitro human BBB model. We discovered that dTAT plays a role in facilitating the delivery of high-molecular-weight proteins across the BBB.

[0052] The advantages of our HSA fusion platform can be enumerated as follows: (1) HSA confers pH-dependent FcRn recycling / transcytosis to the delivery shuttle, resulting in significantly extended circulation residence time in vivo (Sand, 2015); (2) the mechanism by which dTAT enters the central nervous system is primarily through transient disruption of the BBB by reducing the expression of tight junction proteins and altering their distribution or the interaction between dTAT and heparan sulfate proteoglycans on the surface of endothelial cells (Andras, 2005; Xu, 2012; Zhong, 2012; Toschi, 2001); (3) the model protein core in the construct can be replaced by any kind of active protein, including cystatin C (CysC), low-density lipoprotein receptor-related protein 1 cluster IV (LRP1-C4), and phosphatase and tensin analogue of chromosome 10 (phosphatase and tensin analogue). These include the soluble receptor for advanced glycation end products (sRAGE), or myelin basic protein (MBP), which confer additional therapeutic benefits to delivery systems for Alzheimer's disease and other central nervous system disorders (Sundelof, 2008; van Kasteren, 2011; Basurto-Islas, 2013; Tizon, 2010; Sagare, 2013; Zhao, 2016; Zhang, 2006; Zong, 2010; Liao, 2009).

[0053] In summary, we demonstrate for the first time that human serum albumin fusion proteins bearing dTAT, such as AL04, exert inhibitory effects against the deleterious effects of Aβ1-42 treatment in PC12 cells. We also demonstrate that dTAT in AL04 promotes the delivery of high-molecular-weight proteins across the human BBB in an in vitro model, and that this model is a useful proof-of-concept tool as an alternative to in vivo models. These results suggest that human serum albumin fusion proteins can significantly improve neuroprotection and blood-brain barrier permeability, thus providing a useful platform for drug development for the treatment of Alzheimer's disease.

[0054] AL04 has the following nucleic acid sequence (2211 nucleotides) and amino acid sequence (737 amino acid residues). tccagccctggcaagccccctcgcctggtgggcggccccatggacgccagcgtgggaggagggggcgtga ggcgggctctggacttgccgtgggcgagtacaacaaggcctccaatgatatgtatcactctagggctct gcaggtggtgagagcccgcaagcagatcgtgggctggcgtgaactacttcctggatgtggagctgggcagg accacatgcaccaagacacagccaaacctggacaattgtccttttcacgatcagccacatctgaagcgga aggccttctgctcttttcagatctatgctgtgccctggcagggcaccatgacactgtctaagtccacctg tcaggacgctggcggctccgctagcgatgctcacaagtctgaggtggcccataggttcaaggacctgggc gaggagaactttaaggccctggtgctgatcgctttcgcccagtacctgcagcagtgcccttttgaggacc acgtgaagctggtgaacgaggtgaccgagttcgctaagacatgcgtggctgacgagagcgccgagaattg tgataagtctctgcataccctgtttggcgataagctgtgcaccgtggccacactgagagagacatatggc gagatggctgactgctgtgccaagcaggagccagagcgcaacgagtgcttcctgcaggacaaggacgata accccaatctgcctagactggtgcgcccagaggtggacgtgatgtgcaccgctttccacgataatgagga gacatttctgaagaagtacctgtatgagatcgccaggcggcatccttacttttatgctccagagctgctg ttctttgccaagagatacaaggccgctttcaccgagtgctgtcaggccgctgataaggccgcttgcctgc tgcccaagctggacgagctgagagatgagggcaaggcttccagcgccaagcagcgcctgaagtgtgcttc cctgcagaagttcggcgagagagcctttaaggcttgggctgtggctaggctgagccagcggttccctaag gctgagtttgccgaggtgtctaagctggtgaccgacctgacaaaggtgcacaccgagtgctgtcatggcg acctgctggagtgcgccgacgatagggctgatctggccaagtacatctgtgagaaccaggactctatctc ttccaagctgaaggagtgctgtgagaagccactgctggagaagtcccattgcatcgctgaggtggagaac gacgagatgccagctgatctgccctccctggccgctgactttgtggagagcaaggacgtgtgcaagaatt acgccgaggctaaggacgtgttcctgggcatgtttctgtacgagtatgctagacgccaccctgactacag cgtggtgctgctgctgagactggccaagacctatgagaccacactggagaagtgctgtgccgctgccgat ccacatgagtgctatgctaaggtgttcgacgagtttaagcccctggtggaggagcctcagaacctgatca agcagaattgtgagctgtttgagcagctgggcgagtacaagttccagaacgccctgctggtgcgctatac aaagaaggtgccacaggtgtctacccccacactggtggaggtgtccaggaatctgggcaaggtcggcagc aagtgctgtaagcaccctgaggctaagcggatgccatgcgccgaggattacctgtccgtggtgctgaatc agctgtgcgtgctgcatgagaagaccccagtgagcgacagggtgaccaagtgctgtacagagtctctggt gaacaggcggccctgcttttccgctctggaggtggatgagacatatgtgcctaaggagttcaatgctgag accttcacatttcacgccgacatctgtaccctgagcgagaaggagcggcagatcaagaagcagacagccc tggtggagctggtgaagcataagcccaaggctaccaaggagcagctgaaggccgtgatggacgatttcgc tgcctttgtggagaagtgctgtaaggctgacgataaggagacatgctttgccgaggagggcaagaagctg gtggctgcctctcaggctgccctgggactgggcttcctgggatacgctaggaaggctgctaggcaggccc gggcttatgctaggaaggctgctagacaggctcgcgccggc (SEQ ID NO: 5) Amino acid sequence of AL04 (737 amino acids) SSPGKPPRLVGGPMDASVEEEGVRRLDFAVGEYNKASNDMYHSRLQVVRRKQIVAGV NYFLDVELGRTTCTKTQPNLDNCPFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA GGSASDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVA DESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRL VRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAFTECCQADKA ACLLPKLDELRDEGKASSAKQRLKCASLQKFGERFKAWAVARLSQRFPKAEFAEVSKLV TDLTKVHTECCHGDLLECADDRDLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN DEMPADLPSLADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHPDYSVVLLLRLAKTYET TLEKCCWDHECYAKVFDEFKPLVEEPQNL IKQNCELFEQLGEYKFQNALLVRYTKKV PQVSTPTLVEVSRNLGKVGSKCCKHPEAKPJVIPCAEDYLSVVLNQLCVLHEKTPVSDRVTK CCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKH KPKATKEQLKAVMDDFAFVEKCCKADDKEICFAEEGKKLVASQALGLGFLGYARKA RQARYARKARQARG (SEQ ID NO: 6)

[0055] In this study, we investigated the therapeutic potential of AL04, which not only ameliorates Aβ1-42-induced toxicity but also reduces phosphorylated tau levels in PC12 cells. The PC12 cell line has been used as a common in vitro model to evaluate neuronal damage and neurotoxicity in Alzheimer's disease. Furthermore, PC12 cells offer high throughput and retain a mature neuronal phenotype (Tan, 1999). The biochemistry and morphology of nerve growth factor (NGF)-induced PC12 cells resemble neurons, making them particularly sensitive to Aβ peptides and NGF-deficient conditions. Furthermore, several reports suggest that Aβ1-42 not only induces cytotoxicity and cell death in PC12 cells, but also causes excessive ROS production and mitochondrial dysfunction (Hensley, 1994). Therefore, the PC12 cells used in our experiments provide a reasonable approach to determine whether AL04 provides protection against Aβ-induced cytotoxicity and NGF depletion.

[0056] HSA is one of the most abundant proteins circulating in the blood. Its long half-life in the bloodstream, lack of toxicity, and easy cellular uptake make it a popular carrier protein for therapeutic drugs (Chaudhury, 2003; Andersen, 2014). We selected over 10 proteins of interest (POIs), including cystatin C (CysC), low-density lipoprotein receptor-related protein 1 cluster IV (LRP1-C4), phosphatase and tensin homolog on chromosome 10 (Pten), soluble form of receptor for advanced glycation end products (sRAGE), and myelin basic protein (MBP), which may play important roles in either Aβ clearance or p-tau reduction by preventing fibril formation and promoting their clearance. To develop an HSA fusion protein-producing cell line, we constructed the expression vector pOptivec (Invitrogen) containing the HSA fusion protein (AL04, CysC-HSA-dTAT) and the selectable marker dihydrofolate reductase (DHFR). We generated pools of stably transduced DHFR-deficient cells (DG44 cells) and cells expanded with methotrexate (MTX; a DHFR inhibitor). We tested the proof-of-concept using purified AL04 protein. AL04 reduced Aβ-induced cell death, decreased NGF deprivation-induced tau hyperphosphorylation, and improved endogenous tau-tubulin interactions in pheochromocytoma (PC12) cells. We utilized an in vitro human BBB model of the cell line to evaluate the permeability of the high-molecular-weight protein AL04 (80 kDa) through the blood-brain barrier. This BBB-crossing human serum albumin fusion protein platform is useful for drug development for the treatment of Alzheimer's disease.

[0057] The therapeutic effect of HSA fusion proteins depends on their successful delivery into cells. We investigated whether human serum albumin fusion proteins (AL04, CysC-HSA-CPP; AL12, RAGE(V)-HSA-SIRT1 (exon 4)-CPP) can penetrate differentiated PC12 cells. We demonstrated that different versions of the HSA fusion proteins (AL04 or AL12) readily penetrated PC12 cells and effectively delivered their biological activity into the cells.

[0058] In this study, we used differentiated PC12 cells to investigate the role of a human serum albumin-fused therapeutic agent (AL04) in NGF deprivation-induced tau hyperphosphorylation under NGF deprivation. In undifferentiated PC12 cells, tau is expressed at low levels, while stimulation with NGF resulted in increased tau expression beginning after 3 days of treatment (Drubin, 1985; Hanemaaijer, 1991). Tau is known to be required for microtubule stabilization in the late stages of NGF-induced axon outgrowth (Brandt, 1995; Hanemaaijer, 1991). It has been reported that the amount of tau phosphorylated at Thr205 and Thr231 (p-tau) is increased in brains and mouse models of Alzheimer's disease (Wang, 2013). We investigated whether AL04 treatment reduced NGF deprivation-induced tau hyperphosphorylation in PC12 cells. We observed that the levels of p-tau Ser202 / Thr205 and p-tau Thr231 were significantly increased in NGF-depleted PC12 cells (data not shown). In the presence of AL04, we observed a dramatic decrease in the levels of tau phosphorylated at Ser202, Thr205, and Thr231. It has been reported that PP2A activity is inhibited by the interaction of the activated I2PP2A fragment with the catalytic subunit of PP2A (Arnaud, 2011). Because AL04 treatment reduced the levels of the approximately 20 kDa I2PP2A fragment, as well as p-tau Ser202 / Thr205 and p-tau Thr231, these results suggest that AL04 (containing cystatin C) affects the regulation of PP2A activity, which is related to tau dephosphorylation, through downregulation of I2PP2A.

[0059] The mutation sites for the tau protein described herein are those in SEQ ID NO: 7 below.

[0060] Human tau: microtubule-associated tau protein isoform 2 (NCBI reference sequence: NP_005901.2), 441 amino acids MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQT PTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQAN ATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVV RTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQS KCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFK DRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPR HLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:7)

[0061] Several studies have provided evidence that phosphorylation of key sites on tau strongly influences its normal function and may contribute to its pathological role (Ksiezak-Reding, 1992; Augustinack, 2002). It has also been shown that phosphorylation of tau at Thr-231 reduces its affinity for microtubules (Cho, 2004; Sengupta, 1998; Lin, 2007). We have also observed that AL04 treatment stabilizes microtubule-tau interactions by reducing phosphorylated tau levels. Although our studies do not reveal the signaling pathway responsible for the reduction in tau phosphorylation associated with AL04 treatment, treatment with AL04 inhibits tau aggregation and tangle formation in vivo, apparently mediated by inhibition of AMPK activity. In a recent report, we observed a decrease in phosphorylated ULK1 (Unc51-like kinase 1, Ser555) following AL04 treatment, suggesting that AL04 may regulate autophagy through the AMPK signaling pathway. AMPK has been reported to directly mediate autophagy activation by phosphorylating autophagy-initiating protein kinases and phosphorylate its direct substrates, including ULK1 at S555 (Egan, 2011; Kim, 2011).

[0062] In conclusion, our findings indicate that AL04 has a significant protective effect against the reduction of phosphorylated tau under NGF-deficient conditions. These in vitro studies suggest that the protective effect of AL04 may be mediated by regulating tau kinase activity (AMPK), PP2A activity, and stabilizing tau-tubulin interactions. Because tau kinase and PP2A play important roles in NFT formation in Alzheimer's disease, these data suggest that AL04 may alter tau phosphorylation, thereby potentially affecting NFT accumulation in Alzheimer's disease brains.

[0063] Described herein are methods for delivering an effective dose of a therapeutic polypeptide across the BBB to the central nervous system by systemically administering a therapeutically effective amount of a fusion polypeptide, as described herein. The systemic dose suitable for delivery of a fusion polypeptide is based on its central nervous system uptake characteristics and its specific activity, as described herein. Systemic administration of a fusion polypeptide to a subject suffering from a deficiency of a therapeutic protein is an effective approach to non-invasive delivery of a therapeutic protein to the central nervous system.

[0064] The amount of fusion polypeptide that is a therapeutically effective systemic dose of the fusion polypeptide will depend, in part, on the central nervous system uptake characteristics of the administered fusion polypeptide (e.g., the percentage of a systemically administered dose that is taken up in the central nervous system), as described herein.

[0065] In some embodiments, 1% (i.e., about 0.3%, 0.4%, 0.48%, 0.6%, 0.74%, 0.8%, 0.9%, 1.05%, 1.1%, 1.2, 1.3%, 1.5%, 2%, 2.5%, 3%, or any percentage in the range of about 0.3% to about 3%) of a systemically administered fusion polypeptide is delivered to the brain as a result of uptake from the peripheral blood across the BBB. In some embodiments, at least 0.5% of the systemically administered fusion polypeptide dose (i.e., about 0.3%, 0.4%, 0.48%, 0.6%, 0.74%, 0.8%, 0.9%, 1.05, 1.1, 1.2, 1.3%, 1.5%, 2%, 2.5%, 3%, or any percentage in the range of about 0.3% to about 3%) is delivered to the brain within 2 hours or less after systemic administration, i.e., 1.8, 1.7, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.6, 0.5, or other time period between about 0.5 and about 2 hours.

[0066] Thus, in some embodiments, the invention provides methods for systemically administering a therapeutically effective amount of a fusion polypeptide such that the amount of fusion polypeptide that crosses the BBB is at least 3 ng of therapeutic protein per mg of protein in the subject's brain (e.g., 3, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, or any other value (ng) of therapeutic protein between 3 and 50 ng per mg of protein in the subject's brain).

[0067] In one aspect, when 100 μg of AL04 is administered to a mouse, assuming a BBB penetration rate of 0.01%, 10 ng of AL04 can be delivered to 40 mg of mouse brain protein, where the mouse brain weighs 400 mg and its total protein content is 40 mg, which can be extrapolated to humans.

[0068] In some embodiments, a therapeutically effective systemic dose comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 units per brain, or any other systemic dose value between about 50 and 2500 units of therapeutic protein activity per brain.

[0069] In some other embodiments, a therapeutically effective systemic dose comprises at least about 10 units of therapeutic protein activity per Kg of body weight, at least about 10, 12, 15, 18, 25, 30, 50, 75, 100, 150, 200, 250 units, or any other number of units.

[0070] Those skilled in the art will appreciate that the total therapeutically effective systemic dose of a fusion polypeptide will depend, in part, on its specific activity. In some embodiments, the specific activity of the fusion polypeptide is at least 10 U, at least about 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50 U, or any other specific activity value between about 10 and about 50 U per mg of protein.

[0071] Thus, taking into account the specific activity of the fusion polypeptide and the body weight of the subject to be treated, the systemic dose of the fusion polypeptide can be at least 5 mg, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, or any other value between about 5 mg and about 125 mg of fusion polypeptide.

[0072] As used herein, the terms "systemic administration" or "peripheral administration" include any method of administration that does not involve direct administration into the central nervous system (i.e., does not involve physical penetration or disruption of the BBB). "Systemic administration" includes, but is not limited to, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, intranasal, buccal, transdermal, rectal, transalveolar (inhalation), or oral administration. As described herein, any suitable fusion polypeptide can be utilized.

[0073] The fusion polypeptide may be administered as part of a combination therapy. Combination therapy includes administering the composition of the present invention in combination with other therapies to treat or alleviate symptoms typically seen in patients with Alzheimer's disease. When the composition of the present invention is used in combination with other methods or compositions for central nervous system diseases, any combination of the composition of the present invention and the additional methods or compositions may be used. Thus, for example, if the composition of the present invention is used in combination with other therapeutic agents for central nervous system diseases, both may be administered simultaneously, sequentially, overlapping in time, at similar frequencies, at the same frequency, or at different frequencies. In some cases, a composition comprising a combination of one or more other therapeutic agents for central nervous system diseases and the composition of the present invention is used.

[0074] In some embodiments, the composition (eg, fusion polypeptide) is co-administered to a patient with other medications, either in the same formulation or as separate compositions.

[0075] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. The following examples are offered for purposes of illustrating, but not limiting, the present invention.

[0076] Example Example 1: Construction of pOptivector expressing a human serum albumin (HSA) fusion protein The plasmid pOptiVec (Invitrogen, USA) was used as an expression vector. Codon-optimized synthetic gene cDNA sequences were ordered from Genescript; these sequences were: human serum albumin (NM_000477), target protein: MOA1 or MOA2; cystatin C (NM_000099.3); Pten-long (NM_001304718); receptor for advanced glycation end products (RAGE) (NM_001136.4); human transcription factor EB (TFEB) (NM_007162.2); SIR1 (human NAD-dependent protein deacetylase sirtuin-1 (SIRT1) (NM_012238.5); and dTAT (tandem repeat TAT, TAT HV1H2 from UniProt-P04608) as the CPP. The plasmid was digested with restriction enzymes XbaI, NheI, and NotI (Thermo, USA). DNA fragments were separated by electrophoresis on a 0.8% agarose gel. We used a Thermo gel extraction kit (Thermo, USA) to elute the DNA fragments from the gel. The resulting fragments were then ligated together with T4 DNA ligase (NEB, USA) to create the expression vector pOptiVec-AL000.

[0077] We used the Plasmid Midi kit (Thermo, USA) for transfection of highly purified isolated plasmid DNA. Correct construction of the expression vector was confirmed by restriction enzyme analysis. The nucleotide sequences of both the gene and its flanking regions were confirmed by sequencing (Genewiz, USA).

[0078] Example 2: Cultivation of the CHO-DG44 cell line Cell culture was performed in 125 mL Erlenmeyer flasks in a CO2 incubator operating at 125 rpm in an 8% CO2 atmosphere at a temperature of 37°C and 95% humidity. Reseeding was performed at 0.3-0.5 x 10 6 The cells were cultured at a density of 1000 cells / mL every 3–4 days. We used serum-free medium CD-DG44 (Life Technologies, USA) supplemented with 8 mM L-glutamine. Cell counts and cell viability analyses were performed after trypan blue staining using an automated cell counter, Cellometer AutoT4 (Nexcelom Bioscience, USA).

[0079] Example 3: Development of stable cell lines Prior to transduction, the expression vector plasmid was linearized using the restriction enzyme FspI (Thermo, USA). Dihydrofolate reductase (DHFR)-deficient CHO-DG44 cell line was transduced with linearized pOptiVec-AL000 according to the FreeStyle MAX reagent protocol (Invitrogen, USA). 72 hours after transduction, transduced cells were selected for growth in the absence of hypoxanthine and thymidine (HT) using CD OptiCHO® (Life technologies, USA) medium containing 8 mM L-glutamine (complete selection medium). The selection medium was maintained at 0.5x10 cells / mL until the viability of selected cells was greater than 90%. 6The medium was replaced every 3–4 days at a density of 1000 cells / mL, followed by two or three rounds of genome amplification in complete medium containing increasing concentrations of methotrexate (MTX) from 250 nM to 2 μM. HSA fusion protein production was examined at the end of culture in the selection / amplification medium using a human albumin quantification ELISA kit (Bethyl Laboratory, USA) and SDS-PAGE.

[0080] Example 4: Shaking flask culture of AL04 0.5x10 cells amplified with 2000nM MTX were cultured at 1000μg / ml. 6 The cells were cultured at a viable cell density of 1000 cells / ml in 150 ml of CD OptiCHO® medium (Life Technologies, USA) containing 8 mM L-glutamine in a 500 ml disposable Erlenmeyer flask until their viability reached >90%. Cell density and viability were measured every other day using the trypan blue dye exclusion method with an automated cell counter, Cellometer AutoT4 (Nexcelom Bioscience, USA). On day 10, or when cell viability fell below 90%, the culture was harvested for protein purification.

[0081] Example 5: Purification of AL04 Culture medium containing human serum albumin fusion protein secreted from CHO-DG44 cells was filtered (0.2 μm filter) and then purified by Blue dye affinity chromatography followed by ion-exchange column chromatography. Briefly, the filtered fusion protein-containing medium was loaded onto a Blue HP column (GE) equilibrated with 0.05 M Tris buffer (pH 8.0). The column was washed with equilibration buffer to remove unbound protein, and the bound fusion protein was eluted with 10 column volumes of elution buffer (0.05 M Tris, pH 8.0 + 1.0 M NaCl). The eluted fraction was desalted using a centrifugal desalting column (Pierce) with a 40 kDa molecular weight cutoff. Further purification was performed using ion-exchange column chromatography (HiTrap QFF (GE)). Fractions containing the fusion protein were collected and dialyzed against PBS according to the manufacturer's instructions. The purified protein was analyzed by SDS-PAGE and was found to be >90% pure. The human serum albumin fusion protein was then sterilized by filtration (0.2 μm) and stored at −80° C. Protein concentration was determined by the Bradford method (Thermo).

[0082] Example 6: ELISA quantification of human albumin A 96-well ELISA plate (Bethyl Laboratory, USA) was coated overnight at 4°C with purified goat anti-human albumin coating antibody (Bethyl Laboratory, USA) diluted 1:100 in sodium bicarbonate coating buffer (pH 9.0). Blocking was performed with 1% bovine serum albumin prepared in TBS buffer for 1 hour. After incubation, the plate was washed five times with Tris-buffered saline containing Tween-20 (TBST). Samples and controls were diluted with sample buffer (TBS buffer containing 1% BSA), and 100 μL of each sample was added directly to the coated wells. A calibration curve was prepared at 6.5, 12.5, 25, 50, 100, 200, and 400 ng / mL using reference human serum (supplied by Bethyl Laboratory) diluted in sample buffer. The microplate was incubated at room temperature for 1 hour with 100 μL of diluted samples and standards. The plate was then washed five times with TBST buffer and incubated with 100 μL of horseradish peroxidase-conjugated goat anti-human albumin detection antibody (Bethyl) diluted 1:100,000 in sample buffer. The microplate was incubated for 1 hour. After washing the plate five times, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Bethyl) was added, and the plate was incubated for 15 minutes at room temperature in the dark. The reaction was then stopped with ELISA stop solution (Bethyl). Finally, the absorbance was measured at 450 nm using a plate reader (BioTEK). Each measurement was performed in triplicate, and the AL04 (CysC-HSA-dTAT) protein concentration was interpolated from the linear portion of the standard curve.

[0083] Example 7: Preparation for LC-MS / MS analysis Eight micrograms of purified AL04 (CysC-HSA-dTAT) protein was loaded onto a 4%-12% gradient gel (Life Technologies). After staining the gel with Coomassie Brilliant Blue G-250 (Thermo) and rinsing with water, the AL04 (CysC-HSA-dTAT) protein band was excised from the gel for protein identification and analysis by LC-MS / MS (BioSyn). To prepare samples for N-terminal sequencing, after electrophoresis, proteins were transferred to a 0.22 μm PVDF membrane (Bio-Rad) at 100 V for 90 minutes in NuPAGE transfer buffer containing 10% methanol. The transferred PVDF membrane was washed three times with DD₂O, stained with Ponceau S (Bio-Rad), and then destained in DD₂O. The stained AL04 (CysC-HSA-dTAT) protein band was excised from the membrane for N-terminal sequencing (BioSyn, USA).

[0084] Example 8: Toxicity assay for Aβ1-42 in PC12 cells Aβ (AnaSpec, USA) was dissolved in DPBS containing 0.1% NH4OH to a 1 mM stock solution, and a portion was stored at -80°C and pre-incubated at 37°C for 3 days (aging treatment) for peptide aggregation.

[0085] The rat pheochromocytoma cell line (PC12) is a classic in vitro neuroendocrine cell model. Unlike primary neurons, undifferentiated PC12 cells do not require nerve growth factor (NGF) for survival, but they respond to it by extending very long axons and by undergoing other neurospecific changes, such as increased expression of cholinergic receptors (Jumblatt, 1982; Amy, 1983). NGF-treated PC12 cells exhibit many characteristics of differentiated neurons. PC12 cells were cultured in DMEM medium (Gibco) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS), 5% (v / v) heat-inactivated horse serum (HS), and 1% (v / v) penicillin and streptomycin, and then incubated at 37°C in a humidified 5% CO2 atmosphere. Cells were either harvested for various experiments or passaged when they reached 80% plate surface coverage (confluence). Prior to the experiment, PC12 cells were seeded at 15,000 cells per well in 100 μl of poly-D-lysine-coated 96-well plates to allow cells to adhere to the wells. For experiments with undifferentiated cells, 96-well plates were used, and drug treatment was initiated immediately 24 h after cell seeding. NGF-stimulated (neuronally differentiated) PC12 cells were grown in DMEM medium containing 1% horse serum and 100 ng / ml NGF (Sigma, USA) for at least 6 days. Aβ1-42-mediated toxic effects were examined using a WST-8 reduction assay (Dojindo Molecular Technologies, USA). Briefly, cells were treated at 37°C for an additional 3 days in the absence or presence of AL04 and / or Aβ1-42 (10 μM) at different AL04 concentrations (0, 0.01, 0.1, 1, and 10 μM). After this incubation period, 10 μl of WST-8 stock solution was added, and the incubation was continued for another 3–4 h. The A450 of WST-8-formazan produced by dehydrogenase activity in viable cells was measured using a microplate reader (BioTEK, USA) at a reference wavelength of 630 nm.

[0086] Example 9: BBB permeability assay of AL04 An in vitro human BBB model (Neuromics, USA) was established using a coculture of primary human brain endothelial cells (HBECs), human brain pericytes (HBPCs), and human brain astrocytes (HBACs). The in vitro human BBB model kit contains 12 transwell inserts (polyester membrane, 0.4 μm pores, 12 mm diameter, growth area of ​​insert: 1.12 cm) with two sides (luminal, blood side / abluminal, brain side). 2 HBPCs were cultured on the bottom side of the insert, HBECs were grown as a monolayer on the top side of the insert, and HBACs were cultured on the bottom of a 12-well culture plate (Neuromics, USA). The in vitro BBB model was activated for 4 days according to the manufacturer's instructions. Briefly, the medium from both the luminal and abluminal (bottom, brain side) sides of the transwell insert was replaced every other day. Before transport experiments, the abluminal side was filled with permeability assay medium. For the permeability assay, purified AL04 or recombinant HSA (Sigma, USA) was added to the luminal side of the transwell insert (0.3 ml) to a final concentration of 1 or 10 μM. Cultures were performed at 37°C using an orbital shaker (100 rpm). Samples (150 μl) were taken from the abluminal side (1.2 ml) at 60, 120, and 240 min and immediately replaced with fresh permeability assay medium. The concentrations of transported AL04 or rHSA were measured using a human albumin quantification ELISA kit (Bethyl laboratory, USA) and analyzed using a calibration curve method. The permeability coefficient (Pe, cm s) was calculated. -1 ) was calculated using the formula: Pe = (VA / (Axc0)) x (dQ / dt), where VA is the volume of assay buffer on the blood side (inner surface of the insert) and A is the surface volume of the insert (1.12 cm 2 ), c0 is the initial concentration of the protein sample added to the blood side, and dQ / dt is the concentration of the protein sample transported to the brain side at a specific time. The permeability coefficient (Pe, cm s) of purified AL04 or recombinant HSA -1 ) was calculated as described elsewhere ( Prades, 2015 ; Nakagawa, 2009 ).

[0087] Example 10: Results Example 10.1: Gene construct and vector design for recombinant HSA fusion protein (AL04) In the first step, genes encoding human serum albumin, a protein of interest (e.g., cystatin C, Pten-long, as MOA1 or MOA2), and dTAT were synthesized with optimized codon compositions. Gene sequences were obtained from publicly available sources (NCBI, National Center for Biotechnology Information, and UniProt, Universal Protein Resource). The pOptiVec plasmid vector was used as a carrier for the human serum albumin, protein of interest (e.g., cystatin C, Pten-long, as MOA1 or MOA2), and dTAT genes (Figures 1-4).

[0088] Example 10.2: Production of HSA fusion protein (AL04) After suspension, CHO-DG44 cells were transduced with the linearized expression vector, Optivec-CysC-HSA-dTAT, and detected in the culture supernatant using a human albumin quantification ELISA kit (Bethyl). The transduced CHO cells were then selected and MTX-amplified in CD Opti CHO medium containing 8 mM L-glutamine. To achieve high-concentration production of recombinant proteins, we used a methotrexate (MTX) amplification system, a mammalian cell expression system widely used in the biopharmaceutical industry (Ng, 2012). During the MTX-amplified process, the expression of AL04 (Optivec-CysC-HSA-dTAT) increased in the culture supernatant as human albumin, and quantified by ELISA. The cell pool was then adapted to AL04 expression culture at an MTX concentration of 2000 nM.

[0089] Example 10.3: Characterization of purified AL04 protein The identity of the AL04 protein was confirmed by SDS-PAGE analysis (Figure 5). The size (full-length) of the purified AL04 protein band was shown to be approximately 80 kDa under reducing conditions in the eluate from blue column chromatography. To confirm the authenticity of the AL04 protein purified from the blue column, the protein was subjected to LC-MS / MS analysis (Biosyn). At least 69 unique peptides corresponding to AL04 were detected in 92% of the protein coverage. To confirm the N-terminus of the AL04 protein, a unique peptide, SSPGKPPRLV, was observed in the sample (data not shown). The analysis also confirmed that the signal peptide was correctly cleaved from the secreted mature protein. Based on LC-MS / MS analysis and N-terminal sequencing, we confirmed that the AL04 protein purified from the blue column was identical to the expressed sequence.

[0090] Example 10.4: Protective effect of AL04 (CysC-HSA-dTAT) against Aβ1-42-induced cytotoxicity We investigated the potential nonspecific cytotoxicity of AL04 in PC12 cell cultures. No changes in cell viability were observed when AL04 was incubated alone at 0.01 μM, 0.1 μM, and 1 μM, but concentrations as high as 10 μM AL04 reduced cell viability (Figure 6A). In vitro studies have revealed that cystatin C binds to the central domain of Aβ, which is structurally important, inhibiting Aβ aggregate formation and preventing Aβ-induced toxicity (Juszczyk, 2009; Tizon, 2010). We investigated whether AL04 containing cystatin C inhibits Aβ-induced toxicity. Treatment of PC12 cells with 10 μM Aβ1-42 alone resulted in a significant decrease in cell viability, down to 20% at 10 μM soluble Aβ1-42 compared to control cells (without Aβ1-42). However, in the presence of AL04, Aβ1-42-induced cell death was rescued (Fig. 6B).

[0091] Similar to undifferentiated PC12 cells, nerve growth factor (NGF)-treated (differentiated) PC12 cells showed approximately a 40% decrease in cell viability when they were exposed to either soluble or aggregated Aβ1-42 treatment (10 μM) for 3 days. Apparently, 0.01 μM AL04 alone did not cause any adverse effects in differentiated PC12 cells. Taken together, these data indicate that AL04 can protect cells against Aβ1-42-induced cytotoxicity (Figure 7). It is clear that AL04 effectively prevents Aβ aggregation, plaque formation, and neurotoxicity.

[0092] Example 10.5: Evaluation of BBB permeability of AL04 In this study, we utilized a commercially available in vitro human BBB model purchased from Neuromics. We performed a BBB permeability assay using concentrations ranging from 1 to 10 μM. Following this assay, we measured the AL04 concentration from the brain side using a human albumin quantitative ELISA and estimated the permeability coefficient (Pe) calculated using the formula described in Example 9. To confirm the fusion of the TAT peptide in the AL04 delivery vehicle (CysC-HSA-dTAT), we compared the permeability of AL04 and TAT-free recombinant human serum albumin (rHSA) through the BBB model. Several mechanisms by which TAT protein disrupts the BBB have been proposed, including the reduction of tight junction protein expression, induction of vascular permeability in the endothelium, and relocalization (Andras, 2005; Xu, 2012; Zhong, 2012; Toschi, 2001).

[0093] As shown in Figure 8A, the dose-dependent results revealed that AL04 was transported across the model BBB. AL04 containing dTAT tended to be transported to the brain side through the BBB model with higher permeability than rHSA. We also evaluated the effect of assay time on the permeability of 1 μM and 10 μM AL04 through the BBB model. We compared the permeability at three different assay times (60, 120, and 240 minutes). Figure 8B showed that the Pe gradually increased up to 120 minutes, but the Pe of 10 μM AL04 tended to slightly decrease at 240 minutes.

[0094] Example 11: Design for a Dual-Acting Therapeutic Platform (Human Serum Albumin Fusion Protein) Example 11.1: Materials and Methods In this study, we selected a cystatin C sequence to test the HSA fusion protein-CPP platform version 1. To avoid the influence of the large HSA structure on the HSA fusion protein platform version 1, a linker (GGSAS) was inserted between cystatin C and HSA. A cleavable linker (GFLG) was inserted between HSA and CPP, which may facilitate the release of CPP from the permeation of the HSA fusion protein (Figure 1). Four different protein sequences were selected for the HSA fusion protein platform version 2 (MOA1-HSA-MOA2-CPP: AL06, AL07, AL08, AL09, and AL10). The V domain of the receptor for advanced glycation end products (RAGE-V, RAGE-V & C1 domain for AL10) was used as mechanism of action 1 (MOA1) to reduce Abeta in version 2. Three different proteins were used as mechanism of action 2 (MOA2) for loading to reduce phosphorylated tau levels in the version 2 platform: Pten-Long (for AL06), PDZ domain-deleted Pten-Long (for AL09 and AL10); TFEB (transcription factor EB, a master regulator of autophagy and lysosomal degradation) for AL07; and SIRT1 (silent mating signaling regulator 2 homolog 1, which deacetylates tau tangles and marks them for proteasomal degradation) for AL08. In version 2, various linkers were used: a GS linker (GGGSGGGS) was inserted between MOA1 and HSA; a GS linker / cleavable linker (GFLGGGGSAS) was inserted between HSA and MOA2; and a cleavable linker (GFLG) was inserted between MOA2 and CPP (Figure 3). In the HSA fusion protein platform version 2.1 (MOA1-HSA-MOA2-CPP:AL12), the RAGE-V domain was used as mechanism of action 1 (MOA1) to reduce Abeta, and 52 amino acids (exon 4 of SIRT1, part of the catalytic domain of SIRT1) were used as MOA2 to reduce phosphorylated tau. In version 2.1, a GS linker (GGGSGGGS) was inserted between MOA1 and HSA; between MOA2 and CPP; and a GS linker / cleavable linker (GFLGGGGSAS) was inserted between HSA and MOA2.We used various linkers to avoid steric hindrance between HSA and other fusion moieties (Figure 4).

[0095] Example 12: Western Blot PC12 cells were differentiated with 100 ng / ml NGF in DMEM medium containing 1% horse serum. After 4 days, NGF-deprived PC12 cells were treated with various concentrations (0–10 μg / ml) of AL04 (or AL07, AL08, or AL12) for 24 hours. Cells were rinsed twice with ice-cold DPBS (pH 7.5), lysed in lysis buffer, and subjected to SDS-PAGE and immunoblotting, as described elsewhere (Bang, 2012). The following antibodies were used: Tau46 (total tau), phosphorylated AMPKa (Thr172), AMPKoc, phosphorylated AMPKp (Ser182), AMPKp, phosphorylated ULK1 (Ser555), β3-tubulin, cystatin C, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were purchased from Cell Signaling Technology. Phosphorylated tau (AT8: Ser202 / Thr205, Thermo), phosphorylated tau (PHF-6: Thr231, Santa Cruz), and anti-human albumin (Bethyl) were used. Supersignal West Pico chemiluminescent reagent (Thermo) was used for signal detection. Quantification of phosphorylated tau and total tau was performed by densitometry using image analysis software (NIH). The phosphorylated tau level at each time point was normalized to the total tau concentration from the same sample, and each normalized value was expressed as a percentage of the maximum value (defined as 100%). Statistical significance was determined by Student's t-test.

[0096] Example 13: Immunoprecipitation (IP) Treated cells were lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 15% glycerol, phosphatase inhibitor cocktail (Santa Cruz), and protease inhibitor cocktail (BioVision). Total protein (1000 μg) was reacted with 5 μg of β3-tubulin antibody as indicated for 16 hours at 4°C and then precipitated with 60 μL of TrueBlot anti-rabbit Ig immunoprecipitation beads (Rockland) for an additional 3 hours. The immunoprecipitated beads were washed five times with lysis buffer, and sample buffer supplemented with SDS was added. Samples were separated by SDS-PAGE and analyzed by immunoblotting as previously described (Bang, 2014). For tau blot immunoprecipitates, a mouse TrueBlot ULTRA anti-mouse Ig horseradish peroxidase-conjugated secondary antibody (Rockland) was used for detection with SuperSignal West Pico chemiluminescence reagent (Thermo). Quantitation of bound and added tau signals was performed by densitometry using image analysis software (NIH). Statistical significance was determined by Student's t-test.

[0097] Example 14: Results Example 14.1: Characterization of purified AL04 (80 kDa), AL07 (135 kDa), AL08 (129 kDa), and AL12 (88 kDa) AL04 (version 1), AL07 and AL08 (version 2), and AL12 (version 2.1) were expressed and purified using blue dye affinity chromatography followed by ion-exchange column chromatography. The purified proteins were concentrated and buffer exchanged into PBS using an ultrafiltration spin column, then quantified using the Bradford protein assay. The purity of AL04 and AL12 was greater than 90% by Coomassie blue staining. The purity of AL07 and AL08 was confirmed by both Coomassie blue staining and Western blot, where the major bands in the Coomassie-stained gels were consistent with those observed from Western blots using a human albumin antibody (Figures 5 and 11).

[0098] Example 14.2: Either AL04 or AL12 penetrates into differentiated PC12 cells in a dose-dependent manner New strategies are constantly being evaluated for intracellular delivery of functional proteins or peptides; however, they still lack comprehensive efficacy and safety for translation into clinical models (Zhang, 2012). Multiple modes of cellular internalization, including receptor-mediated and endocytic pathways, have been shown to play a role (Gradishar, 2005). Therefore, we investigated whether AL04 or AL12 penetrates into cells. Cells were treated with various concentrations (0–5 μg / ml) of either AL04 or AL12 under NGF deprivation for 24 hours. Western blot analysis was used to assess the degree of penetration of the HSA fusion protein into differentiated PC12 cells. As shown in Figure 9, AL04 or AL12 proteins penetrate into differentiated PC12 cells in a dose-dependent manner (Figure 9). This indicates that different versions of HSA fusion proteins (AL04, cystatin C-HSA-CPP; AL12, RAGE(V)-HSA-SIRT1(exon 4)-CPP) could easily penetrate into PC12 cells and efficiently deliver their biological activities into the cells.

[0099] Example 14.3: HSA fusion proteins (AL04, AL07, and AL08) affect the phosphorylation state of tau in NGF-deprived PC12 cells AL07 and AL08 contain RAGE-V, a common factor that reduces Abeta, and TFEB and SIRT1, which reduce phosphorylated tau. Differentiated PC12 cells were treated with 1 μg / ml AL04 or 2 μg / ml AL04, AL07, or AL08 for 24 hours. Cells were lysed, and total cell lysates (20 μg / lane) were separated by SDS-PAGE. Relative levels of tau epitopes were determined by immunoblotting using the following antibodies: tau-46, a measure of total tau; AT8, which recognizes the phosphorylated Ser202 / Thr205 residues on tau protein; and PHF-6, which recognizes the phosphorylated Thr231 residue on tau protein. Administration of AL04, AL07, and AL08 reduced tau phosphorylation at Thr231, but only AL04 resulted in a decrease in p-tau at Ser202 / Thr205. Treatment with AL07 and AL08 failed to reduce tau phosphorylation at Ser202 / Thr205. Total tau levels, as measured by Tau-46 antibody, were unchanged by AL04 treatment, but were slightly increased by AL07 and AL08 treatment. Immunoblot analysis provided evidence that the ratio of phosphokinase at Thr231 to total tau protein was decreased by treatment with AL04, AL07, or AL08 (Figure 12), while a significant decrease in the ratio of phosphokinase at Ser202 / Thr205 to total protein was observed from AL04 treatment.

[0100] Example 14.4: AL04 reduces levels of phosphorylated tau in NGF deprivation by downregulating I2PP2A (an inhibitor of PP2A phosphatase) PP2A activity is reduced in Alzheimer's disease and is thought to be responsible for tau neurofibrillary pathology. I2PP2A is a potent inhibitor of PP2A, a phosphoserine / phosphothreonine phosphatase that accounts for approximately 70% of tau protein phosphatase activity in adult human brain (Liu, 2005). Because cystatin C is an inhibitor of AEP (Alvarez-Fernandez, 1999; van Kasteren, 2011), AL04 (containing cystatin C) may affect the regulation of PP2A activity, which is involved in tau dephosphorylation, through downregulation of I2PP2A. Therefore, we investigated whether AL04 treatment inhibits I2PP2A cleavage (activation). We treated differentiated PC12 cells with 1–10 μg / ml AL04 for 24 hours under NGF deprivation. As shown in Figure 13, AL04 treatment reduced the levels of the approximately 20-kDa activation fragment of I2PP2A. We also found that AL04 treatment significantly reduced the levels of P-tau Ser202 / Thr205 in a dose-dependent manner. Similarly, the levels of P-tau Thr231 were slightly reduced in AL04-treated cells compared with untreated cells. The levels of total tau measured with a Tau-46 antibody were unchanged by AL04 treatment. No changes were observed in GAPDH. These results suggest that AL04 reduces NGF deprivation-induced tau hyperphosphorylation by rescuing PP2A activity through downregulation of I2PP2A.

[0101] Example 14.5: Effect of decreasing tau phosphorylation on tubulin-tau interactions Tau plays an important role in regulating microtubule dynamics during neural development. It has been shown that phosphorylation of key sites on tau significantly impacts its normal function and may contribute to its pathological role (Cho, 2004; Sengupta, 1998; Lin, 2007). We next investigated whether NGF deprivation-induced tau hyperphosphorylation is necessary for altered tubulin binding by testing in the presence or absence of AL04. We found that AL04 treatment increased tau-tubulin binding levels by twofold compared with those obtained without treatment (Figure 14), indicating that AL04 restores microtubule-tau interactions reduced by NGF deprivation through reducing phosphorylated tau levels.

[0102] Example 14.6: AL04 reduces hyperphosphorylated tau by modulating tau kinase (AMPK) in a dose-dependent manner Because AL04 treatment reduces phosphorylated tau, we investigated whether AL04 treatment also alters the activation of multiple kinases potentially involved in tau phosphorylation. Activation of endogenous AMPK in Alzheimer's disease brains and primary mouse neurons leads to increased tau phosphorylation at multiple sites, and AMPK inhibition rapidly reduces tau phosphorylation (Vingtdeux, 2011; Domise, 2016). Therefore, we investigated the possible involvement of the AMPK signaling pathway in the reduction of hyperphosphorylated tau induced by NGF deprivation due to AL04. Because AMPK activation requires phosphorylation of Thll72 in the activation loop of the α1 and α2 subunits, AMPK activity was examined by Western blot to detect the expression level of phosphorylated AMPK (Bang, 2012; Bang, 2014). We performed the following experiments. First, we performed Western blotting using antibodies against the AMPK-activating catalytic α subunit, p-AMPK (Thr172), and the regulatory subunit β1. As shown in Figure 15, we observed a dose-dependent decrease in the levels of p-AMPK (Thr172) and p-AMPKβ1 (Ser182), suggesting that AL04 treatment reduced AMPK activity. Next, we examined ULK1, an AMPK-dependent substrate and regulator of autophagy (Egan, 2011; Kim, 2011; Guha, 2019), and found that ULK1 phosphorylation at Ser555 was significantly reduced in an AMPK-dependent manner after treatment of cells with AL04. Notably, the decrease in p-ULK1 (Ser555) levels in these cells was similar to that of two bona fide AMPK substrates, namely ACC and Raptor (data not shown). GAPDH was unchanged. AL04 treatment may regulate (inactivate) AMPK, known as tau kinase, suggesting that it contributes to the reduced levels of phosphorylated tau in PC12 cells.

[0103] Example 15: Description of Hippocampal and Entorhinal Experiments Using Tg2576 and JNPL3 Mice In human Alzheimer's disease, memory impairment associated with disease progression may result from pathological changes in the entorhinal cortex (EC) and hippocampus, regions important for new memory formation and most affected by Alzheimer's disease (Knowles, 1998; Alvarez, 1995; Bannerman, 2001; Buckmaster, 2004). The hippocampus is composed of the following subregions: dentate gyrus (DG), CA1, CA2, CA3, and subiculum. The hippocampus and entorhinal cortex have been reported to be the primary sites of Aβ deposition in Tg2576 mice, which overexpress human APP with the Swedish double mutation (K670N, M671L) (Hsiao, 1996; Su, 1998; Reilly, 2003; Dong, 2007; Lauritzen, 2012; Xu, 2015), and also display pathological tau (neurofibrillary tangles containing oligomers / aggregated phosphorylated tau) in JNPL3 mice, which express human tau with the P301L mutation (Lewis, 2000; Lin, 2003; Acker, 2013; Vitale, 2018). To evaluate the efficacy of AL04 against Alzheimer's disease (AD), we investigated whether AL04 reduces the accumulation of amyloid beta (AP) and / or hyperphosphorylated tau in the hippocampus and entorhinal cortex of Tg2576 and JNPL3 mice, respectively. The changes in Aβ deposition or hyperphosphorylated tau following AL04 treatment were compared with those in control (PBS-injected) animals using immunohistochemistry.

[0104] Example 15.1: Animals and Treatments Nine- to ten-month-old female Tg2576 mice and three- to four-month-old JNPL3 mice used in this study were purchased from Taconic Farms (Germantown, NY, USA) and maintained and handled according to a protocol approved by the Animal Care and Use Committee of Noble Life Science, Inc. (Sykesville, MD, USA) (approval number: NLS-511). All animal experiments in this study were planned and performed by L&J Biosciences, Inc. Tg2576 mice were treated with either 10 mg / kg AL04 or PBS via intraperitoneal injection every week for two months. JNPL3 mice were treated with either 5 mg / kg AL04 or PBS via intraperitoneal injection every two weeks for six months. After treatment, animals were anesthetized with isoflurane, and brains were removed. Brains were split at the midline (sagittal) so that only half of the brain was dissected for immunohistochemical analysis.

[0105] Example 15.2: Histology and Immunohistochemistry Analysis Brains were post-fixed for an additional 24 hours and then paraffin-embedded using standard protocols. Coronal sections (5 μm) were cut on a microtome and subjected to immunohistochemistry using the following primary antibodies: 6E10 (residues 1-16 of human Aβ, Biolegend, 1:500), AT8 (phosphorylated tau Ser202 / Thr205, Thermo, 1:1000), and HT7 (total tau, Thermo, 1:1000). After incubation with the primary antibodies, sections were washed in PBS and then incubated with a secondary antibody (HRP-conjugated; Jackson). The slides were incubated with either a fluorescent AlexaFluor antibody (Alexa 488 conjugated; Thermo Scientific, 1:1000) or a fluorescent AlexaFluor antibody (Alexa 488 conjugated; Thermo Scientific, 1:1000). For detection of Aβ deposits in Tg2576 mouse brain samples or total tau in JNPL3 mouse brains, slides treated with HRP-conjugated antibodies were incubated with diaminobenzidine (DAB), rinsed, and counterstained with hematoxylin. For DAB color development, slides were observed using a light microscope (Zeiss). Fluorescent slides were incubated with DAPI for 5 minutes for nuclear staining. Immunofluorescence of phosphorylated tau in JNPL3 mouse brain samples was visualized using a confocal microscope (Zeiss) with excitation filters 340 (DAPI blue) and 488 (p-tau green). Histology / immunohistochemistry and image analysis were performed by L&J Biosciences, Inc., respectively. The study was designed by Biosciences, Inc. and performed by Histoserve (Germantown, MD, USA) and CVPath (Gaithersburg, MD, USA).

[0106] Example 16: Results Example 16.1: AL04 reduces amyloid beta deposition in the hippocampus and entorhinal cortex of Tg2576 mice. Based on the amyloid cascade hypothesis, Aβ accumulation and aggregation induce the pathological and clinical symptoms of Alzheimer's disease (Walsh, 2012; Hardy, 1992; Hardy, 2002). Previous studies have reported that Aβ accumulation occurs in the brains of Tg2576 mice and leads to behavioral impairment (Hsiao, 1996). Alterations in the perforant tract, a neuronal projection from the entorhinal cortex to the hippocampal dentate gyrus, are known to be associated with memory impairment (Hyman, 1984; Hyman, 1986; Gomez-Isla, 1996; van Strien, 2009). Therefore, we investigated whether AL04 treatment reduces Aβ accumulation in the hippocampus and entorhinal cortex of Tg2576 mice. Starting at 9–10 months of age, Tg2576 mice received weekly intraperitoneal injections of either PBS or AL04 (10 mg / kg). Animals received a total of eight doses and were sacrificed at week 9. AL04 treatment was well tolerated. Amyloid-β precursor protein (AβPP) is cleaved by α- and β-secretases. Antibody 6E10 recognizes the first 16 residues of the Aβ domain and therefore theoretically labels full-length PAPP, C99 (a 15 kDa APP fragment produced by β-secretase (BACE1) digestion), and Aβ (Lauritzen, 2012). In PBS-treated Tg2576 mice at 11–12 months of age, 6E10 (Figure 16) demonstrates the presence of dense deposits (brown) in portions of the CA1, CA3, and DG subregions of the hippocampus. Furthermore, 6E10 strongly labeled the entorhinal cortex of PBS-treated animals, suggesting extracellular Aβ deposition (Figure 17). In contrast, reduced Aβ accumulation was observed in each subregion of the hippocampus (CA1, CA3, and DG) and the entorhinal cortex of AL04-treated Tg2576 mice (Figures 16 and 17). These results indicate that AL04 reduces Aβ accumulation in the hippocampus and entorhinal cortex of Tg2576 mice containing human APP mutants.

[0107] Example 16.2: AL04 reduces hyperphosphorylated tau in the hippocampus of JNPL3 mice In Alzheimer's disease and other neurodegenerative diseases, tau becomes hyperphosphorylated and dissociates from microtubules, leading to phosphorylated tau aggregation and neurofibrillary tangle formation in neuronal cell bodies and dendrites. These neurofibrillary tangles (NFTs) are well recognized by multiple phosphorylated tau on serine and threonine antibodies / paired helical filaments (PHFs), such as AT8 (p-tau S202 / T205), AT100, and PHFl (p-tau S396 / 404) (Lewis, 2000; Augustinack, 2002; Lace, 2009).

[0108] NFT formation is closely associated with pathological symptoms in tauopathy models (Braak, 1991, 1997; Duyckaerts, 1997; Rub, 2000; Sassin, 2000; Lewis, 2000; Lace, 2009). JNPL3 mice, which express the human mutant tau P301L, which causes frontotemporal lobe dementia in humans, form NFTs as early as 4.5 months of age and later develop progressive motor decline (Lewis, 2000). We investigated whether AL04 treatment could reduce phosphorylated tau levels in the hippocampus and entorhinal cortex of JNPL3 mice. Starting at 3–4 months of age, JNPL3 mice received intraperitoneal injections of either PBS or AL04 (5 mg / kg) every 2 weeks for 6 months. Animals received a total of 12 injections and were sacrificed at 25 weeks.

[0109] As a proof-of-concept for reducing hyperphosphorylated tau levels, AT8 (phosphorylated tau, S202 / T205) immunofluorescence analysis was performed in the CA1 / CA3 to DG subregions of the hippocampus and the entorhinal cortex. The hippocampus, entorhinal cortex, and perforant tract play a major role in memory formation and are vulnerable to damage in tau pathology, dementia, and mild cognitive impairment that occurs relatively early in aging (Braak and Braak, 1991; Duyckaerts, 1997; Tulving, 1998; Braak, 2006; Lace, 2009).

[0110] We observed phosphorylated tau (S202 / T205)-positive cells in the hippocampus of PBS-treated 9- to 10-month-old animals (Figure 18). The presence of phosphorylated tau (S202 / T205) suggests that axonal tau is abnormally phosphorylated, raising the possibility that axonal dysfunction in projections to the hippocampus may be a common early change in aging (Lace, 2009). In contrast, a significant decrease in phosphorylated tau (S202 / T205) levels was detected in the hippocampal subregions CA1 and CA3 and the entorhinal cortex of AL04-treated 9- to 10-month-old mice (Figure 18). These results suggest that AL04 reduces NFTs (hyperphosphorylated tau) in the hippocampus and entorhinal cortex of JNPL3 mice and attenuates the severity of tau pathology within hippocampal subregions, including CA1, CA3, and the dentate gyrus.

[0111] Cystatin C plays a neuroprotective role in the development of Alzheimer's disease and has clinical relevance as a therapeutic agent (Li, 1996; Tizon, 2010). Investigations into the selection of carriers and / or CPPs for dual-acting therapy (DAT)-based cystatin C-based drugs as potential Alzheimer's disease therapeutics are summarized in Figure 10. Four variants of the AL04 construct (designated AL04-1 to AL04-4) containing HSA (also known as a long-lived protein in vivo) as an Fc or carrier protein and dNP2 (Lim, 2015) or dTAT as a CPP were generated as described in Example 11.1. Using these constructs, protein expression was examined in either CHO-S or CHO-DG44 cells for transient and stable expression, respectively (data not shown). Constructs containing either Fc or HSA with dTAT as a CPP showed higher protein expression than similar constructs containing dNP2. Furthermore, HSA fusions with dTAT were observed to exhibit better expression than Fc fusion constructs in both transient and stable expression (data not shown). Unwanted immune cell activation, in which Fc fusion proteins induced inflammatory cytokine release from human PBMCs, has been reported (Edwards, 2014). CysC-HSA-dTAT is suggested to be a suitable DAT-based construct for the development of Alzheimer's disease therapeutics. The DAT platform was used to generate seven different constructs (versions 1 and 2) that are potential therapeutic candidates for Alzheimer's disease treatment, and their expression and purification are outlined in Figure 11. Seven different constructs were generated as described in Example 11.1, and their expression and purification processes were investigated. The level of fusion protein expression, including transient expression in DHFR-deficient CHO-DG44 cells and gene amplification using MTX (data not shown), as determined by Western blot or Coomassie staining, was molecular weight dependent. AL06 (145 kDa), AL09 (144.5 kDa), and AL10 (156 kDa) expressed little protein and could not produce protein by the MTX / DHFR gene amplification method. To examine the quality of each fusion protein, the protein was purified as described in Example 5 and analyzed by SDS-PAGE (data not shown).The AL07 and AL08 proteins were partially purified because they were degraded during the purification process (Figure 5). Figure 11 shows that AL04 (80 kDa) and AL12 (88 kDa) are the preferred constructs for protein expression and purification. References Acker et al., Sensitive quantitative assays for tau and phospho-tau in transgenic mouse models. Neurobiol Aging. 2013;34(1):338-350. Alvarez P, Zola-Morgan S, Squire LR. 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[0112] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein.

Claims

1. 1. A composition for use in a method for reducing both amyloid beta (Aβ) plaque deposition and aggregation and hyperphosphorylated tau plaque deposition and aggregation in the brain of a subject, the composition comprising a recombinantly produced polypeptide comprising, in N-terminus to C-terminus order, cystatin C (CysC), a first linker, human serum albumin, a second linker, and a specific cell membrane-penetrating peptide represented as residues 715-737 of SEQ ID NO: 6, the method comprising systemically administering to the subject an effective amount of the composition for reducing amyloid beta aggregation and hyperphosphorylated tau.

2. 2. The composition of claim 1, wherein the first linker is GGSAS (SEQ ID NO: 1) or GGGSGGGS (SEQ ID NO: 2).

3. The composition of claim 1 or 2, wherein the second linker is GFLG (SEQ ID NO: 3).

4. The composition of any one of claims 1 to 3, wherein the first linker is GGSAS (SEQ ID NO: 1) or GGGSGGGS (SEQ ID NO: 2), the second linker is GFLG (SEQ ID NO: 3), and the cell membrane penetrating peptide is set forth as residues 715 to 737 of SEQ ID NO:

6.

5. The composition of any one of claims 1 to 4, wherein the polypeptide is the isolated polypeptide set forth in SEQ ID NO:

6.

6. The composition of any one of claims 1 to 5, wherein the method comprises assaying for a reduction in hyperphosphorylated tau aggregation in the brain after the administration.

7. A composition comprising an isolated polypeptide as set forth in SEQ ID NO: 6, for use in a method for reducing both amyloid beta plaques and tau tangles in the brain of a subject.

8. The composition of claim 7 , wherein the method comprises assaying for a reduction in hyperphosphorylated tau plaque deposition after administration of the polypeptide.

9. The composition of any one of claims 1 to 6, wherein the subject is suffering from dementia or Alzheimer's disease.

10. 9. The composition of claim 7 or 8, wherein the subject is suffering from dementia or Alzheimer's disease.

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