AtMC1 for removing protein aggregates

Abstract

The present invention provides polypeptides having disaggregase activity for use in pharmaceuticals, more particularly for use in the prevention or treatment of diseases or disorders associated with protein aggregates in a subject, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity. The present invention also provides expression vectors encoding polypeptides for the same use, as well as uses and methods for eliminating or preventing the formation of protein aggregates in protein-containing compositions. The present invention also provides uses of polypeptides for imaging protein aggregates, and for the diagnosis and prognosis of diseases associated with protein aggregates.

Description

[Technical Field]

[0001] This application claims the benefits of European Patent Application No. 23382624.7, filed on 21 June 2023.

[0002] The present invention belongs to the field of compounds capable of removing protein aggregates, particularly polypeptides having disaggregase activity. The polypeptides of the present invention are particularly useful as pharmaceuticals for treating disorders or diseases associated with protein aggregates. [Background technology]

[0003] Proteins are the most abundant macromolecules in cells and are essential for virtually all physiological processes. To perform their biological functions, most proteins need to fold into and maintain their inherent higher-order structure. While a protein's inherent higher-order structure is determined by its amino acid sequence, the folding process is highly complex and error-prone, and its usefulness can be further limited in situations of gene mutations, biogenetic inaccuracies, and post-translational damage. Proteins with abnormal higher-order structures, and the aggregates they form, pose a certain threat to cell viability and function. Failure to remove these aggregates is closely linked to the pathogenesis of various debilitating human diseases, particularly neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

[0004] Current treatments for these diseases are mostly symptomatic and ineffective. There are no effective therapies to reverse the abnormal protein misfolding and aggregation that underlie the diseases. As the prevalence of these diseases increases with the aging population, the lack of effective therapies is causing considerable anxiety.

[0005] Protein disaggregases, or proteins capable of removing protein aggregates, are hypothesized to be potentially useful tools for treating diseases associated with protein aggregates (Shorter J, "Designer protein disaggregases to counter neurodegenerative disease," 2017, Curr Opin Genet Dev., vol.44, pp.1-8). However, most known disaggregases have several drawbacks that limit their use in clinic or industrial settings. For example, many are bacterial in origin, raising concerns about adverse immune responses upon administration, or are ATP-dependent, limiting their efficiency against extracellular aggregates associated with these diseases.

[0006] Therefore, there remains a need to identify novel protein disaggregases, particularly those useful for treating diseases associated with protein aggregates. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Shorter J., "Designer protein disaggregases to counter neurodegenerative disease", 2017, Curr Opin Genet Dev., vol.44, pp.1-8 [Overview of the Initiative]

[0008] The inventors have discovered the potent and specific disaggregase activity of Arabidopsis thaliana metacaspase-1 (AtMC1), which makes it particularly useful as a protein disaggregase for in vivo and in vitro applications.

[0009] As shown in the following examples, we have discovered that AtMC1 can remove pathological protein aggregates, whether used alone, extracellularly, or when the aggregates originate from evolutionarily distant organisms ranging from yeast to humans (see Figure 4). Prior to this invention, it was hypothesized that endogenous AtMC1 may be involved in some way in maintaining cellular proteostasis in plants. However, it was not known or anticipated that AtMC1 could directly affect aggregate removal, much less act alone as a potent disaggregase both intracellularly and extracellularly on aggregates originating from very distant organisms, including humans.

[0010] Furthermore, the inventors discovered that AtMC1's disaggregase activity is specific; that is, it removes protein aggregates but not the functional form of the protein (see Figure 4D, lines 3 and 4). Thus, AtMC1 exhibits slower, less efficient protease activity against typical metacaspase substrates, while showing proteolytic activity specifically directed towards aggregated proteins. In particular, this specific disaggregase activity was not observed in other plant-derived metacaspases (see Figure 4D, lines 8 and 9).

[0011] The inventors also discovered that variants of AtMC1 lacking the N-terminal prodomain and 360 loop maintain disaggregase activity while improving solubility, which facilitates their production and use (see Figure 3).

[0012] In conclusion, the inventors have found that AtMC1 exhibits an exceptional combination of properties that make it highly suitable for the removal of protein aggregates both in vivo and in vitro. Furthermore, the data provided below demonstrate that AtMC1 can be used as a pharmaceutical agent to treat human diseases associated with pathological protein aggregation in several disease models (e.g., Huntington's disease, transthyretin amyloidosis).

[0013] Accordingly, in a first aspect, the present invention provides a polypeptide having disaggregase activity for pharmaceutical use, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 (i.e., AtMC1) or SEQ ID NO: 2 (i.e., soluble AtMC1 lacking the prodomain and 360 loop), or a variant thereof that retains disaggregase activity.

[0014] In a second aspect, the present invention provides a polypeptide having disaggregase activity for use in the prevention or treatment of a disease or disorder associated with protein aggregates in a subject, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0015] In a third aspect, the present invention provides nucleic acids for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates in a subject, wherein the nucleic acids encode polypeptides as defined in the first and second aspects.

[0016] In a fourth aspect, the present invention provides a gene construct for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates in a subject, the gene construct comprising a nucleic acid as defined in the third aspect, operably linked to an expression promoter.

[0017] In a fifth aspect, the present invention provides an expression vector for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates in a subject, the expression vector comprising a gene construct as defined in the fourth aspect.

[0018] In a sixth aspect, the present invention provides a host cell for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates in a subject, the host cell comprising a nucleic acid as defined in a third aspect, a gene construct as defined in a fourth aspect, or an expression vector as defined in a fifth aspect.

[0019] In a seventh aspect, the present invention provides a pharmaceutical composition comprising: a polypeptide having disaggregase activity, which comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity; a nucleic acid encoding a polypeptide having disaggregase activity; a genetic construct comprising the nucleic acid; or an expression vector comprising the genetic construct, together with at least one pharmaceutically acceptable excipient, diluent or carrier.

[0020] As shown in the examples below, the present inventors have found that AtMC1 can also efficiently remove protein aggregates in vitro, and therefore may be useful for avoiding protein aggregation in non-therapeutic methods, for example methods for the in vitro production of recombinant proteins.

[0021] Accordingly, in an eighth aspect, the present invention provides a method for eliminating or preventing the formation of protein aggregates in a protein-containing composition, the method comprising contacting the protein-containing composition with a polypeptide having disaggregase activity, which polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0022] In a ninth aspect, the present invention provides use of a polypeptide having disaggregase activity for eliminating or preventing the formation of protein aggregates in a protein-containing composition, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0023] In a tenth aspect, the present invention provides a method for eliminating or preventing the formation of protein aggregates in a plant, the method comprising contacting the plant with a polypeptide having disaggregase activity, which polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity, or a nucleic acid encoding the same.

[0024] In an eleventh aspect, the present invention provides use of a polypeptide having disaggregase activity or a nucleic acid encoding the same for eliminating or preventing the formation of protein aggregates in plants, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0025] As shown below, the present inventors have found that AtMC1 exhibits specificity for protein aggregates compared to corresponding non-aggregated proteins, which makes it useful for imaging protein aggregates both in vivo and in vitro, and also for diagnosing diseases or disorders associated with protein aggregates.

[0026] Accordingly, in a twelfth aspect, the present invention provides a method for imaging protein aggregates in a subject, the method comprising the steps of: (i) administering to the subject a detectable amount of a detectably labeled polypeptide having disaggregase activity, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity; and (ii) detecting the polypeptide associated with protein aggregates. This aspect can also be formulated as a labeled polypeptide having disaggregase activity for use in a method of imaging protein aggregates in a subject, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity, and the method comprises the steps of: (i) administering to the subject a detectable amount of the detectably labeled polypeptide; and (ii) detecting the polypeptide associated with protein aggregates.

[0027] In a thirteenth aspect, the present invention provides a method for imaging protein aggregates in a sample, the method comprising contacting the sample with a polypeptide having disaggregase activity, wherein the polypeptide is a detectably labeled polypeptide having disaggregase activity, and the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0028] In a fourteenth aspect, the present invention provides a use for imaging protein aggregates of a polypeptide having disaggregase activity, wherein the polypeptide is a detectably labeled polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0029] In a fifteenth aspect, the present invention provides a polypeptide having disaggregase activity for use in vivo diagnosis of a disease or disorder associated with protein aggregates in a subject, wherein the polypeptide is a detectably labeled polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0030] In a sixteenth aspect, the present invention provides a method for diagnosing a disease or disorder associated with protein aggregates in a sample from a subject (i.e., a sample isolated from a subject), the method comprising contacting the sample with a polypeptide having disaggregase activity, wherein the polypeptide is a detectably labeled polypeptide having disaggregase activity, and the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0031] In the 17th aspect, the present invention provides a kit of parts for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates, the kit of parts comprising a polypeptide as defined in the first and second aspects, a nucleic acid as defined in the third aspect, a gene construct as defined in the fourth aspect, an expression vector as defined in the fifth aspect, or a host cell as defined in the sixth aspect, and instructions for use thereof.

[0032] In the eighteenth aspect, the present invention provides a kit of parts comprising a pharmaceutical composition as defined in the seventh aspect and instructions for use thereof. [Brief explanation of the drawing]

[0033] [Figure 1] MC1 is recruited to stress granules (SGs) under heat stress. A) Kinetics of MC1 cytoplasmic focus aggregation and degradation. The graph shows the mean ± sd of three independent experiments, each containing five seedlings. 8–10 cells were analyzed for SG quantification for each seedling. "HS termination" corresponds to the point in time when the plants were moved from 39°C to 22°C. B) Quantification of MC1-GFP focus in the experiment shown in C. The upper and lower box boundaries represent the first and third quartiles, respectively. The horizontal line shows the median, and the whiskers show the maximum and minimum values. Three independent experiments were conducted, each containing at least five individual measurements. Means with different letters are significantly different at P<0.05 (one-way ANOVA). [Figure 2] MC1 contains regions that are inherently disordered and prone to aggregation. A) Prediction of the inherently disordered regions of MC1. Top, scheme of the MC1 protein structure. Zf: LSD1-zinc finger domain (amino acids 1-77) within the prodomain; H164 and C220 correspond to amino acids in the catalytic dyad within the large p20 catalytic subunit; 360 loop (amino acids 318-346): hydrophobic loop within the small p10 catalytic subunit. Middle and bottom, predictions of the disordered regions by D2P2 (https: / / d2p2.pro) and DISOPRED3 (http: / / bioinf.cs.ucl.ac.uk / psipred), respectively. B) Aggrescan3D structure of MC1. The amino acid sequence is that of the prodomain, the 360 ​​loop is shown, and amino acids with high A3D scores (prone to aggregation) are highlighted. [Figure 3]The prodomain and 360 loop contribute to the insolubility of MC1. A) Schematic diagrams of the domain architectures of MC1 full length, MC1Δ360 (without 360 loop), and ΔNMC1Δ360 (without prodomain and 360 loop). B) SDS-PAGE Coomersie-stained gel (upper panel) and Western blot analysis (lower panel) of lysates from whole fractions, soluble fractions, or insoluble fractions of E. coli cells expressing either MC1Δ360 or ΔNMC1Δ360 (rMC1). Arrows indicate the expected molecular weight of each of the two MC1 variants. C) Size exclusion chromatography of concentrated eluates obtained by nickel affinity chromatography. Inlets show SDS-PAGE Coomersie-stained gels of fractions 14 ml to 16 ml of elution volume from a Superdex 75 column. [Figure 4]MC1 can specifically remove protein aggregates in vitro and in vivo, and the absence of MC1 leads to protein aggregate accumulation. A) Filter trap analysis of protein extracts from 5-day-old Arabidopsis thaliana mc1 or WT seedlings subjected to control conditions (NS) or severe heat stress (HS, 90 min at 37°C, 90 min at 22°C, and 90 min at 45°C). SDS-resistant aggregates were detected using antibodies against actin, HSP90-1, or polyQ protein. rHS / NS represents the ratio of HS to NS at the protein level. Signal intensity in the band was quantified using Image J. Two independent experiments were performed with similar results. B) Turbidity assay of endpoint deaggregation reaction using light scattering at 360 nm. rMC1, the catalyst-inactive form rMC1C220A (rMC1CA), or MC4 were co-incubated with TTR aggregates or natural tetramer TTR for 24 hours at 37°C. Recombinant MC1 protein and TTR aggregates incubated for the same period were further measured as controls. Data represent three individual measurements. The upper and lower box boundaries represent the first and third quartiles, respectively. The horizontal line indicates the median, and the whiskers indicate the maximum and minimum values. Means with different letters are significantly different at P<0.05 (one-way ANOVA). C) Electron microscopy images of the deaggregation reaction at the endpoint of TTR aggregates incubated at 37°C for 24 hours with or without purified rMC1. D) SDS-PAGE analysis of endpoint samples shown in panel C. E) Filter trap analysis showing mRFP-Q74 aggregation levels in HEK293 cells. HEK293 cells were transfected with mRFP-Q74 and GFP-MC1, or with mRFP-Q74 and GFP as a control. Q74 SDS-resistant aggregates were detected using mCherry antibody. [Figure 5]MC1 is involved in the removal of terminally misfolded proteins in yeast. A) Serial dilutions of wild-type (WT), ymca1Δ mutant, and ymca1ΔMC1-supplemented cells expressing ΔssCL* were placed in the indicated medium and incubated at 30°C for 3 days. Enhanced growth on leucine-deficient plates (CM-Leu) indicated stabilization of ΔssCL*, while decreased growth indicated increased degradation. Three independent experiments were performed. B) SDS-PAGE of ΔssCL* levels in the strains shown in A. ΔssCL* was detected using α-myc. [Modes for carrying out the invention]

[0034] All terms used herein are to be understood in the ordinary sense known in the art unless otherwise specified. Other, more specific definitions of any particular terms used herein are given below and are intended to apply uniformly throughout the specification and claims unless a more explicit definition is provided.

[0035] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise indicated, definite articles such as "the" as used herein also include plural nouns.

[0036] As described above, the present invention provides a novel disaggregase for use in pharmaceuticals, particularly for use in the treatment or prevention of diseases or disorders related to protein aggregates.

[0037] The term “polypeptide having disaggregase activity” refers to a polypeptide that can eliminate and / or prevent the formation of protein aggregates, particularly insoluble protein aggregates. As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to a compound consisting of two or more amino acid residues covalently linked by a peptide bond.

[0038] In a particular embodiment of the first aspect, the polypeptide is intended for use as a pharmaceutical. In another embodiment, the polypeptide is intended for use as a therapy or in vivo diagnostic tool.

[0039] In certain embodiments, the polypeptide is intended for use in mammals, particularly humans.

[0040] In a second aspect, the present invention provides polypeptides for use in the treatment or prevention of diseases or disorders associated with protein aggregates.

[0041] This embodiment can also be formulated as the use of the polypeptide of the present invention for the manufacture of a medicament for preventing or treating a disease or disorder associated with protein aggregates in a subject. This embodiment can also be formulated as a method for preventing or treating a disease or disorder associated with protein aggregates in a subject, the method comprising administering a therapeutically effective amount of the polypeptide of the present invention, together with at least one pharmaceutically acceptable excipient and / or carrier, to a subject in need thereof.

[0042] The term “disease or disorder associated with protein aggregates” refers to a disease or disorder characterized by the presence of harmful aggregated proteins, typically abnormal or misfolded proteins. These diseases are often neurodegenerative diseases, but also include diseases of other tissues, including the liver, muscles, and heart, and include some cancers. Various methods for assessing whether a disease is associated with protein misfolding or protein aggregates are known in the art. “Protein aggregates” refer to non-covalent oligomers or polymers of one or more types of proteins, typically characterized by altered three-dimensional higher-order structure of the complexed protein units relative to the monomeric protein units, and low solubility of the complex in aqueous solution. “Disease” is an animal health condition in which the animal is unable to maintain homeostasis, and if the disease is not improved, the animal’s health continues to deteriorate. In contrast, “disorder” in animals is a health condition in which the animal is able to maintain homeostasis, but the animal’s health condition is less desirable than that without disorder. Disorders, if left untreated, do not necessarily lead to further deterioration of the animal’s health condition.

[0043] As used herein, the term “treatment” means any type of therapy aimed at ending, preventing, improving or reducing susceptibility to any clinical condition or pre-existing disease described herein, including the complete cure of the disease and the improvement or mitigation of the disease. Thus, “treatment,” “to treat,” and their equivalents mean obtaining a desired pharmacological or physiological effect and encompass any treatment of a pathological condition or disorder in a subject. As used herein, the terms “prevention” or “to prevent” mean a process of prevention in which the subject is exposed to the polypeptide of the present invention before the onset or development of a disease process.

[0044] In certain embodiments, the protein aggregates of a disease or disorder associated with protein aggregates include at least one protein selected from the group consisting of prion proteins, polyglutamine proteins (e.g., huntingtin, androgen receptor, atropine 1, ataxin 1, ataxin 2, ataxin 3, ataxin 7, TATA box-binding protein), amyloid precursor protein (APP), α-synuclein, superoxide dismutase, transthyretin, tau, immunoglobulin, amyloid-A, transthyretin, low-density lipoprotein receptor, crystallin, β2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, FUS, SOD-1, islet amyloid polypeptide, ANF, gelzolin, insulin, lysozyme, p53, and fibrinogen.

[0045] In one embodiment, the disease or disorder associated with protein aggregates is selected from the group consisting of protein structure disorders (i.e., prion diseases, e.g., bovine spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Streussler-Scheinker syndrome, fatal familial insomnia), alpha-synuclein diseases, polyglutamine diseases (e.g., Huntington's disease, spinocerebellar ataxia type 1 (SCA1), SCA2, SCA3, SCA6, SCA7, SCA17), serpin diseases, tauopathies (e.g., frontotemporal dementia, Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration) or other related diseases or disorders.

[0046] In one embodiment, diseases or disorders caused by protein aggregates include Alzheimer's disease, transthyretin amyloidosis, cerebral β-amyloid angiopathy, retinal ganglion cell degeneration, bovine spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Streussler-Scheinker syndrome, fatal familial insomnia, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, and familial Danish dementia. Dementia, hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, seipinopathy, familial amyloidosis neurological lesions, senile systemic amyloidosis, serpin disease, AL amyloidosis, AA amyloidosis, type II diabetes, aortic medial amyloidosis, ApoAI amyloidosis, ApoII amyloidosis, ApoAIV amyloidosis, Finnish-type familial amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis / myopathy, cataracts, cancer associated with p53 aggregates, medullary thyroid carcinoma, cardiac atrial amyloidosis The following conditions are selected from the group consisting of pituitary prolactinoma, hereditary lattice corneal dystrophy, lichenoid amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic neoplasia amyloidosis, seminal vesicle amyloidosis, cystic fibrosis, sickle cell anemia, severe myopathy, von Hippel-Lindau disease, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Angelman syndrome, giant axonal disorder, and inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD). In another specific embodiment, the disease or disorder associated with protein aggregates is amyloidosis (e.g., transthyretin amyloidosis) or polyglutamine disease (e.g., Huntington's disease).In a more detailed embodiment, amyloidosis is transthyretin amyloidosis and / or polyglutamine disease is Huntington's disease.

[0047] In another embodiment, the disease or disorder is a neurodegenerative disease or disorder. In another embodiment, the disease or disorder associated with protein aggregates is selected from the group consisting of Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0048] In another embodiment, the disease or disorder is associated with insoluble protein aggregates.

[0049] In one embodiment, prevention or treatment includes reducing the number or size of protein aggregates in the subject and / or the rate of protein aggregate aggregation in the subject.

[0050] As shown in the following examples, the polypeptides of the present invention can remove aggregates from evolutionarily distant organisms, including yeast and animals.

[0051] Therefore, in certain embodiments, the subject is an animal. In more detailed embodiments, the subject is a mammal. In even more detailed embodiments, the subject is a human.

[0052] Sequence ID 1 is the amino acid sequence of Arabidopsis metacaspase 1, also known as AtMC1, AtMCP1b, AtMCA-Ia, or MC1 (NCBI accession NP_171719; version: NP_171719.2). Sequence ID 2 is the amino acid sequence of a soluble version that has the exact same sequence as AtMC1, except that it lacks the N-terminal prodomain (amino acids 2-77 of Sequence ID 1) and the 360 ​​loop (amino acids 318-346 of Sequence ID 1). As shown in the following examples, this version maintains the disaggregase activity of the full-length protein and has increased solubility.

[0053] In certain embodiments, the polypeptide comprises or consists of amino acids 78-317 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity. In more detailed embodiments, the polypeptide comprises or consists of amino acids 1, 78-317 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity.

[0054] In certain embodiments, the polypeptide comprises or consists of amino acids 78-317 and 347-367 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity. In more detailed embodiments, the polypeptide comprises or consists of amino acids 1, 78-317 and 347-367 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity.

[0055] In one embodiment, the polypeptide comprises or consists of amino acids 1-317 and 347-367 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity. In another embodiment, the polypeptide comprises or consists of amino acids 78-367 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity. In a more detailed embodiment, the polypeptide comprises or consists of amino acids 1 and 78-367 of SEQ ID NO: 1, or a variant thereof that retains disaggregase activity.

[0056] In certain embodiments, the polypeptide comprises or consists of a) the sequence of SEQ ID NO: 1, which may lack amino acids 2-77 and / or 318-346, or b) a variant of SEQ ID NO: 1, which may lack amino acids 2-77 and / or 318-346, that retains disaggregase activity and / or is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0057] In certain embodiments, the polypeptide includes variants thereof that retain the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or disaggregase activity, and / or are at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the variant retains disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0058] In certain embodiments, variants of SEQ ID NO: 1 or SEQ ID NO: 2 substantially maintain or improve the disaggregase activity of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, variants of SEQ ID NO: 1 or SEQ ID NO: 2 are functionally equivalent variants. In other embodiments, variants of SEQ ID NO: 1 or SEQ ID NO: 2 are catalytically active variants.

[0059] In certain embodiments, the polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity. In more detailed embodiments, the polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity and / or is identical to SEQ ID NO: 1 or SEQ ID NO: 2 by at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In even more detailed embodiments, the polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0060] Polypeptide sequence variants are well understood by those skilled in the art and may include amino acid sequence modifications. Amino acid sequence modifications are typically classified into one or more of three classes: substitution variants, insertion variants, or deletion variants. Since substitution modifications in polypeptide variant sequences are typically limited or conserved, the sequences of the reference peptide and the variant are densely similar overall and identical in many regions. Variants and reference peptides may differ in amino acid sequence by any combination of one or more substitutions, additions, or deletions. In some examples, variants have amino acid differences of 75, 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer compared to the reference sequence. Polypeptide variants may occur spontaneously, like allele variants, or they may be variants that are not known to occur spontaneously. Non-spontaneous variants of polypeptides can be produced by mutagenesis techniques or direct synthesis according to conventional methods.

[0061] In certain embodiments, the variant of Sequence ID No. 1 is encoded by a plant ortholog of the Arabidopsis thaliana MC1 gene.

[0062] In certain embodiments, the polypeptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5, or a variant thereof that retains disaggregase activity.

[0063] Those skilled in the art can easily identify variants of SEQ ID NO: 1 or SEQ ID NO: 2 that retain disaggregase activity by following conventional methods without requiring inventive skills. For example, to test whether a variant retains disaggregase activity, those skilled in the art can perform an in vitro cell-free disaggregation assay as described in the following examples. Briefly, protein aggregates (e.g., TTR aggregates) are co-incubated with the variant at 37°C in the presence of DTT and CaCl2, and the turbidity of the sample is measured by synchronized light scattering. If the turbidity of the sample is maintained or increases over time, the variant does not retain disaggregase activity; however, if the turbidity of the sample decreases over time, the variant retains disaggregase activity. Disaggregation activity can also be measured by a filter trap assay, as described in the following examples.

[0064] In this invention, the term “identical” or “identity” refers to the percentage of positions in two sequences that are identical when the sequences are optimally aligned. Sequences exhibit identity with respect to a position if, in optimal alignment, a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence. The percentage of identity determines the number of identical nucleotides or amino acids over a given length in a given alignment. Therefore, the level of identity between two sequences, or “percent sequence identity” (“percent sequence identity”), is measured as the ratio of the number of identical positions shared by the sequences to the number of positions being compared (i.e., percentage sequence identity = (number of identical positions / total number of positions being compared) × 100). Gaps, i.e., alignment positions where a nucleotide or amino acid exists in one sequence but not in the other, are considered positions where the nucleotide or amino acid is not identical and are counted as comparison positions.

[0065] Several mathematical algorithms are known and incorporated into several available software programs for quickly obtaining optimal alignment and calculating the identity between two or more sequences. For the purposes of this invention, sequence identity between two nucleic acids or amino acid sequences is preferably determined using an algorithm based on global alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), preferably implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), or the BLAST Global Alignment tool (default settings) (Altschul et al., "Basic local alignment search tool", 1990, J.Mol.Biol, v.215, pp. 403-410). Local alignment can also be used if the sequences being compared are substantially the same length.

[0066] In certain embodiments of the first aspect, the polypeptide is an isolated, synthetic, or recombinant polypeptide. "Isolated" means modified or removed from its natural state. The sequences disclosed in this invention are shown in Table 1 below: [Table 1] JPEG2026531509000002.jpg138159

[0067] The polypeptides of the present invention may contain cell-permeable peptides (CPPs) or blood-brain barrier-permeable peptides (BBPs) to improve their functionality, whether used in vivo or in vitro.

[0068] Therefore, in certain embodiments, the polypeptide further comprises a cell-permeable peptide (CPP) and / or a blood-brain barrier-permeable peptide (BBP).

[0069] In the present invention, the term "cell-permeable peptide" or "CPP" refers to short peptides, particularly polypeptides, that facilitate the cellular uptake of various molecular cargoes. The term "blood-brain barrier-permeable peptide" or "BPP" refers to short peptides, particularly polypeptides, that facilitate the cellular transport of various molecular cargoes across the blood-brain barrier.

[0070] The function of CPPs is to deliver cargo into cells, a process typically occurring via endocytosis by cargo delivered to delivery vectors for research and pharmaceutical use. CPPs typically have an amino acid composition containing high relative abundances of positively charged amino acids, such as lysine or arginine, or sequences containing alternating patterns of polar / charged amino acids and nonpolar hydrophobic amino acids. These two types of structures are called polycationic or amphiphilic, respectively. A third class of CPPs are hydrophobic peptides with a low net charge, containing only nonpolar residues, or have hydrophobic amino acid groups important for cellular uptake. The design and synthesis of CPPs are well known in the art (Copolovici DM et al., "Cell-Penetrating Peptides: Design, Synthesis, and Applications", 2014, ACS Nano, 2014, 8(3), pp 1972-1994).

[0071] Substantially, any CPP having the ability to internalize polypeptides within a cell, or any BPP having the ability to deliver polypeptides through the blood-brain barrier (BBB), can be used as polypeptides in the present invention. Nevertheless, in certain embodiments, the carrier peptide is a peptide comprising a "PTD" ("protein transduction domain") segment. Exemplary, non-limiting examples of proteins comprising a protein transduction domain (PTD) include the human immunodeficiency virus 1 (HIV-1) TAT ("transacting translational protein") protein, the Drosophila Antennapedia homeotic transcription factor (Antp), and the herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein. However, it has also been suggested that other proteins, such as influenza virus hemagglutinin, lactoferrin, fibroblast growth factor-1, fibroblast growth factor-2, and Hoxa-5, Hoxb-4, and Hoxc-8 proteins, also possess this property of internalizing peptides within a cell.

[0072] CPP or BPP may be positioned at either the amino or carboxyl terminus of the polypeptide of the present invention.

[0073] CPP or BPP can be linked directly to SEQ ID NO: 1 or 2 or via a spacer peptide. Substantially, any peptide with structural flexibility can be used as a spacer peptide. Nevertheless, exemplary, non-limiting examples of spacer peptides include peptides containing amino acid moieties, e.g., repeats of Gly and / or Ser, or any other suitable repeat of any of the amino acid moieties.

[0074] In one embodiment, the polypeptide further comprises a targeting domain, which targets the polypeptide to a desired location. For example, the targeting domain is directed to bind to a protein or protein aggregate associated with a disease or disorder. In some embodiments, the targeting domain includes peptides, nucleic acids, small molecules, etc., that have the ability to bind to target cells, proteins, or protein aggregates. For example, in one embodiment, the targeting domain includes an antibody or antibody fragment that binds to target cells, proteins, or protein aggregates.

[0075] In another embodiment, the polypeptide further comprises a secretion signal peptide to direct the secretion of the polypeptide into the extracellular environment. The secretion signal peptide is intended to translocate the polypeptide across the endoplasmic reticulum membrane into the secretory pathway.

[0076] Polypeptides comprising SEQ ID NO: 1 or SEQ ID NO: 2 or its variants can be administered directly as polypeptides or expressed in target cells of interest by gene therapy. For this purpose, the present invention also, in a third embodiment, provides nucleic acids encoding polypeptides as defined in the first and second embodiments, for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders associated with protein aggregates in a subject.

[0077] The term "nucleotide encoding polypeptide" should be understood, in particular, as polypeptide-encoding mRNA or cDNA sequences resulting from the reverse transcription (RT-PCR) of polypeptide-encoding mRNA.

[0078] This embodiment can also be formulated as the use of the nucleic acid defined above for the manufacture of a pharmaceutical product for preventing or treating a disease or disorder associated with protein aggregates in a subject. The present invention also relates to a method for preventing or treating a disease or disorder associated with protein aggregates in a subject, the method comprising administering a therapeutically effective amount of the nucleic acid defined above, together with a pharmaceutically acceptable excipient or carrier, to a subject, including a human being in need.

[0079] In a particular embodiment of the second aspect, the nucleic acid sequence includes, essentially consists of, or comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[0080] In one embodiment, the nucleic acid sequence includes the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a variant thereof that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3 or SEQ ID NO: 4, and optionally retains disaggregase activity. Those skilled in the art will understand that a nucleic acid variant of SEQ ID NO: 3 or SEQ ID NO: 4 that retains disaggregase activity refers to a nucleic acid that encodes a protein that retains disaggregase activity.

[0081] In more detailed embodiments, the nucleic acid sequence consists of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a variant thereof that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and optionally retains disaggregase activity. Those skilled in the art will understand that a nucleic acid variant of SEQ ID NO: 3 or SEQ ID NO: 4 that retains disaggregase activity refers to a nucleic acid that encodes a protein that retains disaggregase activity.

[0082] In a fourth aspect, the present invention provides a gene construct for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates in a subject, the gene construct comprising a nucleic acid sequence as defined in the third aspect, operably linked to an expression promoter.

[0083] This embodiment can also be formulated as the use of the gene construct defined above for the manufacture of a pharmaceutical product for preventing or treating a disease or disorder associated with protein aggregates in a subject. The present invention also relates to a method for preventing or treating a disease or disorder associated with protein aggregates in a subject, the method comprising administering a therapeutically effective amount of the gene construct defined above, together with a pharmaceutically acceptable excipient or carrier, to a subject, including a human being in need thereof.

[0084] In a specific embodiment of the fourth aspect, the operably linked expression promoter is selected from the group consisting of constitutive expression promoters, inductive promoters, and tissue or cell-specific expression promoters (e.g., neuron-specific expression promoters). In a more detailed embodiment, the gene construct according to the present invention comprises a promoter, the sequence of SEQ ID NO: 3 or 4, and a poly(A) region.

[0085] All of these gene constructs, once inside a cell, can express the target protein.

[0086] To facilitate the administration of the construct, the present invention also, in a fifth embodiment, provides an expression vector for use in pharmaceuticals, particularly in the prevention or treatment of diseases or disorders associated with protein aggregates in a subject, the expression vector comprising a gene construct comprising a nucleic acid sequence of the second embodiment encoding a polypeptide of the first embodiment, as defined in the third embodiment, and thus operably linked to an expression promoter.

[0087] This embodiment can also be formulated as the use of the expression vector defined above for the manufacture of a pharmaceutical product for preventing or treating a disease or disorder associated with protein aggregates in a subject. The present invention also relates to a method for preventing or treating a disease or disorder associated with protein aggregates in a subject, the method comprising administering a therapeutically effective amount of the expression vector defined above, together with a pharmaceutically acceptable excipient or carrier, to a subject, including a human being in need.

[0088] In a specific embodiment of the fifth aspect, the expression vector is a viral vector.

[0089] In a particular embodiment of the fifth aspect, the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, baculovirus vectors, herpes simplex virus (HSV) vectors, retrovirus vectors, lentivirus vectors, vaccinia virus vectors, and RNA virus vectors. In a more detailed embodiment, the viral vector is an adeno-associated virus. In a particular embodiment, it is an adeno-associated virus of a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, PHPeB, and 9P31. Further delivery and expression systems are known in the art and are intended to be incorporated herein.

[0090] Those skilled in the art know, without the exercise of any inventive skills, how to prepare polypeptides, nucleic acids, gene constructs, and expression vectors for use according to the present invention by conventional methods well known in the art, for example, by chemical synthesis, by recombinant expression techniques, or by cleavage from longer polypeptides or nucleic acids. The sequences of peptides or nucleic acids can be confirmed by sequencing.

[0091] The polypeptides of the present invention can be post-translation modified. For example, post-translation modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolysis processing.

[0092] In any one embodiment of the above-described aspects, the polypeptide, nucleic acid, gene construct, or expression vector is administered in the form of a composition.

[0093] In another embodiment of any of the above aspects, the polypeptide, nucleic acid, gene construct, or expression vector is administered in the form of a pharmaceutical composition together with at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0094] As described above, in the seventh embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide, a nucleic acid, a gene construct, or an expression vector.

[0095] The term "pharmaceutical composition" encompasses compositions intended for both humans and non-human animals. Those skilled in the art will understand that a pharmaceutical composition must contain a therapeutically effective amount of the active compound. As used herein, "therapeutic amount" refers to an amount of polypeptide, nucleic acid, gene construct, or expression vector sufficient, when administered, to prevent or, to some extent, alleviate the onset of one or more symptoms of the disease being addressed. The specific dose of a compound administered in accordance with the present invention will, naturally, be determined by the specific circumstances surrounding the case, including the compound administered, the route of administration, the specific condition being treated, and similar considerations.

[0096] The expression "pharmaceutically acceptable excipient, diluent or carrier" refers to a pharmaceutically acceptable material, composition or vehicle. Each component must be pharmaceutically acceptable in the sense that it is compatible with the other components of the pharmaceutical composition. It must also be free from excessive toxicity, irritation, allergic reactions, immunogenicity or other problems or complications and suitable for use in contact with human and non-human animal tissues or organs in proportion to a reasonable benefit-risk ratio.

[0097] Examples of suitable pharmaceutically acceptable excipients include solvents, dispersions, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, and lubricants. Any conventional excipient medium is considered to be within the scope of the present invention unless it is incompatible with the substance or its derivatives, for example, by producing some undesirable biological effect or otherwise interacting adversely with any other component of the pharmaceutical composition.

[0098] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition of the present invention vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route through which the composition is administered.

[0099] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as colorants, coatings, sweeteners, and flavorings may be present in the composition at the discretion of the compounder.

[0100] Pharmaceutical compositions containing polypeptides, nucleic acids, gene constructs, or expression vectors can be provided in any dosage form, e.g., solid or liquid, and can be administered by any suitable route, e.g., oral, parenteral, rectal, topical, intranasal, intraocular, intraperitoneal, sublingual, or intraventricular routes, and for this purpose, they may contain pharmaceutically acceptable excipients necessary for the formulation of the desired dosage form, e.g., topical formulations (ointments, creams, lipogels, hydrogels, etc.), eye drops, aerosol sprays, hydrogels for injection, solutions for injection, osmotic pumps, etc.

[0101] Examples of diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

[0102] Examples of granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0103] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghati gum, isapol husk mucus, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum), larch arabinogalactan, alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, waxes, water, alcohols, and combinations thereof.

[0104] Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcoholic preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate.

[0105] Examples of buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0106] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0107] In certain embodiments, the composition or pharmaceutical composition further comprises nanoparticles that encapsulate polypeptides, nucleic acids, gene constructs, or expression vectors. In more detailed embodiments, the nanoparticles are lipid nanoparticles. Those skilled in the art will understand that the nanoparticles are biocompatible and protect the active ingredient from degradation.

[0108] As used herein, the term “nanoparticle” refers to a particle having at least two nanoscale dimensions, particularly all three dimensions, in the nanoscale range of approximately 1 nm to approximately 300 nm. In particular, if the nanoparticle is substantially rod-shaped with a substantially circular cross-section, for example, a nanowire or nanotube, then “nanoparticle” refers to a particle having at least two nanoscale dimensions, where these two dimensions are the cross-section of the nanoparticle.

[0109] Biodegradable nanoparticle delivery systems that increase intracellular uptake, such as polymers and surface-modified nanoparticles, can also be used. Examples include poly-DL-lactide-coglycolide (PLGA) nanoparticles, for example, those surface-modified with known surface modifiers, such as heparin, dodecylmethylammonium bromide (DMAB), DEAE-dextran, lipofectin, and fibrinogen.

[0110] As used herein, the term “lipid nanoparticles” refers to nanoparticles whose membranes are entirely composed of lipids. Suitable lipids include, but are not limited to, phospholipids such as phosphatidylcholine ("PC"), phosphatidylethanolamine ("PE"), phosphatidylylserine ("PS"), phosphatidylglycerol ("PG"), phosphatidylinositol ("PI"), and phosphatidic acid ("PA"). Such phospholipids generally have two acyl chains, which may be both saturated, both unsaturated, or one saturated and one unsaturated. Chains include, but are not limited to, myristrate, palmitate, stearate, oleate, linoleate, linolenate, arachidate, arachidonate, behenate, and lignocerate chains. Phospholipids can also be derivatized by attaching a suitable reactive group thereto. Since such a group is generally an amino group, a derivatized phospholipid is typically phosphatidylethanolamine. Different moieties suitable for binding to PE include, but are not limited to, acyl chains useful for improving the fusibility of liposomes to biological membranes, peptides useful for destabilizing liposomes near target cells, biotin and maleimide moieties useful for linking targeted portions such as antibodies to liposomes, and various molecules such as gangliosides, polyalkyl ethers, polyethylene glycols, and organic dicarboxylic acids. Other lipids that can constitute nanoparticle membranes include, but are not limited to, cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0111] In one embodiment, the lipid nanoparticles are selected from the group consisting of liposomes and solid lipid nanoparticles. In another embodiment, the lipid nanoparticles are liposomes.

[0112] The term "solid lipid nanoparticles" refers to typically spherical particles having an average diameter of 10 to 1000 nanometers. Solid lipid nanoparticles have a solid lipid core matrix that can solubilize lipophilic molecules. The lipid core is stabilized by a surfactant (emulsifier). The term lipid is used herein in a broader sense and includes triglycerides (e.g., tristearin), diglycerides (e.g., glycerol behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). Lipid dispersions were stabilized using emulsifiers of all classes (in terms of charge and molecular weight).

[0113] In this invention, the term “liposome” should be understood as a self-assembling structure comprising one or more lipid bilayers, each comprising two monolayers containing relative-oriented amphiphilic lipid molecules. The amphiphilic lipids include polar (hydrophilic) head regions covalently bonded to one or two nonpolar (hydrophobic) acyl chains. Energically unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous medium induces the amphiphilic lipid molecules to reorient themselves such that their polar head groups are oriented toward the surface of the bilayer and the acyl chains toward the interior of the bilayer. Thus, an energetically stable structure is formed that effectively shields the acyl chains from contact with the aqueous environment. Liposomes may have a single lipid bilayer (monolayer liposome, “ULV”) or multiple lipid bilayers (multilayer liposome, “MLV” or “SPLV”). Each bilayer surrounds or encapsulates an aqueous compartment. By encapsulating an aqueous volume within a protective barrier of lipid molecules, liposomes can sequester the encapsulated molecules, such as nucleic acids, from degradation factors present in the external environment, such as nuclease enzymes. Liposomes can have a variety of sizes, for example, small ones as small as 25 nm or large ones with an average diameter of over 10,000 nm. The size is influenced by several factors, such as lipid composition and preparation methods, which are usually determined and explained within the knowledge of those skilled in the art, and is also determined by several techniques, such as quasi-elastic light scattering, which is also within the scope of knowledge of those skilled in the art. Smaller liposomes can be prepared from larger ones using various methodologies, such as sonication, homogenization, and grinding, which are also well known to those skilled in the art. Liposomes can be reduced in size using extrusion, i.e., by passing liposomes through a filter pore of a predetermined selected size under pressure, thereby producing liposomes with a predetermined average size. Liposome size can also be adjusted using tangential flow filtration, i.e., a population of liposomes with a defined size distribution, lower size heterogeneity, and greater homogeneity.

[0114] Polypeptides, nucleic acids, gene constructs, or expression vectors for use according to the present invention can be encapsulated in particles using well-known methods of the latest technology.

[0115] In one embodiment, the nanoparticles are also release-controlled formulations, including, for example, implants and microencapsulation delivery systems, that protect polypeptides, nucleic acids, gene constructs, or expression vectors for use according to the present invention from rapid elimination from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations may be prepared using standard techniques or purchased.

[0116] In another embodiment, polypeptides, nucleic acids, gene constructs, or expression vectors are administered in combination with another activator. Suitable activators administered in combination with the compounds of the present invention are, for example, drugs for treating neurodegenerative diseases.

[0117] As described above, the present invention provides further uses of AtMC1 as a disaggregase in the eighth, ninth, tenth, and eleventh embodiments. All embodiments disclosed above, in particular relating to polypeptide sequences and aggregates, are also applicable to these embodiments.

[0118] In one embodiment of the 8th, 9th, 10th, and 11th aspects, the protein aggregate is either a pathogenic protein aggregate or a non-pathogenic protein aggregate. In another embodiment, the protein aggregate is an insoluble protein aggregate. In a more detailed embodiment, the protein aggregate is a pathogenic insoluble protein aggregate.

[0119] In one embodiment of the eighth aspect, the method is an in vivo or ex vivo method. In one embodiment of the ninth aspect, the use is an in vivo or ex vivo use. In another embodiment, the protein-containing composition is a biological or non-biological sample. In another embodiment, the sample is a sample isolated from a subject. In another embodiment, the sample is a cell culture. As used herein, “protein-containing composition” refers to any composition containing at least one protein. The protein-containing composition may contain one protein or multiple proteins.

[0120] In one embodiment of the eighth and ninth aspects, the method is a cell-free method. In another embodiment, the protein-containing composition is a sample, and in particular a cell-free sample.

[0121] As described above, the present invention provides, in embodiments 10 to 16, a method for imaging (i.e., detecting the presence of) protein aggregates and for diagnosing diseases or disorders associated with protein aggregates, comprising the use of a detectably labeled polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof. All embodiments disclosed above, in particular those relating to polypeptide sequences, aggregates, and diseases, are also applicable to these embodiments.

[0122] As used herein, “detectable labeled polypeptide” refers to a polypeptide comprising one or more detectable labels. Those skilled in the art will understand that various labels known in the art can be conjugated to polypeptides using standard techniques for labeling proteins. Examples of labels include fluorescent labels and radioactive labels. While there are many types of radioactive labels that can be used, generally, labels are not limited to these. 1 S F, 11 C and 123Radioactive labels containing l are often selected. These, and other radioisotopes, can be bound to proteins using well-known chemistry. In one embodiment, the label is detected using positron emission tomography (PET). However, any other suitable technique can be used for the detection of the label, particularly the radioisotope.

[0123] In one embodiment of the twelfth aspect, the subject is a mammal, particularly a human.

[0124] In one embodiment of the thirteenth aspect, the sample is an in vitro or ex vivo sample. In another embodiment, the sample is a cell-free sample.

[0125] In one embodiment of the 15th aspect, the in vivo diagnosis comprises (i) administering a detectable amount of a detectably labeled polypeptide having disaggregase activity to a subject, and (ii) detecting the polypeptide associated with protein aggregates, the subject being diagnosed with a protein aggregate-related disease or disorder if the detectably labeled polypeptide is bound to the protein aggregates.

[0126] In one embodiment of the sixteenth aspect, the method further comprises detecting polypeptides that associate with protein aggregates in a sample, and if detectably labeled polypeptides are bound to protein aggregates, the subject is diagnosed with a disease or disorder associated with protein aggregates.

[0127] Throughout the specification and claims, the word “comprise” and variations thereof are not intended to exclude other technical features, additives, components, or processes. Furthermore, the word “comprise” encompasses the case of “consisting of.” Further objects, advantages, and features of the present invention may become apparent to those skilled in the art by considering the specification or may be acquired through the practice of the invention. The following examples and drawings are provided for illustrative purposes only and are not intended to limit the invention. Furthermore, the invention encompasses all possible combinations of the particular preferred embodiments described herein.

[0128] For completeness, various aspects of the present invention are described in the following numbered clauses.

[0129] 1. A polypeptide having disaggregase activity for use in pharmaceuticals, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0130] 2. A polypeptide having disaggregase activity for use in the prevention or treatment of diseases or disorders related to protein aggregates in a subject, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0131] 3. A polypeptide for use as described in Clause 2, wherein the protein aggregate comprises at least one protein selected from the group consisting of prion protein, polyglutamine protein, amyloid precursor protein, α-synuclein, superoxide dismutase, transthyretin, tau, immunoglobulin, amyloid-A, low-density lipoprotein receptor, crystallin, β2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, FUS, islet amyloid polypeptide, ANF, gelzolin, insulin, lysozyme, p53, and fibrinogen.

[0132] 4. Diseases or disorders caused by protein aggregates include Alzheimer's disease, transthyretin amyloidosis, cerebral β-amyloid angiopathy, retinal ganglion cell degeneration, bovine spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Streussler-Scheinker syndrome, fatal familial insomnia, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, and familial Danish dementia. Dementia, hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, seipinopathy, familial amyloidosis, senile systemic amyloidosis, serpinopathy, AL amyloidosis, AA amyloidosis, type II diabetes, aortic medial amyloidosis, ApoAI amyloidosis, ApoII amyloidosis, ApoAIV amyloidosis, Finnish-type familial amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis / myopathy, cataracts, cancer associated with p53 aggregates, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactin Polypeptides for use as described in Clause 2 or 3, selected from the group consisting of: cerebrospinal amyloidosis, hereditary lattice corneal dystrophy, lichenoid amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic neoplastic amyloidosis, seminal vesicle amyloidosis, cystic fibrosis, sickle cell disease, severe myopathy, von Hippel-Lindau disease, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Angelman syndrome, giant axonal disorder, and inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD).

[0133] 5. Polypeptides for use as described in any one of clauses 2-4, wherein the subject is a mammal, in particular human.

[0134] 6. A polypeptide for use as described in any one of Clauses 1 to 5, comprising a variant thereof that retains the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or disaggregase activity, and is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0135] 7. A polypeptide for use as described in any one of clauses 1 to 6, wherein the polypeptide consists of SEQ ID NO: 1 or SEQ ID NO: 2.

[0136] 8. An expression vector for use in pharmaceuticals, particularly for the prevention or treatment of diseases or disorders related to protein aggregates in a subject, comprising a nucleic acid encoding a polypeptide defined in any one of clauses 1 to 7, which is operably linked to an expression promoter.

[0137] 9. A polypeptide for use as described in any one of Clauses 1 to 7 or an expression vector for use as described in Clause 8, administered in the form of a pharmaceutical composition together with at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0138] 10. Together with at least one pharmaceutically acceptable excipient, diluent, or carrier - A polypeptide having disaggregase activity, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity. - Nucleic acids encoding polypeptides having disaggregase activity, - Genetic constructs containing nucleic acids, or - A pharmaceutical composition comprising an expression vector containing a gene construct.

[0139] 11. A method for eliminating or preventing the formation of protein aggregates in a protein-containing composition, comprising contacting the protein-containing composition with a polypeptide having disaggregase activity, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity.

[0140] 12. Use of a polypeptide having disaggregase activity, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity, for the purpose of eliminating or preventing the formation of protein aggregates in a protein-containing composition.

[0141] 13. A method for imaging protein aggregates in a subject, comprising the steps of (i) administering a detectable amount of a detectably labeled polypeptide having disaggregase activity, the polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity, to the subject, and (ii) detecting the polypeptide associated with the protein aggregates.

[0142] 14. Use of a detectably labeled polypeptide having disaggregase activity, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity, for imaging protein aggregates.

[0143] 15. A disaggregase-active polypeptide for use in vivo diagnosis or prognosis of a disease or disorder associated with protein aggregates in a subject, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity, and which is detectably labeled.

[0144] Examples 1. Materials and Methods Materials used and growth conditions All experiments were performed using the *Arabidopsis thaliana* Columbia-0 (Col-0) ecotype. The single mutant mc1 has been previously described (GK-096A10, Coll et al., 2010). All seeds were surface-sterilized with 35% NaClO for 5 minutes and washed five times with sterile dH2O for 5 minutes each. Sterilized seeds were sown in solid 1 / 2 Murasigeskoog (MS) medium containing vitamins and stratified at 4°C for 48 hours. Plants were grown vertically under long-day (LD) conditions (16 hours light / 8 hours dark) at 22°C. For dark-induced aging studies, one-week-old seedlings were transplanted to soil and grown for a further two weeks under LD conditions.

[0145] Plasmid construction To generate the Pro35S::MC1-GFP construct, the coding sequence and natural promoter (approximately 1kb) of Arabidopsis thaliana MC1 (AT1G02170) were amplified from Col-0 cDNA and genomic DNA, respectively. Plasmids were constructed by GreenGate cloning.

[0146] For the purification of recombinant MC1 (rMC1), MC1 coding sequences lacking 360 loops and containing or not containing the prodomain (MC1Δ360 [SEQ ID NO: 5] or ΔNMC1Δ360 / rMC1 [SEQ ID NO: 2]) (Figure 2) were synthesized using codon optimization for expression in E. coli (Twist Bioscience). The synthesized sequences contained NdeI and XhoI restriction sites at the 5' and 3' ends, respectively. Both the synthesized gene and the target vector pET28b(+) were cleaved at NdeI and XhoI, and subsequently ligated so that the N-terminal 6xHis tag preceded the start site of the MC1 variant. Point mutations were induced in the catalytic site of rMC1 using QuickChange Site-Directed Mutagenesis (Agilent Technologies) to generate rMC1CA (SEQ ID NO: 6).

[0147] To complement the yeast metacaspase mutant ymca1Δ with Arabidopsis thaliana MC1 (AT1G02170), a gene substitution cassette was constructed by PCR-induced homologous recombination as previously described (Gardner and Jaspersen, 2014). The gene substitution cassette containing Arabidopsis thaliana MC1 was transformed into the ymca1Δ mutant KanMX4.

[0148] HEK293T cell transfection and protein extraction The MC1 (AT1G02170) gene was codon-optimized and synthesized for expression in animal cells (SEQ ID NO: 3) (Twist Bioscience). To generate ProCMV::GFP-MC1, the synthetic gene was cloned into the pDEST-CMV-N-GFP vector using Gateway technology (Addgene). ProCMV::mRFP-Q74 (Balaji et al., 2022), ProCMV::GFP-MC1, and ProCMV::GFP (Llamas et al., 2022) were used to transfect HEK cells (CRL-1573) according to a previously described protocol (Llamas et al., 2022). After 72 hours of incubation, cells were lysed in undenatured intrinsic lysis (300 mM NaCl, 100 mM Hepes pH 7.4, 2 mM EDTA, 2% Triton X-100) supplemented with 1× plant protease inhibitor (Merck), scraped from tissue culture plates, and homogenized by passing through a 27G needle. The sample was centrifuged at 10,000×g for 10 minutes at 4°C, and the supernatant was collected. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher).

[0149] Yeast strains and spot dilution assays Yeast medium preparation and molecular biology techniques were performed using standard methods (Lazaro-Silva et al., 2015). All experiments were conducted using the genetic background of the budding yeast (Saccharomyces cerevisiae) strain BY4741. To test the ability of each yeast strain to remove misfolded proteins, yeast cells were transformed with plasmid pFE15 encoding the fusion construct ΔssCL*myc (Eisele and Wolf, 2008). Growth phenotypes were evaluated by spot dilution assays. Undiluted ~10 4 Tenfold serial dilutions were performed within the dilution range. 5 μl of each dilution was placed in the corresponding selective medium (-ura or -ura-leu plate), and the plate was incubated for at least 3 days before images were taken.

[0150] Protein purification E. coli OverExpress C41 (DE3) chemically competent cells from BioCat GmbH (Heidelberg, Germany) or E. coli BL21 strain containing the pBB542 vector (de Marco et al., 2007) were transformed with expression plasmids and grown in either autoinducible medium or LB, respectively. The cells were then OD 600 Cells were initially grown at 37°C with continuous shaking until the pH reached 0.6, and then transferred to 25°C for overnight growth. For expression in E. coli Chaperone Competent Cells BL21, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to 400 ml of cell culture during the transfer to 25°C to induce protein expression. The pellet from the overnight culture was resuspended in 20 mM HEPES, pH 7.5, 500 mM NaCl and sonicated on ice. The lysate was centrifuged at 25,000 × g for 20 minutes to remove cell debris and insoluble proteins. The soluble lysate was filtered through a 0.45 μM sterile filter and loaded onto a 5 mL nickel ion HisTrap purification column (Cytiva, Marlborough, MA, USA). Column washing was performed using 20 mM HEPES, pH 7.5, 500 mM NaCl, and 20 mM imidazole. Protein elution was performed by increasing the imidazole concentration up to 250 mM. The cleanest eluate was concentrated using an Amicon filter and loaded onto a Superdex 75 size exclusion chromatography column (GE Healthcare Life Sciences, Chicago, IL, USA) connected to an AKTA FPLC system. The Superdex 75 column was equilibrated in 20 mM HEPES, pH 7.5, 500 mM NaCl. Proteins were separated using a flow rate of 0.75 ml / min. Samples belonging to the most prominent peaks were retained and loaded onto an SDS-PAGE gel to verify sample purity. MC4 (SEQ ID NO: 7) is described in Vercammen et al., 2004.

[0151] Enzyme activity assay Protease activity was measured by quantifying the fluorescence intensity emitted from the AMC (7-amino-4-methylcoumarin) group of the fluorescence-generating substrate Z-FR-AMC (PeptaNova, Sandhausen, Germany) at excitation and emission wavelengths of 383 nm and 455 nm, respectively, using a Tecan Infinite M200 Microplate Reader System (Mannedorf, Switzerland). All proteolytic assays were performed in 20 mM HEPES (pH 7.0) containing 150 mM NaCl, various CaCl2 concentrations, and 5 mM DTT. Buffers containing 100 mM acetate (pH 4–pH 5.5), 100 mM MES (pH 6–pH 6.5), 100 mM HEPES (pH 7.0–pH 8.0), 100 mM Tris (pH 8.5–pH 9), and 100 mM CAPS (pH 9.5–pH 11) were used to estimate the optimal pH. 0.2 μg of recombinant protease was used, and the concentration of the fluorescence-generating substrate was 5 μM.

[0152] Preparation of TTR aggregates TTR was expressed and purified according to a previously described procedure (Pinheiro et al., 2021). Briefly, TTR aggregation was induced by mixing 7 μM purified TTR with an equal volume of 400 mM sodium acetate, 200 mM KCl, pH 4.4 to obtain a final TTR concentration of 3.5 μM. The sample was incubated under static conditions at 37°C for 72 hours. The aggregated sample was centrifuged at 20,000 × g for 1 hour to recover the insoluble material, which was then resuspended in 20 mM HEPES, 150 mM NaCl, pH 7.5 to a concentration of 100 μM.

[0153] In vitro cell-free disaggregation assay Endpoint deaggregation was performed by co-incubating 7 μM TTR aggregates with 0.25 mg mL-1 of protease at 37°C in the presence of 5 mM DTT and 5 mM CaCl2. Protease deaggregation was monitored using sample turbidity, SDS-PAGE, and transmission electron microscopy.

[0154] Turbidity assay The turbidity of the sample was monitored using synchronized light scattering as an indicator of the amount of aggregated material. Spectra were recorded using a JASCO Spectrofluorometer FP-8200 with an excitation wavelength of 360 nm and an emission range of 340–380 nm. The excitation and emission bandwidths were set to 5 nm. Light scattered at 360 nm was used as a measure of turbidity.

[0155] Protein extraction and immunoblotting 500 milligrams of leaf material were mixed with 2 ml of extraction buffer (50 mM HEPES pH 7.3, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA pH 8, 5 mM DTT, 1% PVPP, and 1× protease inhibitor cocktail (Sigma, P599)) and centrifuged at 14,000 × g for 10 minutes at 4°C. 100 μl of 5× Laemmli sample buffer was added to the supernatant and boiled for 5 minutes. An equal volume of supernatant was loaded onto a 12% SDS-PAGE gel. The antibodies used for immunoblotting were as follows: α-GFP-HRP (1:5,000 Milteny Biotec), α-RFP-HRP (1:5,000 Abcam), α-myc (1:10,000 Sigma-Aldrich), α-actin (dilution 1:5,000 Agrisera), α-Hsp90-1 (1:2,000 Abcam), and α-polyQ (1:1,000 Merck).

[0156] Filter trap assay Protein extracts were obtained using a natural lysis buffer (300 mM NaCl, 100 mM HEPES pH 7.4, 2 mM EDTA, 2% Triton X-100) supplemented with a protease inhibitor cocktail that did not contain EDTA. When processing the plant protein extracts, 1× plant protease inhibitor (Merck) was added to the natural lysis buffer. In experiments using HEK cells, the cells were homogenized by passing them through a 27G needle seven times. Cell debris was removed by several centrifugation steps at 8,000×g for 10 minutes at 4°C. The supernatant was collected and the protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). A cellulose acetate membrane filter (GE Healthcare Life Sciences) was placed in a slot blot apparatus (Bio-Rad) connected to a vacuum system. The membrane was equilibrated by three washes with equilibration buffer (natural buffer supplemented with 0.5% SDS). Approximately 150 μg of protein extract was supplemented with 0.5% SDS at a final concentration, loaded through the membrane, and filtered. The membrane was then washed three times with 0.2% SDS. The membrane was blocked with 3% BSA in TBST for 30 minutes, followed by three washes with TBST. The membrane was incubated with the indicated antibody, then washed three times over 5 minutes, and incubated with the secondary antibody in 3% BSA in TBST for 30 minutes. The membrane was developed using Odissey DLx (Licor). The extract was also analyzed by SDS-PAGE and Western blotting to determine the loading control.

[0157] heat treatment A heat tolerance assay was performed using 5-day-old seedlings. Seedlings were grown at 22°C for 5 days, placed in a hot air incubator set to 37°C for 90 minutes, placed in a growth chamber set to 37°C for 90 minutes, incubated at 45°C for 90 minutes, and recovered at 22°C for 8 days. The percentage of seedlings in each phenotypic class was calculated based on the results from three biological replications. At least 50 seedlings of each genotype were used in each biological replication.

[0158] Bioinformatic analysis The inherently disordered area of ​​MC1 is represented in the D2P2 database (D 2 P 2 Predictions were made using Oates et al. (2013) and DISOPRED3 (Jones and Cozzetto, 2015). LLPS predisposition was assessed using the PSPredictor tool (Chu et al., 2022). MC1 amino acid aggregation tendencies were analyzed using Aggrescan3D (Zambrano et al., 2015) with Alphafold2 (Jumper et al., 2021) predicted MC1 structures as input files.

[0159] statistical analysis All quantitative analyses and statistical tests were performed using R software. T-tests were used to compare the significance of differences between two experimental groups. One-way ANOVA or Kruskal-Wallis one-way ANOVA was performed to compare the significance of differences between multiple experimental groups, as shown in each experiment. Different letters indicate statistically significant differences between samples.

[0160] 2.Results MC1 dynamically localizes to cytoplasmic stress granules when subjected to acute proteotoxic stress. To gain a deeper understanding of the function of MC1 (AtMC1), we generated transgenic Arabidopsis thaliana lines expressing green fluorescent protein-tagged MC1 under the control of the 35S promoter in the mc1 mutant background (mc1 Pro35S::MC1-GFP) and evaluated its subcellular localization. Under basal conditions, MC1 showed a diffuse pattern in both the cytoplasm and the nucleus. Dynamic cytoplasmic puncta were rapidly formed upon heat stress treatment (39°C for 40 min), which grew in size over time and disappeared immediately after returning plants to non-stress conditions (Figure 1A). When MC1-GFP was expressed under the control of its native promoter, the same heat-responsive relocalization pattern was observed. To assess whether such puncta correspond to stress granules, we used cycloheximide, an inhibitor of translational elongation that blocks SG assembly and promotes disassembly of existing SGs. Application of cycloheximide blocked the appearance of the observed heat stress-induced puncta (Figure 1B).

[0161] In summary, these data demonstrate that MC1 dynamically relocalizes to SGs upon heat treatment, which disappears upon stress removal.

[0162] The intrinsically disordered region of MC1 is prone to aggregation, leading to insolubility in vitro. Using a combination of two prediction software (D 2 P 2Using https: / / d2p2.pro and DISOPRED3 (http: / / bioinf.cs.ucl.uk / psipred; Oates et al., 2013; Jones and Cozzetto, 2015), we identified potential intrinsically disordered regions (IDRs) within the MC1 amino acid sequence (SEQ ID NO: 1). Based on these predictions, MC1 contains two major IDRs (Figure 2A), one in the N-terminal prodomain and the other in the predicted C-terminal p10 catalytic domain region, known as the 360 ​​loop. Since aggregation tendency is also considered an intrinsic determinant of phase separation, we used AGGRESCAN3D (A3D) to predict the structural aggregation tendency of MC1 based on its α-fold predicted structure (http: / / biocomp.chem.uw.edu.pl / A3D / ; Zambrano et al., 2015). Interestingly, MC1 exhibits a strong aggregation tendency in the predicted IDR (Figure 2B). In particular, the 360 ​​loop exhibits the longest amino acid stretch, along with a high aggregation tendency score.

[0163] The 360 ​​loop of MC1 is a highly hydrophobic sequence present only in plant type I metacaspases. In fungi, protozoa, and red algae, type I metacaspases do not contain the 360 ​​loop, and interestingly, these proteins are soluble when their full length is recombinantly produced in vitro. In contrast, previous efforts to produce recombinant plant type I metacaspases have proven unsuccessful due to the fact that their full-length versions are highly insoluble. Removal of the 360 ​​loop (amino acids 318-346 of SEQ ID NO: 1) and the prodomain (amino acids 2-77 of SEQ ID NO: 1) was necessary to express soluble MC1 (ΔNMC1Δ360 or rMC1, SEQ ID NO: 2) in E. coli (Figures 3A and 3B). MC1Δ360 (SEQ ID NO: 5), produced by removing only the 360 ​​loop, was not sufficient to solubilize MC1 (Figure 3B). A soluble MC1 variant (SEQ ID NO: 2) lacking the prodomain and 360 loop, possessing an N-terminal hexahistidine tag (6xHis), was homogeneously purified by nickel affinity chromatography and further isolated by size exclusion chromatography to remove trace impurities (Figure 3C). Importantly, rMC1 was catalytically active, as demonstrated by its ability to cleave the previously reported inhibitor *Arabidopsis thaliana* SERPIN1 in plant substrates. rMC1 functioned as a standard type I metacaspase, exhibiting a calcium ion dependence at low millimolar concentrations (1–10 mM) and neutral pH (pH 7) for maximum cleavage of the fluorescence-generating substrate Z-FR-AMC. Consistent with the observed trypsin-like activity of metacaspases, rMC1 cleaved the trypsin substrate β-casein (rβ-casein). We also prepared and purified rMC1 (rMC1CA, SEQ ID NO: 5) possessing a point mutation from catalytic cysteine ​​(C220) to alanine. Importantly, rMC1CA was unable to cleave SERPIN1 or rβ-casein. In summary, these data indicate that MC1 contains two distinct IDRs, which are prone to aggregation and result in high insolubility for protein overexpression and in vitro isolation. Upon removal, proteolytically active and soluble rMC1 could be readily expressed and isolated.

[0164] MC1 can specifically degrade aggregated proteins. First, we investigated whether Arabidopsis thaliana plants lacking MC1 exhibited disadvantages in protein aggregate removal and survival after prototoxic stress. To monitor changes in protein aggregation, we used filter trap analysis, a robust method for detecting and quantifying protein aggregates, in 5-day-old WT and mc1 seedlings after heat stress. Seedlings were subjected to moderate heat shock at 37°C for 90', followed by recovery at 22°C for 90' and severe heat shock at 45°C for 90'. Unstressed seedlings were used as controls, and samples were collected after 1 day of recovery at 22°C. Analysis of the accumulation of aggregated forms of actin, Hsp90, and polyQ stretch-containing proteins showed that they all aggregated in Arabidopsis thaliana after heat stress (Llamas et al., 2022). Under basal conditions, protein aggregates (actin, Hsp90-tagged or polyQ-containing proteins) can be efficiently removed by the PQC mechanism, making them barely detectable in both wild-type (WT) and mc1 mutants (Figure 4A). Stress, such as heat shock, leads to a rapid over-accumulation of misfolded proteins, which often exceeds the cell's PQC capacity, resulting in protein aggregation detectable by filter traps (Figure 4A). Plants lacking MC1 accumulate more aggregated proteins than WT after proteotoxic stress, indicating a reduced ability to manage protein aggregation and proteotoxic stress in mutants.

[0165] Next, we evaluated the ability of MC1 to degrade pathological protein aggregates in a cell-free state. Equimolar concentrations of rMC1 (SEQ ID NO: 2) were co-incubated with aggregates of human transthyretin (TTR). TTR is a homotetrameric thyroxine transporter protein whose tetrameric dissociation event leads to aggregation (Quintas et al., 2001). Extracellular insoluble deposits of TTR in some human organs cause a distinctly progressive and fatal clinical syndrome known as transthyretin amyloidosis (Rapezzi et al., 2010). Monitoring protein aggregation using turbidity measurements revealed that rMC1 treatment reduced TTR aggregates by 90% (Figure 4B). This protein aggregate removal activity is dependent on MC1 catalytic activity, as evidenced by the absence of disaggregation in the catalytically dead mutant rMC1CA (SEQ ID NO: 6). Indeed, a 3-fold higher turbidity signal was observed in samples treated with rMC1CA, suggesting that inactive MC1 aggregates when TTR-insoluble aggregates are present in the reaction. Visual inspection of TTR samples by transmission electron microscopy (TEM) (Figure 4C) confirmed the deaggregation activity of MC1. The need for catalytic activity for deaggregation is consistent with observations that aggregated TTR is significantly degraded in the presence of rMC1 (lane 3), as demonstrated by SDS-PAGE (Figure 4D). Notably, rMC1 acts as a specific disaggregase, removing protein aggregates but failing to degrade soluble TTR in its innate tetrameric state (nTTR), thus not removing the functional form of the protein (lanes 3 and 4 in Figure 4D, respectively). The obtained data indicate that rMC1 specifically targets and degrades the aggregated form and pathogenicity of TTR. Notably, this disaggregase activity against TTR aggregates was not observed in samples incubated with MC4 (Figures 4B and 4D). As previously mentioned, the activity of the MC4 protease was confirmed by rapid self-processing in the presence of calcium (Figure 4D, lane 10).

[0166] Finally, the ability of MC1 to degrade protein aggregates was tested in in vitro cultured cells. For this purpose, two well-established model systems were used: i) human embryonic kidney (HEK) cells expressing poly-Q elongated huntingtin morphology (Q74) that causes aggregation and prototoxicity and is used as a surrogate for neurodegenerative Huntington's disease (Jimenez-Sanchez et al., 2015), and ii) yeast expressing constitutively misfolded carboxypeptidase (ΔssCPY*) that forms insoluble protein aggregates in response to stress (Park et al., 2007). Co-expression of mCherry-fused Q74 and full-length MC1 (SEQ ID NO: 1) in HEK cells reduced protein aggregates compared to expression of mCherry-fused Q74 alone, as demonstrated by filter-trap analysis using anti-mCherry antibodies (Figure 4E). In yeast, ΔssCL* fused with the prototrophic marker Leu2 and a C-terminal myc tag was expressed in wild-type (WT), a mutant lacking the single yeast metacaspase MCA1 (ymca1Δ), and ymca1Δ supplemented with a WT copy of Arabidopsis thaliana MC1. All strains grew normally in control medium, but in leucine-deficient selective medium, WT yeast showed reduced growth capacity due to the degradation of misfolded ΔssCL* by the PQC system (Figure 5A, right panel and Figure 5B). In contrast, ymca1Δ was unable to degrade ΔssCL* and therefore grew normally in leucine-selective medium. This phenotype could be fully complemented by AtMC1 (SEQ ID NO: 1), which restored yeast WT growth levels by its ability to degrade ΔssCL* (Figure 5A, right panel and Figure 5B). In summary, these data demonstrate that MC1 can degrade protein aggregates, particularly pathogenic protein aggregates, both under cell-free and intracellular conditions.

[0167] List of References Shorter J., "Designer protein disaggregases to counter neurodegenerative disease", 2017, Curr Opin Genet Dev., vol.44, pp.1-8

[0168] Oates, M.E. et al. (2013) "D2P2: database of disordered protein predictions", Nucleic Acids Research, 41(Database Issue)

[0169] Chu, X. et al. (2022) "Prediction of liquid-liquid phase separating proteins using machine learning", BMC Bioinformatics, 23(1), pp.1-13

[0170] Zambrano, R. et al. (2015) "AGGRESCAN3D(A3D): server for prediction of aggregation properties of protein structures", Nucleic Acids Research, 43(W1), pp.W306-W313

[0171] Jumper, J. et al. (2021) "Highly accurate protein structure prediction with AlphaFold", Nature 2021 596:7873, 596(7873), pp.583-589

[0172] Llamas, E. et al. (2022) "Chloroplast protein import determines plant proteostasis and retrograde signaling", bioRxiv, p.2022.03.19.484971

[0173] Quintas,A.et al.(2001)「Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants」,The Journal of biological chemistry,276(29),pp.27207-27213

[0174] Rapezzi,C.et al.(2010)「Transthyretin-related amyloidoses and the heart:a clinical overview」,Nature reviews.Cardiology,7(7),pp.398-408

[0175] Jimenez-Sanchez,M.et al.(2015)「siRNA screen identifies QPCT as a druggable target for Huntington’s disease.」,Nature Chemical Biology,11(5),pp.347-354

[0176] Park,S.H.et al.(2007)「The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system」,Molecular Biology of the Cell,18(1),pp.153-165

[0177] Gardner,J.M.and Jaspersen,S.L.(2014)「Manipulating the yeast genome:deletion,mutation,and tagging by PCR」Methods in molecular biology(Clifton,N.J.),1205,pp.45-78

[0178] Balaji,V.et al.(2022)「A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing」Molecular cell,82(17),pp.3239-3254.e11

[0179] Lazaro-Silva,D.N.et al.(2015)「The Use of DNA Extraction for Molecular Biology and Biotechnology Training:A Practical and Alternative Approach」,Creative Education,6(8),pp.762-772

[0180] Eisele,F.and Wolf,D.H.(2008)「Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1」,FEBS letters,582(30),pp.4143-4146

[0181] de Marco,A.et al.(2007)「Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E.coli’,BMC Biotechnology」7,p.32

[0182] Vercammen,D.et al.(2004)「Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine」,The Journal of biological chemistry,279(44),pp.45329-45336

[0183] Pinheiro,F.et al.(2021)「Tolcapone,a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis」,The FEBS Journal,288(1),pp.310-324

[0184] Coll,N.S.et al.(2010)「Arabidopsis type I metacaspases control cell death」,Science,330(6009),pp.1393-1397

[0185] Copolovici D.M.et al.,「Cell-Penetrating Peptides:Design,Synthesis,and Applications」,2014,ACS Nano,2014,8(3),pp 1972-1994

[0186] Altschul et al.,「Basic local alignment search tool」,1990,J.Mol.Biol,v.215,403-410ページ

[0187] EMBOSS:The European Molecular Biology Open Software Suite,Rice et al.,2000,Trends Genet.16:276-277

Claims

1. A polypeptide having disaggregase activity for use in pharmaceuticals, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

2. A polypeptide having disaggregase activity for use in the prevention or treatment of diseases or disorders related to protein aggregates in a subject, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

3. The polypeptide for use according to claim 2, wherein the protein aggregate comprises at least one protein selected from the group consisting of prion protein, polyglutamine protein, amyloid precursor protein, α-synuclein, superoxide dismutase, transthyretin, tau, immunoglobulin, amyloid-A, low-density lipoprotein receptor, crystallin, β2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, FUS, islet amyloid polypeptide, ANF, gelzolin, insulin, lysozyme, p53, and fibrinogen.

4. The polypeptide for use according to claim 3, wherein the protein aggregate comprises a polyglutamine protein or transthyretin.

5. The polypeptide for use according to claim 4, wherein the polyglutamine protein is huntingtin.

6. The diseases or disorders caused by protein aggregates include Alzheimer's disease, transthyretin amyloidosis, cerebral β-amyloid angiopathy, retinal ganglion cell degeneration, bovine spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Streussler-Scheinker syndrome, fatal familial insomnia, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, and familial Danish dementia. Dementia, hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, seipinopathy, familial amyloidosis neurological lesions, senile systemic amyloidosis, serpin disease, AL amyloidosis, AA amyloidosis, type II diabetes, aortic medial amyloidosis, ApoAI amyloidosis, ApoII amyloidosis, ApoAIV amyloidosis, Finnish-type familial amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis / myopathy, cataracts, cancer associated with p53 aggregates, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma, genetic Polypeptides for use according to any one of claims 2 to 5, selected from the group consisting of lattice corneal dystrophy, lichenoid amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic tumor amyloidosis, seminal vesicle amyloidosis, cystic fibrosis, sickle cell disease, severe myopathy, von Hippel-Lindau disease, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Angelman syndrome, giant axonal dysfunction, and inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD).

7. The polypeptide for use according to claim 6, wherein the disease or disorder caused by the protein aggregates is Huntington's disease or transthyretin amyloidosis.

8. A polypeptide for use according to any one of claims 2 to 7, wherein the target is a mammal, particularly a human.

9. The polypeptide for use according to any one of claims 1 to 8, wherein the polypeptide includes variants thereof that retain the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or disaggregase activity, and are identical to SEQ ID NO: 1 or SEQ ID NO: 2 by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

10. The polypeptide for use according to any one of claims 1 to 9, wherein the polypeptide comprises SEQ ID NO: 1 or SEQ ID NO:

2.

11. An expression vector for use in pharmaceuticals, particularly in the prevention or treatment of diseases or disorders associated with protein aggregates in a subject, comprising a nucleic acid encoding a polypeptide as defined in any one of claims 1 to 10, which is operably linked to an expression promoter.

12. A polypeptide for use according to any one of claims 1 to 10 or an expression vector for use according to claim 11, administered in the form of a pharmaceutical composition together with at least one pharmaceutically acceptable excipient, diluent, or carrier.

13. Together with at least one pharmaceutically acceptable excipient, diluent, or carrier, A polypeptide having disaggregase activity, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO:

2. Nucleic acids encoding the polypeptide having disaggregase activity, Gene constructs containing the aforementioned nucleic acids, or Expression vector containing the aforementioned gene construct A pharmaceutical composition containing the following:

14. An in vitro or ex vivo method for eliminating or preventing the formation of protein aggregates in a protein-containing composition, the method comprising contacting the protein-containing composition with a polypeptide having disaggregase activity, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

15. In vitro or ex vivo use of a disaggregase-active polypeptide for eliminating or preventing the formation of protein aggregates in a protein-containing composition, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

16. A disaggregase-labeled polypeptide having disaggregase activity for use in a method for imaging target protein aggregates, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the method comprises (i) administering a detectable amount of the detectably labeled polypeptide to the target, and (ii) detecting the polypeptide associated with the protein aggregates.

17. Use of a disaggregase-active polypeptide for imaging protein aggregates, wherein the polypeptide is a detectably labeled polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

18. A polypeptide having disaggregase activity for use in vivo diagnosis or prognosis of a disease or disorder associated with protein aggregates in a subject, wherein the polypeptide is detectably labeled and comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof that retains the disaggregase activity and is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

Images