Cathepsin d peptides as therapeutic agents in alzheimer's disease

Abstract

Cathepsin D peptides are provided that are capable of interacting with and / or inhibiting the formation of beta amyloid aggregates, thereby leading to a competitive reduction of amyloid- amyloid interactions. Also provided are recombinant expression vectors encoding said peptides as well as of pharmaceutical formulations comprising said peptide-analogues. Said peptides, compositions and recombinant vectors are useful as therapeutic agents in the treatment and / or amelioration of the symptoms of amyloidoses such as Alzheimer's disease.

Description

[0001] CATHEPSIN D PEPTIDES AS THERAPEUTIC AGENTS IN ALZHEIMER’S DISEASE

[0002] TECHNICAL FIELD

[0003] The present invention relates to Cathepsin D peptides for use as therapeutic agents in amyloidoses such as Alzheimer’s disease. It also relates to the provision of recombinant expression vectors encoding said peptides as well as of pharmaceutical formulations comprising said peptides.

[0004] BACKGROUND OF THE INVENTION

[0005] Alzheimer’s disease (AD) is a progressive, age-associated neurodegenerative disease and is known as the most common form of dementia worldwide. In its early stages, it is characterized by memory loss and can ultimately lead to cognitive decline, affecting the patient’s behavior and speech patterns, the sense of orientation, as well as the motor system. The two key pathological hallmarks of AD are the extracellular amyloid plaques and the neurofibrillary tangles of the microtubule binding protein tau. Amyloid plaques are mainly constituted by the amyloid-P peptide (AP), which is a proteolytic product of the amyloid precursor protein (APP). The processing of APP for the generation of Ap is executed by two aspartyl proteases, namely, P- and y-secretase. Particularly, y- secretase cleavage determines the length of Ap, whose principal forms comprise of either 38, 40 or 42 amino acid residues. The latter, Ap42, has been characterized as the most aggregation-prone of all Ap forms. Ap monomers have the propensity to self-assemble into a spectrum of intermediate aggregation states, including dimers, trimers, toxic oligomers, protofibrils, and ultimately mature amyloid fibrils, which concentrate within extracellular amyloid plaques, a defining pathological hallmark of AD. Despite its pivotal role in AD, in the brains and cerebrospinal fluid of healthy humans, Ap is physiologically active and does not promote neurodegeneration. Neuronal damage is believed to be caused by the interaction, self-association and ultimately aggregation of Ap molecules. Specifically, within the amyloid deposits, Ap is organized into insoluble amyloid fibrils. It is now known that amyloid fibrils have a common morphology in terms of their basic characteristics, as they appear as straight, unbranched, with a diameter ranging between 70 - 120A. Depending on the conditions, however, Ap and other amyloidogenic proteins can form both amorphous and ordered aggregates.

[0006] A number of other proteins co-localize in the Ap fibrillar deposits, including serum amyloid P component (SAP), clusterin, cathepsin D and apolipoprotein E (apoE) (Nastou KC et al. (2019) Amyloid. 26, 112-117). These co-localized proteins are known as amyloid signature proteins (Buxbaum et al., Amyloid. 2022 Dec;29(4):213-219). Although the precise roles of these proteins in either promoting or inhibiting oligomerization and the processes of amyloid formation remain incompletely understood, it is established that some of them possess the capability to form amyloid fibrils or can undergo partial unfolding to adopt an amyloid fibril-like structure under specific conditions (Biza et al., PloS one. 2017;12:e0173163). Many studies have focused on the elucidation of the possible self-assembly mechanisms of amyloidogenic proteins and several investigations have studied the role of amino acid sequence in guiding protein aggregation, concluding that not all regions of a polypeptide chain have the same importance. Specifically, experimental analyses showed that the amyloidogenic potential of these proteins is concentrated in specific regions of the polypeptide chain. These regions are short in length (Ivanova et al., Proc Natl Acad Sci USA. 2004;101: 10584-10589; Ventura et al., Proc Natl Acad Sci USA. 2004;101:7258-7263) and are referred to as beta-aggregating regions, aggregation-prone regions or amyloidogenic determinants. It was later confirmed that small amino acid segments guide the selfassembly of proteins into amyloids, without requiring the rest of the protein backbone to participate in the amyloidogenic core of the fibrils (Teng and Eisenberg, Protein Eng Des Sei 2009;22:531- 536).

[0007] Cathepsin D (CathD), a predominant lysosomal protease, plays a crucial role in cellular processes. Structurally, it exists in single-chain and double-chain forms, predominantly found in endosomes and lysosomes, respectively. The three-dimensional structure of CathD is characteristic of aspartic proteases, featuring amino-terminal and carboxy-terminal domains with P-sheet fold and an active center. It functions as an endopeptidase, responsible for degrading misfolded proteins, long-lived proteins, and denatured proteins. Additionally, CathD regulates various cellular processes, including antigen processing, programmed cell death, and cell signaling. CathD has been associated with diseases characterized by amyloid fibril formation, such as AD and AA amyloidosis (van der Hilst et al., Eur. J. Clin. Invest. 2009;39:412-416). CathD is implicated in processing APP, apolipoprotein E, and tau protein, all significant factors in AD pathogenesis. Moreover, studies in AD-affected mice have indicated that A peptide aggregates can disrupt lysosomal pH, reducing CathD activity and causing lysosomal damage. Another mechanism suggests that Ap42 peptide inhibits CathD function, potentially preventing the degradation of tau protein by this protease.

[0008] Despite the fact that Cathepsin D has been identified as a key component in the pathology of amyloidoses such as AD, the exact interplay between cathepsin D and pathology is still elusive. Notably, the localization of CathD within amyloid deposits in AD patients (Urbanelli et al., Neurobiol Aging. 2008;29: 12-22) opens up the possibility that CathD localization and / or balance may be deregulated in AD.

[0009] Therefore, the need remains to further elucidate the role of CathD in amyloidogenesis and provide novel therapeutic tools for the treatment of AD and other amyloidoses specifically targeting this protein. SUMMARY OF THE INVENTION

[0010] The invention described herein relates to novel means for the inhibition of amyloid protein aggregation by providing peptides that exhibit a strong amyloidogenic profde. Specifically, the present invention provides isolated Cathepsin D peptides for use in the treatment or prevention of amyloidosis, wherein each peptide comprises at least one beta-aggregating region (aggregation- prone region). Each peptide is identical to or has at least 80% identity to a contiguous stretch of 50 amino acids or less, naturally occurring in Cathepsin D.

[0011] In certain embodiments, the peptide for use in the treatment or prevention of amyloidosis comprises at least 5 contiguous amino acids. In a preferred embodiment, said peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a peptide having an amino acid sequence that is at least 80% identical to any of the amino acid sequences of SEQ ID NO: 1 to 10. In a more preferred embodiment, said peptide is selected for the group consisting of a peptide having an amino acid sequence that is at least 90% identical to any of the amino acid sequences of SEQ ID NO: 1 to 10.

[0012] In a certain embodiment, said peptide is selected for the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7, or a peptide having an amino acid sequence that is at least 80% identical to any of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7.

[0013] The peptides disclosed herein are capable of interacting with and / or inhibiting the formation of beta amyloid aggregates, thereby leading to a competitive reduction of amyloid-amyloid interactions.

[0014] In certain embodiments, said amyloidosis is Alzheimer’s disease.

[0015] The invention also provides pharmaceutical compositions comprising at least one peptide as defined herein; and a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, said pharmaceutical composition is for use in the treatment of an amyloidosis such as Alzheimer’s Disease.

[0016] Provided are also recombinant vectors comprising a polynucleotide encoding at least one peptide according to the present invention, wherein said vector, upon transformation to a cell or organism, produces said peptide in said cell or organism.

[0017] The present invention also discloses an isolated Cathepsin D peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. In a preferred embodiment, said peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10.

[0018] The disclosure herein addresses the needs in the art by providing for peptides, compositions and recombinant vectors for use in the treatment of an amyloidosis such as AD. To date, no currently approved drugs are available which can prevent protein aggregation formation, disrupt existing protein aggregates and reduce the overall amount of protein aggregates in a patient suffering from an amyloidosis. The disclosed peptides, compositions and recombinant vectors can achieve these outcomes by disrupting or preventing the formation of amyloidogenic aggregates.

[0019] Other aspects and benefits of the present invention will become apparent from the detailed description to follow.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will now be described with reference to certain embodiments thereof which are illustrated in the accompanying drawings. It should be noted that the accompanying drawings illustrate preferred embodiments of the invention, therefore should not be considered as limiting the scope of the invention.

[0022] Fig. 1 is a diagrammatic presentation of the Amyloid propensity histogram of Homo sapiens CathD.

[0023] Fig. 2 shows experimental results of self-aggregation assays for the CathD peptides. (A, C, E, G, I, K, M, O, Q, S): ThT fluorescence emission spectra; (B, D, F, H, J, L, N, P, R, T): Transmission electron micrographs and negative staining.

[0024] Fig. 3 shows the ThT fluorescence emission spectrum (A) and transmission electron micrograph (B) of the Ap42peptide.

[0025] Fig. 4 shows the ThT fluorescence emission spectra (A, C, E, G, I, K, M, O, Q and S) and transmission electron micrographs (B, D, F, H, J, L, N, P, R and T) illustrating the coincubation of Ap42peptide with each CathD peptide.

[0026] Fig. 5 is a diagrammatic presentation of the delay of paralysis in the AD temperature-inducible C. elegans CL4176 strain under the effect of different concentrations of the CathD [26-56] peptide. Fig. 6 presents the paralysis curves of AD C. elegans CL4176 strain after continuous exposure to different concentrations of CathD[28-32] or control sample (H2O).

[0027] Fig. 7 shows the paralysis curves of AD C. elegans CL4176 strain after continuous exposure to different concentrations of CathD[51-55] or control sample (H2O).

[0028] Fig. 8 is a diagrammatic presentation of amyloid deposits in the anterior area of the CL2331 strain after continuous exposure to a specific concentration of CathD[26-56] (0.05 pM), CathD[28-32] (0.5 pM), CathD[51-55] (0.5 pM) or control sample (H2O).

[0029] Fig. 9 presents the percent cellular survival of SH-SY5Y human neuroblastoma cells being exposed to exogenously added Ap42oligomers in the presence or absence of Cath[26-56] peptide (A, B), Cath[28-32] (C, D) and Cath[51-55] (E, F).

[0030] Fig. 10 schematically presents the results from the molecular dynamics simulations examining the binding between CathD peptides and the Ap42peptide.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] The present disclosure provides peptides and peptide-analogues of CathD that exhibit a strong amyloidogenic profile.

[0033] The term “beta-aggregating region” or “aggregation-prone region” as used throughout the description and claims refers to the short stretches (5-30 residue long) within a protein that have the tendency to self-assemble into ordered intermolecular beta-sheet structures called “cross-beta” spines, guiding a globular protein to misfold, thus leading to amyloid fibril formation.

[0034] The term “isolated peptide” is used interchangeably with the term “peptide” throughout the claims and description to refer to peptides that are created by chemical synthesis methods and are designed to correspond to naturally occurring protein regions.

[0035] Human Cathepsin D is among others also known as CTSD, CLN10, CPSD, Ceroid-Lipofuscinosis, Neuronal 10; EC 3.4.23.5, Epididymis Secretory Sperm Binding Protein Li 130P, Cathepsin D (Lysosomal Aspartyl Protease) and Lysosomal Aspartyl Protease. Database IDs for the gene and / or protein are HGNC: 2529; NCBI Gene: 1509; Ensembl: ENSG00000117984; OMIM®: 116840 and UniProtKB / Swiss-Prot: P07339.

[0036] The term “polyethylene glycol (PEG)” modification as used throughout the description and claims, refers to the modification resulting from the covalent attachment of a PEG polymer chain to a peptide. The PEG polymer may be attached at the N-terminus, C-terminus of the peptide, the side chain of Lys and / or the thiol group of Cys.

[0037] The term “non-natural amino acid” as used throughout the description and claims, also known as “noncanonical amino acid” or “nonstandard amino acid” refers to an organic compound that is not among those encoded by the standard genetic code, or incorporated into proteins during translation. Therefore, non-natural amino acids include amino acids or analogs of amino acids, but are not limited to, the D-isostereomers of amino acids, the beta-amino-analogs of amino acids, homocitrulline, homoarginine, hydroxyproline, homoproline, ornithine, 4-amino-phenylalanine, cyclohexylalanine, a-aminoisobutyric acid, N-methyl-alanine, N-methyl-glycine, norleucine, N- methyl-glutamic acid, tert-butylglycine, a-aminobutyric acid, tert-butylalanine, 2-aminoisobutyric acid, a-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid, selenomethionine, dehydroalanine, lanthionine, y-amino butyric acid, and derivatives thereof wherein the amine nitrogen has been mono- or di-alkylated.

[0038] The term “beta-amino acid” as used throughout the description and claims refers to an analogue of an alpha-amino acid where the amino group is attached to the beta-carbon instead of the alphacarbon. Common and well known beta-amino acids include beta-alanine (3 -aminopropionic acid), beta-phenylalanine, beta-tyrosine, and their derivatives, among others.

[0039] The term “cyclic peptide” as used throughout the description and claims refers to a peptide with a cyclic ring structure. A cyclic peptide can be formed through a connection between the amino- and carboxyl- ends of the peptide; between the amino end and a side chain; the carboxyl end and a side chain (such as via an ester bond between the C-terminal and a serine or threonine sidechain); or two side chains (such as via disulfide bridging of cysteine residues, via amide formation between and aspartic acid or glutamic acid residue and one of the basic amino acids, via biaryl ethers of tyrosine or hydroxyphenylglycine, or other methods); or more complicated arrangements.

[0040] The term “peptidomimetic” as used throughout the description and claims refers to a peptide-like polymer that mimics the basic structure of the peptide and may include non-natural peptide linkages. The amino acids forming all or a part of a peptidomimetic may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-natural amino acids, post- translationally modified amino acids, enzymatically modified amino acids, and the like. A peptidomimetic may for instance be formed from a backbone that is suitably modified, such as extending the chain and adding heteroatoms in such a way that the peptide is protected from proteolytic degradation. The term “stapled peptide” as used throughout the description and claims refers to a modified peptide, that is constrained by a synthetic brace that forces the peptide structure into an a-helical one. The cross-link is obtained by a covalent linkage between two amino acid side-chains, forming a peptide macrocycle.

[0041] The inventors have unexpectedly found that certain regions of the Cathepsin D amino acid sequence exhibit a high propensity for self-aggregation. Specifically, a computational analysis of the amyloidogenic profile of mature Homo sapiens Cathepsin D (Uniprot AC: P07339, Release 2023_05) (SEQ ID NO: 12) was utilized in order to examine the presence of aggregation-prone regions (APRs) in this protein sequence. The analysis utilized the consensus algorithm AMYLPRED2 (Tsolis et al., PloS one. 2013;8:e54175, http: / / aias.biol.uoa.gr / AMYLPRED2 / ), which combines information from up to eleven prediction algorithms, using a threshold of n / 2 (where n represents the number of algorithms utilized in each iteration, rounded down to the nearest integer). The algorithm results revealed increased aggregation propensity in specific sequence segments, typically spanning between 5 to 30 amino acid residues. Other suitable publicly available algorithms for predicting amyloid aggregation can be used instead of AMYLPRED2, such as those reviewed in Belli M. et al (EM BO reports. 2011; 12(7), 657-663).

[0042] Aggregation prone regions are mainly hydrophobic. However, other structural properties such as beta-propensity, side chain size and charge are equally important for accurate prediction of aggregation propensity (De Baets G et al. Essays Biochem. 2014:56:41-52; Louros N et al. Nat Commun 2020; 11, 3314). That means that the aggregation interactions are sequence dependent. Protein aggregation is the outcome of highly specific, sequence- and structure dependent interactions between aggregation prone amino acid stretches.

[0043] Using the aforementioned algorithm, the inventors identified four significant regions: one encompassing 31 amino acid residues (CathD[26-56]), another comprising 24 amino acid residues (CathD[123-146]), a smaller region containing 12 amino acid residues (CathD[160-171]), and a segment consisting of 18 amino acid residues (CathD[318-335]). Within these regions, six oligopeptides were predicted by more than 5 AMYLPRED2 methods. These oligopeptides include two pentapeptides (CathD[28-32] and CathD [51-55]), a seven amino acid residue-long peptide (CathD[163-169]), and two hexapeptides (CathD[322-327] and CathD[328-333]). The amyloidogenic profile of CathD is presented in Figure 1.

[0044] In line with the strategy of designing inhibitors that mimic the site of self-association, and based on the analysis of the amyloidogenic profde of CathD, ten peptides were designed, shown in Table 1. Six of them were chosen to align with the aggregation hot-spots of CathD (CathD[28-32], CathD[51-55], CathD[124-128], CathD [163 -169], CathD[322-327] and CathD[328-333]), with the primary objective of directly targeting regions where amyloid formation is most prominent, thus effectively inhibiting Ap42aggregation. The remaining four peptides (CathD[26-56], CathD[123- 146], CathD[160-171] and CathD[318-335]) were designed to the length and physicochemical properties of Ap42. This choice was made in order to capture and mimic specific characteristics of Ap42associated with aggregation and amyloid formation, aiming to enhance the effectiveness of the approach disclosed herein in inhibiting these processes.

[0045] Table 1. The sequences of the predicted APRs of CathD.

[0046] The ten peptides resulting from the above analysis were chemically synthesized and were tested for their capacity to form beta-sheet-rich structures. As detailed in Example 1, the kinetics of amyloid formation were examined using the ThT Kinetic Assay and the results showed that all ten peptides bound ThT and displayed a strong ThT fluorescence signal, confirming their amyloid nature. The same conclusion was reached when the inventors used Transmission Electron Microscopy (TEM) and negative staining, as they observed fibrils with characteristics similar to amyloid fibrils. The results suggest that the full-length Cathepsin D may have amyloidogenic properties.

[0047] The inventors have also studied the inhibitory capacity of these peptides on the aggregation of Ap42, with the aim of shedding light on their potential to obstruct Ap42fibrillogenesis, a critical factor in AD pathology. To assess their effectiveness, in vitro experiments utilizing Transmission Electron Microscopy (TEM) and Thioflavin T (ThT) fluorescence measurements were conducted as explained in Example 2. The results showed that all peptides either inhibited or delayed Ap42fibril formation. Consequently, these peptide-analogues are able to disrupt the aggregation of Ap42. Furthermore, the impact of these peptides on Ap42-related neurotoxicity was assessed through in vivo experiments using an accepted as AD model transgenic Caenorhabditis elegcms strain that expresses the human Ap42peptide under a muscle-specific promoter and upon temperature upshift, they accumulate Ap peptide in their body wall muscle cells and become paralyzed in a few hours (Link et al., Neurobiol Aging 2003;24:397-413). The results shown in Example 4 revealed that CathD[26-56], CathD[28-32] and CathD[51-55] decelerated the rate of paralysis of CL4176 strain.

[0048] To evaluate the Ap aggregation state in vivo, the inventors took advantage of the CL2331 strain that expresses the human Ap3.42peptide fused to green fluorescent protein (GFP) in its body wall muscle cells. The animals are gradually filled with Ap aggregates that are visible through confocal microscopy (Link et al., Neurobiol. Dis. 2008;32:420-425). The results, detailed in Example 5, showed that treatment with CathD[26-56] significantly reduced the Ap aggregates. A similar tendency (that did not however reach statistical significance at the tested concentration) was revealed for CathD[28-32],

[0049] Additionally, in vitro cytotoxicity assays were performed using human neuroblastoma cells exposed to exogenously added Ap42peptide. The results shown in Example 6 revealed that all three peptides tested, CathD[26-56], CathD[28-32] and CathD[51-55], enhance survival of SH-SY5Y human neuroblastoma cells being exposed to exogenously added Ap42oligomers.

[0050] To unravel the molecular complexity of these interactions, molecular dynamics simulations were employed to explore the binding between CathD peptides and the Ap42peptide. The Molecular Docking simulations shown in Example 7 revealed that all peptides tested interacted with the Ap42oligomeric state, and after the 500 ns simulation, they induced changes to its secondary structure.

[0051] Thus, the present invention provides an isolated peptide of 5 to 50 amino acids in length, comprising the sequences of one or more of Regions 1 to 6, for use in the treatment or prevention of an amyloidosis, wherein Region 1 consists of amino acid residues 28-32 of human Cathepsin D (SEQ ID NO: 2); Region 2 consists of amino acid residues 51-55 of human Cathepsin D (SEQ ID NO: 3); Region 3 consists of amino acid residues 124-128 of human Cathepsin D (SEQ ID NO: 5); Region 4 consists of amino acid residues 163-169 of human Cathepsin D (SEQ ID NO: 7); Region 5 consists of amino acid residues 322-327 of human Cathepsin D (SEQ ID NO: 9); Region 6 consists of amino acid residues 328-333 of human Cathepsin D (SEQ ID NO: 10). The reference sequence of human Cathepsin D is defined by SEQ ID NO: 12. Each Region 1 to 6 allows up to one conservative amino acid substitution, In a preferred embodiment, the isolated peptide has a length of 5 to 45 amino acids. In a more preferred embodiment, the isolated peptide has a length of 5 to 40 amino acids. Even more preferably, the isolated peptide has a length of 5 to 35 amino acids. The term “conservative amino acid substitution” refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their amides and esters: Asp, Asn, Glu, Gin, cysteic acid and homocysteic acid; III. Polar, positively charged residues: His, Arg, Lys; Ornithine (Om) IV. Large, aliphatic, nonpolar residues: Met, Leu, He, Vai, Cys, Norleucine (Nle), homocysteine V. Large, aromatic residues: Phe, Tyr, Trp, acetyl phenylalanine.

[0052] As mentioned above, the APRs mainly contain hydrophobic amino acid residues. The APRs are not expected to lose their properties if the amino acids in the APRs are replaced by conservative residues, as it seems that the distribution of these residues in the protein sequence is important.

[0053] The skilled person can ascertain the capacity of a peptide with one or more conservative amino acid substitutions to form beta-sheet-rich structures by analyzing the kinetics of amyloid formation using the ThT Kinetic Assay as detailed in Example 1.

[0054] In a preferred embodiment, the isolated peptide comprises the sequence of SEQ ID NO: 1 (amino acid residues 26-56 of human Cathepsin D). This sequence comprises both Region 1 and Region 2. Region 1 and Region 2 may include up to one conservative amino acid substitution each. The portion of the peptide outside of Region 1 and Region 2 may include up to three amino acid deletions, insertions, or substitutions.

[0055] In another preferred embodiment, the isolated peptide comprises the sequence of SEQ ID NO: 4 (amino acid residues 123-146 of human Cathepsin D). This sequence comprises Region 3. Region

[0056] 3 may include up to one conservative amino acid substitution. The portion of the peptide outside of Region 3 may include up to two amino acid deletions, insertions, or substitutions.

[0057] In yet another preferred embodiment, the isolated peptide comprises the sequence of SEQ ID NO: 6 (amino acid residues 160-171 of human Cathepsin D). This sequence comprises Region 4. Region

[0058] 4 may include up to one conservative amino acid substitution. The portion of the peptide outside of Region 4 may include up to one amino acid deletions, insertions, or substitutions.

[0059] In a further preferred embodiment, the isolated peptide comprises the sequence of SEQ ID NO: 8 (amino acid residues 318-335 of human Cathepsin D). This sequence comprises both Region 5 and Region 6. Region 5 and Region 6 may include up to one conservative amino acid substitution each. The portion of the peptide outside of Region 1 and Region 2 may include up to two amino acid deletions, insertions, or substitutions. The present invention discloses isolated Cathepsin D peptides, each comprising at least one betaaggregating region naturally occurring in Cathepsin D, for use in the treatment or prevention of amyloidosis. Preferably, the peptides disclosed herein are identical to a contiguous stretch of 50 amino acids or less, naturally occurring in Cathepsin D. However, in some instances, it is envisaged that non-identical, but closely related, sequences can be used. In order to maintain specificity, it is envisaged that, particularly for non-conservative substitutions, a sequence identity that is at least 80% would be tolerated. Thus, provided are peptides comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, 99%) or more identical to the sequence of human CathD with the aforementioned accession number. For instance, the amino acid sequence of a ten-amino acid long peptide may differ from the corresponding sequence of CathD in one or more amino acids. Such substitutions include, as an example, conservative amino acid substitutions, where the acid substitution does not alter the nature or function of the peptide. The term "sequence identity" as used herein refers to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which an identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0060] In a preferred embodiment, the present invention provides an isolated Cathepsin D peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NON, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a peptide having an amino acid sequence that is at least 80% identical to any of the amino acid sequences of SEQ ID NO: 1 to 10, for use in the treatment or prevention of an amyloidosis.

[0061] The peptides of the invention may comprise natural, synthetic or modified amino acids, such as methylated amino acids and amino acids analogs. In certain embodiments, the peptides of the present invention further comprise a modification. Possible chemical modifications include disulfide bridges, methylation, glycosylation, acetylation, formylation, amidation, biotinylation, to name of few. If present, modifications to the amino acid structure may be imparted before or after assembly of the peptide. Peptides are often modified for clinical use to enhance their stability, bioavailability, and overall therapeutic efficacy. These modifications are crucial because natural peptides have certain limitations, such as rapid degradation by enzymes, poor stability, and limited bioavailability. In more detail, below are some common modifications made to peptides for clinical applications:

[0062] • Chemical Modifications: (i) N-terminal Acetylation: Protects the peptide from enzymatic degradation and increases stability; (ii) C-terminal Amidation: Increases the peptide's resistance to proteolytic enzymes, prolonging its half-life; (iii) PEGylation: Attaching polyethylene glycol (PEG) to peptides improves solubility, reduces renal clearance, and decreases immunogenicity; (iv) Cyclization: Cyclizing the peptide structure (e.g., head-to-tail cyclization) reduces susceptibility to enzymatic degradation and increases binding affinity to targets; (v) Non-natural Amino Acids: Incorporating D-amino acids or other non-standard amino acids improves stability and resistance to enzymatic breakdown; (vi) Sumoylation: The conjugation of small ubiquitin-like modifiers (SUMOs) to Lysine residues may have a protective functionality against proteolysis.

[0063] • Structural Modifications: (i) P-amino acids or peptidomimetics: Using these can significantly increase the metabolic stability and therapeutic potential; (ii) Lipidation: Adding fatty acids (e.g., palmitoylation) to peptides enhances their ability to bind to albumin, prolonging circulation time; (iii) Stapled Peptides: Introducing hydrocarbon “staples” between two amino acids to lock peptides in a helical structure, which improves stability, cell penetration, and binding affinity.

[0064] Examples of Clinically Modified Peptides include: Liraglutide (Victoza): A GLP-1 analog used in diabetes treatment, modified by attaching a fatty acid chain to increase its half-life; Semaglutide (Ozempic): Modified with a side-chain for albumin binding and increased stability, allowing weekly dosing. Substitutions are made at semaglutide positions 2 and 28, where alanine and lysine are replaced by 2-aminoisobutyric acid and arginine, respectively. The substitution of the alanine prevents chemical breakdown by dipeptidyl peptidase-4; Octreotide: A somatostatin analog that is cyclized to enhance stability and has prolonged action compared to the natural peptide. These modifications are crucial in making peptide-based therapies more viable for clinical use, ensuring that they remain effective after administration.

[0065] Generally, the peptides are at least 2 amino acids long. In some embodiments, the peptide is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, or at least 40 amino acids long. In some embodiments, the peptide is at least 5 amino acids long.

[0066] The peptides or compositions thereof can be administered by any suitable delivery route, such as, intramuscular (i.m.), subcutaneous (s.c.), intracerebroventricular, intradermal (i.d.), oral, intranasal, sublingual, rectal, to name a few.

[0067] For instance, the peptides may be administered i.m., s.c., i.d., using suitable devices such as a syringe and needle and jet injection devices. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Suitable solutions may be prepared using, for example, isotonic vehicles such as sodium chloride, Ringer's solution, lactated Ringer's and others. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, if required.

[0068] The peptides may be administered orally, in the form of a tablet, a capsule, a powder or a liquid to name a few. Suitable excipients include, but are not limited to, salts, pH modifying agents, such as buffers, preservatives, binders, fdlers, solubilizers, disintegrants, sorbents, solvents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof.

[0069] The peptides or compositions thereof may be administered by a mucosal route, e.g., intranasally. Many intranasal delivery devices such as spray devices, aerosols, or droppers, are available and well known in the art.

[0070] The amount administered is preferably an amount that is effective to treat the amyloidosis, prevent or delay the onset of the disease or ameliorate a symptom of the disease, such as lessening the severity of disease or symptoms, slowing or halting disease progression, causing a remission, or limiting the frequency and / or severity of the symptoms of the disease.

[0071] The actual amount of the peptide administered and the dosing scheme applied will depend on the nature and severity of the amyloidosis being treated, as well as other parameters such as the potency of the peptide, its concentration in the formulation, the condition and age of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

[0072] The peptides or compositions disclosed herein can be administered as sole treatment or provided in combination with other treatments (medical and non-medical), either simultaneously or sequentially dependent upon the condition to be treated. For instance, for the treatment of AD the peptides disclosed herein may be administered in combination with cholinesterase inhibitors such as donepezil and rivastigmine, glutamate regulators or antibodies such as Aducanumab or Lecanemab.

[0073] In a preferred embodiment, the peptides disclosed herein are encoded by a recombinant vector (e.g. a plasmid, cosmid, viral vector) and can be synthesized inside a host. According to this preferred embodiment, a recombinant vector is provided, comprising a polynucleotide encoding a peptide as disclosed herein, wherein said vector, upon transformation to a cell or organism, produces said peptide in said cell or organism. Recombinant adenovirus vectors (AdV), adeno-associated virus type 2 (rAAV-2) vectors and herpes simplex virus (HSV) vectors have been widely investigated for gene transfer to neurons in several animal models of human neurodegenerative diseases. A viral vector refers to a polynucleotide molecule including the viral genome or part thereof and a transgene operably linked to a promoter. AAV vectors, particularly serotype 2, are the predominant viral vectors of use to treat neurological diseases and have emerged to be less cytotoxic and less immunogenic than HSV and AdV. Other delivery technologies known to the skilled person may be used instead, for instance nanoparticles such as polylactic-co-glycolic acid-based nanoparticles. In an alternative embodiment the peptides can be introduced into a cell through carrier-mediated delivery, e.g. by liposomal carriers or nano-particles or by injection.

[0074] For the treatment of amyloidoses such as AD, it is important to ascertain that the peptide will efficiently cross the blood-brain barrier (BBB). Technologies for delivering peptides through the BBB include brain-homing and brain-penetrating molecular transport vectors, such as brain permeable peptides or BBB shuttle peptides. Thus, in a preferred embodiment, the peptides disclosed herein are further modified through the recombinant or synthetic attachment of a BBB shuttle peptide such as Angiopep-2, GSH, RVG29 or a peptide belonging to the cell-penetrating peptide (CPP) family, to name a few. Still other commonly used BBB shuttle peptides (both natural and artificial peptides) are reviewed in Sanchez-Navarro M. and Ernest Giralt E. (Pharmaceutics. 2022 Sep; 14(9): 1874).

[0075] EXAMPLES

[0076] Hereinafter, the present invention is described in more detail with reference to examples and comparative examples. It will be apparent to one of ordinary skill in the art that these examples are for illustrative purposes only and should not be construed as limiting or altering the scope of the present invention.

[0077] EXAMPLE 1

[0078] CathD peptides exhibit amyloidogenic potential

[0079] To evaluate the amyloidogenic potential of the ten CathD peptides, their capacity to form beta- sheet-rich structures was examined by studying the kinetics of amyloid formation using the ThT Kinetic Assay (see Materials & Methods section below). All ten peptides tested formed aggregates. The majority of these peptides demonstrated aggregation kinetics resembling those of amyloid fibrils (Figure 2A, 2C, 2E, 2G, 21, 2K, 2M, 20, 2Q and 2S). More specifically, two peptides, CathD[51-55] (Figure 2E) and CathD [163 -169] (Figure 20), displayed the highest propensity for amyloid fibril formation, characterized by a remarkably short lag phase of 2.5 hours, followed by rapid growth to peak ThT fluorescence intensity at approximately 4 hours. A more moderate aggregation kinetic was observed for CathD[26-56] (Figure 2A), CathD[28-32] (Figure 2C) and CathD[160-171] (Figure 2M), reaching their peak fluorescence signals at 6-8 hours. Following them CathD[124-128] (Figure 2G), CathD [322-327] (Figure 21) and CathD[328-333] (Figure 2S) peptides reached their highest signals at 10-12 hours. The slowest kinetics were observed for CathD[123-146] (Figure 2K), featuring a lag phase of 11 hours, with peak fluorescence occurring at 30 hours. Of particular interest is the behavior of the CathD[318-335] (Figure 2Q) peptide, which deviated from the observed patterns. This peptide exhibited maximum fluorescence intensity from the very first measurement, followed by a sharp decline. This behavior suggests the presence of two aggregate types in solution: amyloid fibrils and amorphous aggregates. Presumably, the CathD[318-335] peptide demonstrates sufficient amyloidogenic properties to initiate immediate fibril formation upon solubilization, resulting in the initial high fluorescence. However, as fibrinogenesis progresses, the formation of higher order structures from individual fibrils, along with the growth and prevalence of amorphous aggregates, leads to a reduction in the observed fluorescence signal.

[0080] Moreover, the fibrils formed by the ten peptides were examined using Transmission Electron Microscopy (TEM) and negative staining (as detailed in the Materials & Methods section) and the results are shown in Figure 2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P, 2R and 2T). Peptides CathD[26-56] (Figure 2B) and CathD[51-55] (Figure 2F) displayed similar morphology, forming amyloid-like fibrils with a diameter of 100 A, while some protofibrils with a diameter of 50-70 A were also observed. Peptide CathD[126-146] (Figure 2L) formed similar fibrils. In contrast to the fibril morphology of CathD[26-56] (Figure 2B) and CathD[51-55] (Figure 2F), the peptide CathD[28-32] (Figure 2D) formed fibrils with a diameter of 70 - 120 A, which interacted with each other to form tapes. CathD[160-171] (Figure 2N) and CathD [163 -169] (Figure 2P) peptides produced fibrils with the typical amyloid characteristics: they were straight, unbranched, of indefinite length and approximately 100 A in diameter. The fibril network of CathD[163-169] was denser than that of CathD[160-171], which may be attributed to their differences in the kinetics of amyloid formation, with CathD [163 -169] forming fibrils more rapidly. CathD[124-128], CathD[318-335], CathD[322- 327] and CathD[328-333] did not form amyloid fibrils at a concentration of 100 pM. At a concentration of 500 pM, CathD[124-128] (Figure 2H) and CathD[328-333] (Figure 2T) formed tapes composed of fibrils with a diameter ranging from 90-120 A. Peptide CathD[318-335] (Figure 2R) formed fibrils that exhibited the typical characteristics of amyloids. These fibrils interacted laterally to form ribbons as well as loosely twisting ribbons. The fibrils of the peptide CathD [322- 327] (Figure 2J) were less well-defined, and the presence of amorphous aggregates was also observed.

[0081] All peptides bind Congo Red, as seen under bright field illumination in a polarizing stereomicroscope. When the polars are crossed, the peptides exhibit the apple-green birefringence that amyloids typically exhibit. EXAMPLE 2

[0082] CathD peptides impact the aggregation and fibrillogcncsis of the AP42peptide

[0083] Given the self-assembling capability of CathD peptides to form amyloid-like fibrils, the inventors investigated their interactions with Ap42peptide aggregates. Each CathD peptide was co-incubated with equimolar amounts of Ap42, and the aggregation kinetics were assessed through ThT fluorescence measurements over time and TEM. ThT is known to specifically recognize and bind to amyloid fibrils. Upon binding, it exhibits a strong fluorescence signal at approximately 484 nm when exposed to 444 nm.

[0084] ThT fluorescence measurements shown in Figure 3A revealed that individual Ap42aggregation kinetics followed the typical sigmoidal curve of the amyloid fibrillogenesis. The Ap42lag phase lasted approximately 6 to 7 hours, followed by a rapid increase in the ThT fluorescence signal, peaking around 17 hours. Subsequently, the signal continued to rise, reaching its highest point by the end of the experiment. These results support the idea that molecules capable of forming beta sheets, such as the CathD peptides, can interact with structurally similar amyloid aggregates rich in beta structures. This interaction would lead to a competitive reduction of amyloid-amyloid interactions, effectively preventing amyloid aggregation (Ren et al., J Mater Chem B. 2022;10: 1754-1762).

[0085] Figure 4 (A, C, E, G, I, K, M, O, Q and S) shows ThT fluorescence emission spectra for the Ap42peptide and for the combination of the Ap42peptide with a CathD peptide. The degree of inhibition or delay of aggregation varied among the peptides. Notably, CathD[26-56] peptide, which is the only one approaching the length of Ap42peptide, exhibited the most significant inhibitory effect (Figure 4A), since no fluorescence was observed, indicating the absence of amyloid fibrils. CathD[160-171] (Figure 4K), CathD[51-55] (Figure 4E), CathD [123 -146] (Figure 4G), CathD[318-335] (Figure 40), CathD[322-327] (Figure 4Q) and CathD[328-333] (Figure 4S) appeared to inhibit Ap42fibril formation, though their potential as inhibitors was less pronounced than that of CathD[26-56], Among them, CathD[160-171] achieved the most effective delay (Figure 4K), prolonging the lag phase, with the co-incubation signal reaching half of the value of individual Ap42by the end of the assay. In contrast, despite an initial delay, CathD[124-128] (Figure 41) and CathD[328-333] (Figure 4S) reached the signal of individual Ap42during the last 10 hours of the measurements. Finally, CathD[28-32] (Figure 4C) and CathD [163 -169] (Figure 4M) demonstrated no significant effect on amyloid aggregation, as their curves were comparable to that of individual Ap42. If the fluorescence measurement had been continued for additional time, a drop in the signal may have been observed due to the delayed action of the peptide, while the Ap42signal would have likely stabilized or possibly increased. This assumption could explain why we do not observe the same extent of fibril formation between the Ap42-CathD peptide co-incubation samples and Ap42alone in TEM as shown below. Interestingly, none of the peptides promoted Ap42amyloidogenesis.

[0086] The results from TEM experiments shown in Figure 4 (B, D, F, H, J, L, N, P, R and T) confirm that after 7 days of incubation, all peptides either inhibited or delayed Ap42fibril formation. Ap42was incubated individually as a control to assess the influence of the CathD peptides on its aggregation process. In the control sample, straight, unbranched fibrils with indefinite length and a diameter of approximately 100 A were formed (Figure 3B). In line with the ThT results, CathD[26-56] peptide appeared to inhibit the formation of Ap42amyloid fibrils (Figure 4B). Upon thorough examination of the entire grid, no fibrils were observed. In all other cases, the impact of the peptides was evident, since the dense fibril network of Ap42was absent. CathD[124-128] (Figure 4J), CathD[160-171] (Figure 4L), CathD[163-169] (Figure 4N), CathD[318-335] (Figure 4P), CathD [322-327] (Figure 4R) and CathD[328-333] (Figure 4T) induced the formation of extensive amorphous aggregates. TEM micrographs revealed that, except for CathD[160-171] (Figure 4L), thin fibrils (60 - 90 A in diameter), which were mainly fractured, were observed, suggesting a delaying effect rather than complete inhibition. Finally, peptides CathD[28-32] (Figure 4D), CathD[51-55] (Figure 4F) and CathD [123 -146] (Figure 4H) also delayed the formation of Ap42amyloid fibrils, as scattered thin fibrils were visible.

[0087] EXAMPLE 3

[0088] Phenotypic characterization of the peptides in C. elegants

[0089] The inventors performed a phenotypic analysis of wild-type nematodes treated with each examined peptide or the diluent (H2O) to investigate any differences in the fitness of a multicellular organism at day 1 of adulthood in the presence of the peptides. For all assays, N2 animals were allowed to lay eggs for 20-30 min on NGM plates containing either the tested peptide or H2O (control sample). The following phenotypic characteristics were monitored: a) Developmental timing: The progeny was frequently observed to record the needed time to reach the L4 larval stage from egg hatching. The experiment was repeated three times. b) Fecundity assay: Single N2 L4 larvae were transferred on NGM plates containing either each peptide or H2O (Control). Each animal was transferred every two days to a fresh NGM plate containing each peptide or H2O (Control). Progeny of each animal was scored at the L2-L3 larval stage. At least 5 animals per condition were examined. c) Egg lethality: Single N2 L4 larvae were transferred on NGM plates containing either each peptide or H2O (Control). Each animal was transferred every two days to a fresh NGM plate containing each peptide or H2O (Control) and non-hatched eggs were measured. At least 5 animals per condition were examined. d) Pharyngeal pumping: At day 1 of adulthood, the pharyngeal pumping rate (pumps per minute) was measured. Fifty animals per condition were examined. e) Defecation assay: At day 1 of adulthood, the defecation rate (period in seconds from defecation to defecation) was measured. Fifty animals per condition were examined.

[0090] Each peptide was dissolved in sterile, deionized water at the final concentration indicated in Table 1 (0.05 pM for CathD[26-56], 0.5 pM for CathD[28-32] and CathD[51-55]). The control sample consisted of sterile, deionized water. The exposure to the peptide under study was carried out from the time of egg laying of the wild-type strain.

[0091] CathD[26-56] peptide: The phenotypic characteristics of wild-type animals are not largely affected upon treatment with 0.05 pM CathD [26-56] as compared to control treated animals (Table 2). The observed delay in defecation rate might suggest a slight toxicity.

[0092] ****p<0.0001;aNumber of offspring per worm;bNumber of non-hatched eggs per worm

[0093] Table 2. Phenotypic characteristics of wild-type N2 animals exposed to 0.05 pM CathD[26-56] or H2O (Control).

[0094] CathD [28-32] peptide: The phenotypic characteristics of wild-type animals are not largely affected upon treatment with 0.5 pM CathD 1 as compared to control treated animals (Table 3). The observed delay in defecation rate might suggest a slight toxicity.

[0095] **p<0.01;aNumber of offspring per worm;bNumber of non-hatched eggs per worm

[0096] Table 3. Phenotypic characteristics of wild-type N2 animals exposed to 0.5 pM CathD[28-32] or H2O (Control).

[0097] CathD[51-55] peptide: The phenotypic characteristics of wild-type animals are not largely affected upon treatment with 0.5 pM CathD[51-55] as compared to control treated animals (Table 4). The observed delay in defecation rate might suggest a slight toxicity.

[0098] ***p<0.001;aNumber of offspring per worm;bNumber of non-hatched eggs per worm

[0099] Table 4. Phenotypic characteristics of wild-type N2 animals exposed to 0.5 pM CathD[51-55] or H2O (Control).

[0100] EXAMPLE 4

[0101] C. elegans Paralysis assays

[0102] For the paralysis assays the CL4176 strain was used, accepted as an Ap C. elegans model. The CL4176 strain expresses the human Ap42peptide under a muscle-specific promoter and upon temperature upshift, they accumulate A peptide in their body wall muscle cells and become paralyzed in a few hours (Link et al., Neurobiol. Aging. 2003;24:397-413).

[0103] For the CL4176 strain, 150-300 animals were seeded on different concentrations of each peptide or the control sample (H2O). When they reached the L3 stage, they were shifted from 16°C to 25°C and 16 hours later they were scored over the span of 10-14 hours for paralysis until the entire population was paralyzed. For the GMC101 strain: 80-120 animals were seeded on different concentrations of each peptide or the control sample (H2O). When they reached the stage of young adult, the animals were shifted from 20°C to 25°C. Scoring of paralyzed nematodes was initiated 12 hours after temperature upshift and lasted until the entire population was paralyzed. For both strains, the animals were scored as paralyzed if they failed to undergo a full body wave propagation upon prodding and each paralysis assay was repeated at least three times by two blinded experimenters. The results are shown in Tables 5-7 and Figures 5 to 7. Mean paralysis time values in hours are expressed as mean ± SEM (error of the mean). The number n represents the number of individuals measured and paralyzed during the tests.

[0104] CathD[26-56] peptide As shown in Table 5 below and Figure 5, CathD[26-56] peptide at the concentration of 0.05 pM slows down the rate of paralysis in the CL4176 strain that serves as an AD model. Concentrations of 0.1 and 0.5 pM CathD[26-56] did not reach a statistically significant slowing of the rate of paralysis compared to the administration of the control sample but a tendency to slow down is evident, while the concentration of 5 pM CathD[26-56] causes an acceleration of the rate of paralysis in the CL4176 strain, thus suggesting a toxicity at high concentrations.

[0105] **p<0.01; ****p<0.0001, ns: not significant, 2 independent experiments, n represents the number of animals that got paralyzed during the assays.

[0106] Table 5. Median paralysis time of CL4176 strain in the presence of CathD[26-56] peptide.

[0107] Figure 5 shows the paralysis curves of the C. elegans CL4176 strain after continuous exposure to different concentrations of CathD[26-56] or control sample (H2O). (A) 0.05 pM CathD[26-56] or Control, (B) 0.1 pM CathD[26-56] or Control, (C) 0.5 pM CathD[26-56] or Control, and (D) 5 pM CathD[26-56] or Control. Median time of paralysis in hours is reported as the mean of 2 independent experiments ± SEM. n represents the number of animals that got paralyzed during the assays. (**p<0. 01, ****p<0.0001 (log-rank Mantel-Cox test), ns: not significant, curves are the pooled result of 2 independent experiments).

[0108] CathD[28-32] peptide: As shown in Table 6 and Figure 6, all concentrations (0.5, 5 and 10 pM) of CathD[28-32] peptide tested slow the rate of paralysis in the CL4176 strain.

[0109] ****pO .0001, 3 independent experiments, n represents the number of animals that got paralyzed during the assays.

[0110] Table 6. Median paralysis time of CL4176 strain in the presence of CathD[28-32] peptide.

[0111] Figure 6 shows the paralysis curves of C. elegans CL4176 strain after continuous exposure to different concentrations of CathD[28-32] or control sample (H2O). (A) 0.5 pM CathD[28-32] or Control, (B) 5 pM CathD[28-32] or Control, and, (C) 10 pM CathD[28-32] or Control. p<0.000 1. Median time of paralysis in hours is reported as the mean of 3 independent experiments ± SEM. n represents the number of animals that got paralyzed during the assays. (****p<0.0001(log-rank Mantel-Cox test), curves are the pooled result of 3 independent experiments).

[0112] CathD[51-55] peptide: As shown in Table 7 and Figure 7, all concentrations (0.5, 5 and 10 pM) of CathD[51-55] peptide tested slow the rate of paralysis in the CL4176 strain.

[0113] ****pO .0001, 3 independent experiments, n represents the number of animals that got paralyzed during the assays.

[0114] Table 7. Median paralysis time of the CL4176 strain in the presence of CathD[51-55] peptide.

[0115] Figure 7 shows the paralysis curves of C. elegans CL4176 strain after continuous exposure to different concentrations of CathD[51-55] or control sample (H2O). (A) 0.5 pM CathD[51-55] or Control, (B) 5 pM CathD[51-55] or Control, and, (C) 10 pM CathD[51-55] or Control. Median time of paralysis in hours is reported as the mean of 3 independent experiments ± SEM. n represents the number of animals that got paralyzed during the assays. (****p<0.0001 (log-rank Mantel-Cox test), curves are the pooled result of 3 independent experiments). EXAMPLE 5

[0116] Visualization of the cellular distribution of AB aggregates in vivo

[0117] To evaluate the Ap aggregation state in vivo, the inventors took advantage of the CL2331 strain that expresses the human A3.42 peptide fused to green fluorescent protein (GFP) in its body wall muscle cells. The animals are gradually filled with A aggregates that are visible through confocal microscopy (Link et al., Neurobiol. Dis. 2008;32:420-425).

[0118] For Ap 3-42 deposit measurements, synchronized CL2331 animals were exposed to each examined peptide or H2O (Control) and grown at 20°C (to induce the expression of the Ap3.42peptide) until day 1 of adulthood. Animals were mounted on 2% agarose pads on glass slides, anesthetized with 100 mM levamisole and observed at room temperature using a Leica TSC SPE confocal laser scanning microscope (Leica Lasertechnik GmbH, Heidelberg, Germany). The LAS AF software was used for image acquisition. Images focused in the anterior area of the nematodes were acquired with a 20 / 0.70 objective. The number of aggregates was counted in at least 25 animals / condition.

[0119] Figure 8 shows the number of amyloid deposits in the anterior area of C. elegans CL2331 strain after continuous exposure to specific concentrations of CathD[26-56], CathD[28-32], CathD[51- 55] or H2O (Control). Three independent experiments, ns: not statistically significant, *p<0.05 (unpaired T-test). The results showed that treatment with CathD[26-56] significantly reduced the Ap aggregates. A similar tendency (that did not however reach statistical significance at the tested concentration) was revealed for CathD[28-32], Treatment with the specific concentration of CathD[51-55] did not induce a positive outcome in CL2331 strain. Given the positive results with this peptide in the abovementioned assays, more concentrations need to be investigated.

[0120] EXAMPLE 6

[0121] Effects of the peptide inhibitors on cells of neural origin being exposed to exogenously added AP42oligomers

[0122] The inventors sought to evaluate whether treatment with the peptides may protect human cells of neuronal origin against Ap toxicity. To that end, the SH-SY5Y human neuroblastoma cells were exposed to exogenously added Ap42peptide and cell survival was scored.

[0123] The human dopaminergic neuroblastoma cell line SH-SY5Y was maintained in RPMI supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine. SH-SY5Y cells were treated with 5 pM Ap42peptide. SH-SY5Y cells were exposed to each peptide or H2O (Control) for 24 h before being subjected to Ap treatment (in parallel exposure to the relative peptide or H2O) for another 24 h. Cell death / viability of SH-SY5Y cultures was assessed through scoring of (a) the dead SH-SY5Y cells through trypan blue staining and (b) attached Ap42-treated SH-SY5Y cells (cell viability) in triplicates using a Coulter Z2 counter. All cultures were maintained at 37°C in a 5% CO2humidified incubator.

[0124] The percentage of viable SH-SY5Y neuroblastoma cells after pre-incubation with the peptide inhibitors or Control for 24 h and subsequent incubation with medium containing or not 10 pM Ap42peptide for an additional 24 h is shown in Figure 9. As 100% was set the sample in which the Control was administered per case (with (Ap) or without (No Ap) peptide) and the statistical comparisons are made with respect to the corresponding control, (ns: not statistically significant, ***p<0.001. 2 experimental replicates).

[0125] CathD [26-56] peptide: 0.05 pM CathD[26-56] peptide enhances survival of SH-SY5Y human neuroblastoma cells being exposed to exogenously added Ap42oligomers. Figure 9 (A, B) shows the percentage of live SH-SY5Y human neuroblastoma cells following pre-incubation with 0.05 pM CathD [26-56] or control for 24 h and subsequent incubation with medium containing 5 pM Ap42peptide (Figure 9B) or DMSO (Control; Figure 9A) for additional 24 h in the presence of 0.05 pM CathD [26-56] or control. The number of control cells without Ap42(no AP) or with Ap42(AP) was arbitrarily set to 100%. All statistical comparisons were performed against the relative control, ns: not significant, ***p<0.001, 3 independent experiments.

[0126] CathD[28-32] peptide: 0.5 pM CathD[28-32] peptide enhances survival of SH-SY5Y human neuroblastoma cells being exposed to exogenously added Ap42oligomers. Figure 9 (C, D) shows the percentage of live SH-SY5Y human neuroblastoma cells following pre-incubation with 0.5 pM CathD[28-32] or Control for 24 h and subsequent incubation with medium containing 5 pM Ap42peptide or DMSO (Control) for additional 24 h in the presence of 0.05 pM CathD[28-32] or control. The number of control cells without Ap42(no AP) (Figure 9C) or with Ap42(AP) (Figure 9D) was arbitrarily set to 100%. All statistical comparisons were performed against the relative control. *p<0.05, ***p<0.001, 3 independent experiments. This peptide appears to positively affect cell proliferation even in the absence of exposure to increased concentrations of Ap42peptide. This may be related to the fact that the these cells endogenously produce anyway a small amount of Ap peptide but the observation needs further investigation.

[0127] CathD[51-55] peptide: 0.5 pM CathD[51-55] peptide enhances survival of SH-SY5Y human neuroblastoma cells being exposed to exogenously added Ap42oligomers. Figure 9 (E, F) shows the percentage of live SH-SY5Y human neuroblastoma cells following pre-incubation with 0.5 pM CathD [51-55] or Control for 24 h and subsequent incubation with medium containing 5 pM Ap peptide or DMSO (Control) for additional 24 h in the presence of 0.05 pM CathD[51-55] or control. The number of control cells without Ap42(no AP) (Figure 9E) or with Ap42(AP) (Figure 9F) was arbitrarily set to 100%. All statistical comparisons were performed against the relative control. **p<0.01, ***p<0.001, 3 independent experiments. This peptide appears to positively affect cell proliferation even in the absence of exposure to increased concentrations of Ap42peptide. This may be related to the fact that these cells endogenously produce a small amount of Ap42peptide but the observation needs further investigation.

[0128] EXAMPLE 7

[0129] Computational insight into the inhibition of AP42fibril formation by CathD[26-561, CathD[28-321 and CathD[51-551

[0130] The molecular dynamics simulations shed light on the effect of the three peptides on the conformation of the Ap42peptide pentamer (Figure 10). Notably, the smallest conformational changes were observed in the case of CathD[26-56] peptide, the only peptide that docked outside the groove of the Ap42peptide pentamer. Specifically, CathD[26-56] (Figure 10A), throughout the simulation, maintained its most favorable docking position near the region of Ap42that corresponds to residues 30-42, suggesting a potential advantage in preventing interactions with neighboring Ap42peptides. This observation aligns with its inhibitory effect, as evidenced in TEM micrographs. In contrast, peptides CathD[28-32] (Figure 10B) and CathD[51-55] (Figure 10C) were docked within the pentamer groove, contributing to more efficient changes regarding the secondary structure of the Ap42in its oligomeric state. Particularly, in the case of the CathD[28-32] peptide, a partial shift of chain E in regard to the initial conformation of the Ap42oligomer and the formation of a helical structure were observed after 500 ns of simulation.

[0131] Materials & Methods

[0132] Peptide synthesis. The ten selected peptides were chemically synthesized by GeneCust©, Europe, France, with both the amino-terminal and the carboxy-terminal ends free and they exhibit a purity exceeding 98%. In the case of the peptide CathD[26-56], an intrachain disulfide bond was formed by linking Cysteine (Cys) residues at positions 46 and 53 (amino acids 21 and 28 of SEQ ID No. 1), mimicking the native protein where they contribute to the formation of a disulfide bridge. Conversely, Cys at position 27 (amino acid 2 of SEQ ID No. 1) was methylated to prevent the formation of unintended intermolecular disulfide bonds. Similarly, the Cys residue in the CathD[51-55] (amino acid 3 of SEQ ID No. 1) was methylated to achieve the same objective.

[0133] The Ap42peptide DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID No. 11) corresponding to amino acids 672 to 713 of Homo sapiens protein APP (Accession number P05067) was also synthesized and lyophilized by GeneCust© Europe, France. The purity of the peptide was higher than 95%, with the N- and C-terminals being free.

[0134] Sample preparation - Disaggregation of pre-formed aggregates. Each lyophilized peptide was dissolved in l,l,l,3,3,3-Hexafluoro-2-propanol (HFIP; Sigma-Aldrich) at a concentration of 1 mg / mL. CathD peptides were mixed with equimolar amounts of Ap42. The resulting solutions, both individual peptide solutions and Ap42-CathD peptide solutions, were left to evaporate overnight in a fume hood, at room temperature, until thin peptide-containing films were formed. These films were subsequently stored at -20 °C.

[0135] In vitro fibril formation. The peptide-containing films were initially left at room temperature for 30 min, and then they were dissolved in 0.1 M HEPES (Sigma, St. Louis, MO, USA) buffer at pH = 7.4. The concentrations of the peptide solutions varied, ranging from 10 pM to 500 pM, depending on the specific experimental assay. The peptide solutions were incubated for 1 week at 37 °C, unless stated otherwise.

[0136] Transmission Electron Microscopy (TEM) and negative staining. TEM was utilized to investigate the formation of amyloid fibrils by CathD peptides, as well as Ap42peptide, both when coincubated with CathD peptides and when not. Each 100 pM or 500 pM peptide-solution (10 pL) was applied to 400-mesh glow-discharged and carbon-coated copper grids for 20 minutes. Subsequently, the grids were placed on a droplet of 2% (w / v) aqueous uranyl acetate for 50 seconds, followed by washing by contact with three drops of distilled water to remove excess stain. After blotting with Whatman filter paper, the grids were air-dried and examined using a Morgagni™ 268 transmission electron microscope, operated at 80 kV. Digital images were captured with an 11 Mpixel side mounted Morada CCD camera (Soft Imaging System, Muenster, Germany).

[0137] Thioflavin T Kinetic Assay. Thioflavin T (ThT) fluorescence measurements were carried out at 37 °C in black 96-well plates with flat, clear bottoms, using a Tecan Spark microplate reader. The tops of the plates were securely sealed with microplate covers, and fluorescence readings were performed from the bottom. For excitation, a 444 nm filter was used, and for emission, a 484 nm filter was used. The HFIP peptide films were dissolved in DMSO, followed by dilution in HEPES to achieve a final concentration of less than 5% v / v. These solutions were used immediately without prior incubation or agitation. The reaction solution consisted of freshly prepared 10 pM CathD peptides, 10 pM Ap42co-incubated with CathD peptides and 25 pM ThT (Sigma) in distilled water. As a control for the co-incubation experiments, a freshly prepared 10 pM Ap42solution was utilized. ThT fluorescence was also measured in the absence of peptide solutions. Each experiment was performed in triplicate, with measurements taken over 50 hours at 15 -minute intervals. Prior to each measurement, the plate was briefly agitated for 10 seconds at 270 rpm. The ThT background fluorescence was subtracted from the sample readings at each time point. Standard deviation was calculated, and the data was normalized on a scale of 0 to 100 arbitrary units, with 100 arbitrary units corresponding to maximum individual Ap42fluorescence intensity in the case of co-incubation experiments. Error bars in ThT fluorescence emission spectra represent the standard deviation of the triplicates. Data visualization was performed using the R statistical language and the packages ggplot2, dplyr, ggthemes, extrafont, and ggpmisc via the integrated development environment RStudio.

[0138] Congo Red Birefringence assay. A droplet (3 pL) of each 100 pM or 500 pM peptide solution was applied to a glass slide and allowed to air-dry at room temperature, until a peptide film on top of the slide was produced. Subsequently, the films were stained using a 0.01 M Congo red solution in PBS (137 mmol / L NaCl, 27 mmol / L KC1, 100 mmol / L Na2HPO4, 18 mmol / L KH2PO4, pH = 7.4) or a 1% (w / v) Congo Red solution in distilled water (pH 5.75) for approximately 20-30 min. Excess stain was removed by several washes with either 90% ethanol or tap water, and the samples were left to air-dry approximately for 10 min. The samples were observed under bright field illumination and between crossed polars, using a Leica MZ7.5 polarizing stereomicroscope (Leica CameraAG, Weltzar, Germany), equipped with a Sony a6000 camera (Sony, Tokyo, Japan).

[0139] Molecular Dynamics Simulation

[0140] Molecular docking. Molecular docking experiments were performed to explore how different CathD peptides preferentially bind to the oligomerized Ap42, determining their binding positions. To achieve this, the NMR-derived three-dimensional (3D) structure of the pentameric Ap42(PDB ID: 2BEG) composed of five chains was used (Luhrs et al., Proc Natl Acad Sci U S A. 2005;102: 17342-17347). This structure corresponds to the smallest detectable oligomer in solution (Wolff et al., Sci Rep. 2017;7:2493). The structures of the CathD peptides up to 11 residues long were built utilizing the Builder tool in PyMOL (DeLano and Lam, Abstr Pap Am Chem Soc. 2005;230:U1371-U1372). Lor peptides longer than 11 residues, the structure was extracted from the experimentally determined structure of Cathepsin D (PDB ID: 4OD9) (Gradler et al., Bioorg Med Chem Lett 2014;24:4141-4150). Each peptide was docked to Ap42oligomer by utilizing the automated protein docking server ClusPro (Kozakov et al., Proteins. 2013;81:2159-2166, Kozakov et al., Nat protoc. 2017;12:255-278, Porter et al., Bioinformatics. 2017;33:3299-3301, Vajda et al., Proteins. 2017;85:435-444).

[0141] Molecular dynamics simulations. In order to investigate the interactions between the oligomerized Ap42and the CathD peptides, molecular dynamics simulations were conducted employing the GROMACS software package, version 2018.1 (University of Groningen, Groningen, Netherlands) (Kutzner et al., J Comput Chem. 2019; 40:2418-2431). These simulations used the AMBER99SB- ILDN protein, nucleic AMBER94 force-field (Lindorff-Larsen et al., Proteins. 2010;78: 1950- 1958). Each complex was placed within a 1.2 nm cubic box of 3-point model (TIP3P) water (Jorgensen et al., J Chem Phys 1983;79:926-935) and ionized using NaCl molecules to mimic neutral pH conditions. Each simulation system underwent energy minimization, with a maximum of 2000 steps, employing the steepest descent algorithm. Following this, two stages of equilibration simulations with position restraints on protein coordinates were conducted. The first stage involved a 100 ps simulation in the canonical (NVT) ensemble to equilibrate temperature at 310 K, utilizing the Berendsen-thermostat (Berendsen et al., J Chem Phys. 1984; 81:3684-3690). After the first equilibration, a 100 ps simulation in the isothermal-isobaric (NPT) ensemble was performed to control pressure isotopically at 1.013 bar (1 atm), using the Berendsen weak coupling algorithm (Berendsen, In Computer Simulation in Materials Science: Interatomic Potentials, Simulation Techniques and Applications . M. Meyer and V. Pontikis, editors. Springer Netherlands, Dordrecht. 1991; 139-155) and the Berendsen-thermostat at 310 K. Subsequently, a 500 ns MD simulation with position restraints removed was conducted at 310 K. Periodic boundary conditions were applied in all directions. Bond constraints were modeled using the LINCS algorithm (Hess et al., J. Comput. Chem. 1997;18: 1463-1472), enabling the use of a 2 fs time-step. Short-range non-bonded interactions employed a twin-range cutoff at 0.8 nm, while long-range electrostatic interactions were modeled using the Particle Mesh Ewald (PME) method, with a Fourier grid spacing at 0.12 nm (Essmann et al., J Chem Phys. 1995;103:8577-8593). The results of the simulations were examined using a variety of GROMACS utilities, as well as Visual Molecular Dynamics (VMD) (Humphrey et al., J Mol Graphics. 1996;14:33-38). To assess the structural stability, the “rms” tool was utilized. Secondary structure analysis was performed using the “do_dssp” tool. Frames were extracted at 100 ps intervals for further analysis. Images were captured with PyMOL.

Claims

CLAIMS1. An isolated peptide of 5 to 50 amino acids in length, comprising the sequences of one or more of Regions 1 to 6, with each Region allowing up to one conservative amino acid substitution, for use in the treatment or prevention of an amyloidosis, wherein- Region 1 consists of amino acid residues 28-32 of human Cathepsin D (SEQ ID NO: 2);- Region 2 consists of amino acid residues 51-55 of human Cathepsin D (SEQ ID NO: 3);- Region 3 consists of amino acid residues 124-128 of human Cathepsin D (SEQ ID NO: 5);- Region 4 consists of amino acid residues 163-169 of human Cathepsin D (SEQ ID NO: 7);- Region 5 consists of amino acid residues 322-327 of human Cathepsin D (SEQ ID NO: 9);- Region 6 consists of amino acid residues 328-333 of human Cathepsin D (SEQ ID NO: 10); and wherein the reference sequence of human Cathepsin D is defined by SEQ ID NO: 12.

2. The isolated peptide for use according to claim 1, comprising at least the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10.

3. The isolated peptide for use according to Claim 1 or Claim 2, comprising the sequence of SEQ ID NO: 1, wherein Region 1 and Region 2 may include up to one conservative amino acid substitution each; and the portion of the sequence outside of Region 1 and Region 2 may include up to three amino acid deletions, insertions, or substitutions.

4. The isolated peptide for use according to Claim 1 or Claim 2, comprising the sequence of SEQ ID NO: 4, wherein Region 3 may include up to one conservative amino acid substitution; and the portion of the sequence outside of Region 3 may include up to two amino acid deletions, insertions, or substitutions.

5. The isolated peptide for use according to Claim 1 or Claim 2, comprising the sequence of SEQ ID NO: 6, wherein Region 4 may include up to one conservative amino acid substitution; and the portion of the sequence outside of Region 4 may include up to one amino acid deletions, insertions, or substitutions.

6. The isolated peptide for use according to Claim 1 or Claim 2, comprising the sequence of SEQ ID NO: 8, wherein Region 5 and Region 6 may include up to one conservative amino acid substitution each; and the portion of the sequence outside of Region 5 and Region 6 may include up to two amino acid deletions, insertions, or substitutions.

7. The isolated peptide for use according to claim 1 or 2, comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

8. The isolated peptide for use according to any one of the preceding claims, further comprising at least one modification.

9. The isolated peptide for use according to claim 8, wherein the modification is an N- terminal acetylation; a C-terminal amidation; a polyethylene glycol (PEG); a non-natural amino acid; a beta-amino acid; or a fatty acid.

10. The isolated peptide for use according to any one of the preceding claims, wherein the peptide is a cyclic peptide or a peptidomimetic.

11. The isolated peptide for use according to any one of the preceding claims, wherein the peptide is a stapled peptide.

12. The isolated peptide for use according to any one of the preceding claims, wherein said peptide is capable of interacting with and / or inhibiting the formation of beta amyloid aggregates, thereby leading to a competitive reduction of amyloid-amyloid interactions.

13. The isolated peptide for use according to any one of the preceding claims, wherein said amyloidosis is Alzheimer’s disease.

14. A pharmaceutical composition comprising at least one peptide as defined in any one of claims 1 to 13; and a pharmaceutically acceptable carrier or excipient; for use in the treatment of an amyloidosis such as Alzheimer’s Disease.

15. A recombinant vector comprising a polynucleotide encoding the isolated peptide according to any one of claims 1 to 13, wherein said vector, upon transformation to a cell or organism, produces the peptide in said cell or organism.

16. An isolated peptide comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9 or SEQ ID NO: 10.

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