Use of the p3 peptide to treat alzheimer's disease

Abstract

Certain embodiments of the invention provide methods for treating an amyloid beta dependent neurodegenerative disease by administering to a patient in need there of a therapeutically effective amount of a peptide that comprises a P3 peptide, or that comprises a therapeutically effective variant of a P3 peptide.

Description

[0001] RU: [P2025-040-01] / VHPM: 08035.154WO1

[0002] USE OF THE P3 PEPTIDE TO TREAT ALZHEIMER’S DISEASE CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 746,512 that was filed on January 17, 2025. The entire content of the application referenced above is hereby incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under R00AG065441 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Alzheimer’s disease (AD) is the most common form of dementia in the elderly and a major cause of morbidity and mortality worldwide. It is characterized by progressive neuronal loss and the histopathological appearance of two canonical lesions: extracellular senile plaques, composed of aggregated amyloid beta (AP), and intracellular neurofibrillary tangles, composed of hyperphosphorylated tau. Mutations in the Amyloid Precursor Protein (APP- human, App -rodent), a type-I transmembrane protein that undergoes sequential proteolysis to produce Ap, cause familial forms of AD3. Recently, three anti-Ap antibodies have been shown to effectively reduce senile plaque deposits4'6, but, despite this reduction, AD patients still invariably develop dementia. As such, new and improved methods of treating AD are needed.

[0008] SUMMARY

[0009] Accordingly, certain embodiments of the invention provide methods for treating an amyloid beta dependent neurodegenerative disease by administering to a patient in need there of a therapeutically effective amount of a peptide that comprises a P3 peptide, or that comprises a variant of a P3 peptide.

[0010] Certain embodiments provide a method for treating a neurodegenerative disease in a patient, comprising administering to a patient in need thereof a peptide that comprises the following amino acid sequence, or a therapeutically effective variant of the sequence:

[0011] LVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO:1).

[0012] Certain embodiments provide a method for treating a neurodegenerative disease in a patient, comprising administering to a patient in need thereof a peptide that comprises theRU: [P2025-040-01] / VHPM: 08035.154WO1

[0013] following amino acid sequence, or a therapeutically effective variant of the sequence:

[0014] LVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:2).

[0015] In certain embodiments, peptide comprises a therapeutically effective variant of SEQ ID NO:1 or SEQ ID NO:2 that has at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:1 or SEQ ID NO:2.

[0016] In certain embodiments, the disease is an amyloid beta dependent neurodegenerative disease.

[0017] In certain embodiments, the disease is Alzheimer's disease (AD) or cerebral amyloid angiopathy (CAA).

[0018] In certain embodiments, the disease is cerebral amyloid angiopathy (CAA).

[0019] In certain embodiments, the disease is Alzheimer's disease (AD).

[0020] In certain embodiments, the disease is early-onset AD.

[0021] In certain embodiments, the disease is late-onset AD.

[0022] Certain embodiments provide a P3 peptide or variant thereof as described herein for the prophylactic or therapeutic treatment of an amyloid beta dependent neurodegenerative disease, such as Alzheimer's disease (AD) or cerebral amyloid angiopathy (CAA) in a subject.

[0023] Certain embodiments provide the use of a P3 peptide or variant thereof as described herein to prepare a medicament for the treatment of an amyloid beta dependent neurodegenerative disease, such as Alzheimer's disease (AD) or cerebral amyloid angiopathy (CAA) in a subject.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] Figures 1A-1F. Isolation and characterization of App-EVs. FIG. 1A. Schematic of App-EV isolation from filtered and concentrated Apphand App' rat primary neuronal conditioned media, using anti-App 4G8 antibody directed against the extracellular-facing juxtamembranous region of App full length and App-CTFs. FIG. IB. Immunocapture of App-EVs with 4G8 or control IgG, followed by western analysis with anti-App Y188 antibody directed against the C-terminus of App. Input samples were diluted 20X. FIG. 1C. Mass spectrometry analysis of App-EVs immunoisolated from primary neuronal conditioned media vs control IgG. Spectral Counts and Normalized Spectral Abundance Factors are shown. FIG. ID.

[0026] Co-immunoprecipitation of Vcp in immunocaptured App-EVs. App-EVs were immunoisolated by 4G8 or control IgG, followed by western analysis with anti-Vcp antibody. Input samples were diluted 20X. n=3. FIG. IE. App-EVs were immunoisolated by 4G8 or control IgG,RU: [P2025-040-01] / VHPM: 08035.154WO1

[0027] followed by western analysis with flotillin- 1 , Alix, and CD9. Input samples were diluted 20X.

[0028] FIG. IF. TEM images of immunoisolated App-EVs at 100k X (left) and 150k X (right). Scale bars indicate 200 nm.

[0029] Figures 2A-2H. Effect of Vcp inhibition on App processing and EV release. FIG. 2A. Western analysis of Apphprimary neurons treated with 2.5 pM NMS-873 for 8h. App full length, App P- and a-CTFs, and Gapdh are indicated. Levels of App a-CTF relative to App full length are represented as mean ± S.E.M. and were analyzed by Student’s / -test. *** / ? < .001, n=6. FIG. 2B. Western analysis of conditioned media from NMS-873 -treated Apphprimary neurons. sAppa was detected with anti- App 6E10 directed against the Ap 3-8 region. Red Ponceau is shown below western blot. Levels of sAppa relative to Ponceau stain are represented as mean ± S.E.M. and were analyzed by Student’s / -test. *p < .05, n=6. FIG. 2C. Western analysis of Apphprimary neurons treated with 2.5 pM NMS-873 and / or 50 pM chloroquine for 8h. LC3 I and LC3 II were detected with an antibody against LC3B. LC3 IVI ratios are represented as mean ± S.E.M. and were analyzed by one-way ANOVA with Tukey’s multiple comparison test when ANOVA showed significant differences. ***p < .001, ****p < .0001, n=3. FIG. 2D. MSD electrochemiluminescent assay of conditioned media from NMS-873-treated Apphprimary neurons. Ap40 and p3-40 were detected with a capture antibody against the C-terminus of Ap40 and a 4G8 detection antibody. In the same well, AP42 and p3-42 were detected with a capture antibody against the C-terminus of Ap42 and a 4G8 detection antibody. Ap and p3 levels are represented as mean ± S.E.M and were analyzed by Student’s / -test.

[0030] *p < .05, n=6. FIG. 2E. Western analysis of total (left) and cell-surface (right) protein levels of Apphprimary neurons treated with 2.5 pM NMS-873 for 8h. App a-CTF and Gapdh are indicated. Total App a-CTF levels normalized to surface App a-CTF are represented as mean ± S.E.M and were analyzed by Student’s / -test, n=6. FIG. 2F. Western analysis of total EVs from conditioned media of NMS-873 -treated Apphprimary neurons. Alix and App a-CTF are indicated. App a-CTF and Alix levels are represented as mean ± S.E.M. and were analyzed by Student’s / -test. *p < .05, n=3. G. Nanoparticle tracking analysis of conditioned media from Apphprimary neurons treated with NMS-873. Total particle levels are represented as mean ± S.E.M. and were analyzed by Student’s / -test. ***p < .001, n=6. FIG. 2H. Summary schematic of NMS-873 -induced EV release.

[0031] Figures 3A-3E. Effect of App8mutation on App processing and EV levels. FIG. 3A.

[0032] Western analysis of Apphand App' rat primary neuronal lysate. App full length, App P- and a-CTFs, and Gapdh are indicated. Levels of App a- and P-CTF relative to App full length areRU: [P2025-040-01] / VHPM: 08035.154WO1

[0033] represented as mean ± S.E.M. and were analyzed by Student’s / -test. *p < .05, **p < .01, n=4.

[0034] FIG. 3B. Western analysis of total EVs isolated from Apphand App' rat primary neuronal conditioned media, harvested after 24h. App full length, App a- and P-CTF are indicated, with an additional overexposure of App-CTFs shown below. App full length and App a-CTF levels are represented as mean ± S.E.M. and were analyzed by Student’s / -test. *p < .05, **p < .01, n=4. FIG. 3C. Nanoparticle tracking analysis of conditioned media from Apphand App' primary neurons. Total particle levels and 0-40 nm particle levels are represented as mean ± S.E.M. and were analyzed by Student’s / -test. n=6. FIG. 3D. Proteinase protection assay of pO Apphand App' rat brain post mitochondrial membranous fractions. Samples were treated with proteinase K (PK) and / or sodium dodecyl sulfate (SDS). App full length and App-CTFs were detected by western analysis with Y188, against App C-terminal epitopes (left), with an additional overexposure of App-CTFs shown below. App full length N-terminal epitopes were detected with 6E10 (right, top). Grp78 (right, middle) and Vamp2 (right, bottom) are indicated. The percentages of PK-protected App metabolites are represented as mean ± S.E.M. and were analyzed by Student’s / -test (for Apphand App' comparisons) or one-way ANOVA (for App metabolite comparisons) with Tukey’s multiple comparison test when ANOVA showed significant differences. **p < .01; ***p < .001, ****p < .0001, n=3. FIG. 3E. Nanoparticle tracking analysis of conditioned media from 24h p 3 -treated App' primary neurons. Total particle levels (left) and 0-40 nm particle levels (right) are represented as mean ± S.E.M. and were analyzed by one-way ANOVA with Tukey’s multiple comparison test when ANOVA showed significant differences. *p < .05, n=6.

[0035] Figure 4. Co-immunoprecipitation of App and Vcp in Apphtotal brain lysate. App and Vcp were immunoprecipitated from total brain lysate, with 4G8 and MA3-004, respectively. Eluate was analyzed by western blot against App (Y188) and Vcp (CST 2649).

[0036] Figure 5. Effect of Vcp activation on App a-CTF levels. Western analysis of lysate from Apphrat primary neurons treated with Smer28 and / or VAI for 24h. App full length (left) and App-CTFs (middle) are indicated. App a-CTF levels relative to App full length levels (right) are represented as mean ± S.E.M. and were analyzed by one-way ANOVA with Tukey’s multiple comparison test when ANOVA showed significant differences. *p < .05, **p < .01, n=6.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0037] DETAILED DESCRIPTION

[0038] A new therapeutic approach to treating Alzheimer’s Disease by using the P3 peptide, a metabolite of Amyloid Precursor Protein, to drive extracellular vesicle activity and promote clearance of Amyloid P in order to support neuronal function and slow cognitive decline is described herein.

[0039] Described herein is a description of how the P3 peptide, a metabolite of the Amyloid Precursor Protein (APP), can have direct therapeutic value in treatment of Ap-dependent neurodegenerative diseases, including Alzheimer’s Disease. P3 is produced by a pathway often referred to as the ‘non-amyloidogenic pathway’, where APP is cleaved by a-secretase into the fragments soluble a- APP fragments (sAPPa) and C83. Subsequent cleavage of C-terminal fragment a (C83) by y-secretase produces APP intracellular domain (AICD) and the P3 peptide (LVFFAEDVGSNKGAIIGLMVGGVV; SEQ ID NO:1) (see Sun et al., (2015)

[0040] Front. Pharmacol .6 : 221. doi : 10.3389 / fphar .2015.00221 ).

[0041] As described herein, it has found that P3 increases extracellular vesicle (EV) secretion in an Alzheimer’s model, thereby indicating that EVs are a means by which cells can clear away misfolded proteins, which would be useful in treating Alzheimer's disease.

[0042] These discoveries are useful for treatments for Alzheimer’s Disease and other Ap-related neurodegenerative diseases. For example, a therapy may include delivering P3 or a modified PS-like peptide in sufficient quantity to have a therapeutic effect.

[0043] APP undergoes extensive intracellular processing, which results in multiple APP metabolites3. The majority of APP processing occurs via the nonamyl oidogenic pathway, in which APP is initially cleaved in the juxtamembranous region by a-secretase to produce a large soluble ectodomain (sAPPa), and the membrane bound C-terminal fragment (a-CTF). APP a-CTF undergoes further processing by y-secretase to release the non-aggregating p3 peptide and an APP intracellular domain (AICD). Amyloidogenic processing of APP begins with P-secretase cleavage to produce a soluble APP ectodomain (sAPPP) and a membrane bound C-terminal fragment (P-CTF). APP P-CTF is further cleaved by y-secretase to release Ap and AICD. The length of Ap can vary and determines its propensity to aggregate, with shorter forms, such as AP40, less likely to aggregate and longer forms, such as AP42 and longer, more likely to aggregate. Given the numerous APP metabolites that neurons produce, the typical changes (increase in total Ap, increase in Ap42:40 ratio) that track with plaque formation do not adequately capture the full spectrum of APP metabolism. Moreover, familial APP mutations that increase amyloidogenic processing of APP also result in the dysregulation of these APPRU: [P2025-040-01] / VHPM: 08035.154WO1

[0044] metabolites, many of which exert functions within the cell independent of plaque pathology.

[0045] Here, the immunocapture and analysis of purified App-EVs derived from rat primary neuronal conditioned media is described. App-EVs contain as molecular cargo the valosin-containing protein (Vcp), a ubiquitin-dependent segregase / molecular unfoldase and the genetic cause of autosomal dominant forms of AD related dementias24. As described herein, a new ability of Vcp to regulate App metabolism and global EV secretion in primary neurons has been discovered. Using a genetically faithful rat knock-in model of an AD-associated App mutation, App-EV biogenesis is linked to the nonamyl oidogenic App processing pathway. Together, these results point to a new function of App and its processing that may relate to the clearance of aggregated proteins via Vcp-containing App-EVs.

[0046] Nucleic Acids, Expression Cassettes, Vectors and Cells

[0047] Certain embodiments of the invention provide nucleic acid(s) (e.g., an isolated nucleic acid) encoding a peptide as described herein. In certain embodiments, the nucleic acid further comprises a promoter. In certain embodiments, the isolated nucleic acid encoding a peptide as described herein is DNA. In certain embodiments, the isolated nucleic acid encoding a peptide described herein is mRNA.

[0048] Certain embodiments of the invention provide an expression cassette comprising a nucleic acid as described herein and a promoter.

[0049] Certain embodiments of the invention provide a vector (e.g, a plasmid or phagemid) comprising a nucleic acid or an expression cassette as described herein.

[0050] Certain embodiments of the invention provide a host cell (e.g, mammalian cell, such as CHO cell) comprising a nucleic acid, expression cassette or vector as described herein.

[0051] Compositions and Administration

[0052] Certain embodiments of the invention provide a pharmaceutical composition comprising a peptide described herein, and a pharmaceutically acceptable carrier.

[0053] In certain embodiments, the protein is present in a liquid composition (e.g., saline, D5W, or buffered solution).

[0054] In certain embodiments, the protein is present in a solid composition. In certain embodiments, the protein is present in a lyophilized composition, which may be reconstituted with proper solution prior to administration as a liquid. In certain embodiments, the lyophilized composition further comprises one or more excipients selected from the group consisting of aRU: [P2025-040-01] / VHPM: 08035.154WO1

[0055] cryo-lyoprotectant (e.g., trehalose, sucrose) and a bulking agent e.g., mannitol, glycine).

[0056] For in vivo use, a peptide of the invention is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, the peptide may be present as active ingredient(s) (z.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of a relevant disease, as measured using a representative assay). A pharmaceutical composition may comprise a peptide in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition.

[0057] Compositions for injection will commonly comprise a solution of the peptide in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g, Ringer’s solution. Pharmaceutical compositions desirably are sterile and generally free of undesirable matter. These pharmaceutical compositions can be sterilized by conventional, well known sterilization techniques. The pharmaceutical compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, e.g, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The pharmaceutical compositions may contain salt, sugar, preservative, surfactant, or any other suitable excipient.

[0058] The pharmaceutical composition may contain any suitable concentration of the peptide. The concentration of the peptide in the pharmaceutical composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient’s needs. In certain embodiments, the concentration of peptide in a solution formulation for injection will range from about 0.1% (w / w) to about 10% (w / w), or more such as 100 mg of peptide per milliliter of the formulation.

[0059] In certain embodiments, the peptides of the present invention may be systemically administered, e.g., intravenously, in combination with a pharmaceutically acceptable carrier. In certain embodiments, the peptide may be administered intravenously, subcutaneously, intradermally, intramuscularly, intraperitoneally, intranasally, or intrathecally by infusion or injection. In certain embodiments, the peptide of the present invention may be locally administered into a particular tissue, structure, or organ.

[0060] Frequency of administration can range from multiple doses to a single dose per week, orRU: [P2025-040-01] / VHPM: 08035.154WO1

[0061] less frequently (e.g., single dose per month or every two to three months). In certain embodiments, the peptide of the present invention may be administered, e.g., intravenously or subcutaneously, to a mammal in need of, for example, about once every two weeks, once every three weeks, once every month, once every five weeks, or once every six weeks. In certain embodiments, the peptide may be administered about once every month or once every two or three months. In certain embodiments, the peptide may be administered about once every week. In some embodiments, the peptide is administered from about once per month to about five times per week.

[0062] In one embodiment, the peptide is administered to the patient parenterally. Dosing of the peptide can be by any suitable route, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. The peptide dose can range from about 1 mg / kg (body weight) to about 50 mg / kg, from about 10 pg / kg to about 5 mg / kg, or from about 100 pg / kg to about 1 mg / kg. The peptide dose can be about 100, 200, 300, 400, or 500 pg / kg. The peptide dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg, or more. The peptide dose can also be outside of these ranges, depending on the type and severity of the disorder being treated. The peptide dose for a patient can be adjusted by physician or pharmacist.

[0063] In certain embodiments, the peptide itself is administered. In certain embodiments, the peptide is administered by means of a nuclic acid sequence encoding the peptide.

[0064] In certain embodiments, the peptide may be delivered using a device (e.g., a device comprising one or two chambers, for example, containing the peptide, and / or liquid). In certain embodiments, the device is an injector (e.g., a self-injector device comprising the peptide).

[0065] The term “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of a peptide either alone or as contained in a pharmaceutical composition that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0066] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration orRU: [P2025-040-01] / VHPM: 08035.154WO1

[0067] palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean reducing risk of death, reducing the severity of the disorder, or prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0068] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal X, Clustal W, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0069] The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein.

[0070] The term “variant” polypeptide refers to a polypeptide derived from the native protein but has substitution of one or more amino acids at one or more sites in the native protein, or deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C -terminal end of the native protein; or deletion or addition of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0071] Thus, the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0072] Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).

[0073] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991); Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 (1994). A "nucleic acid fragment" is a fraction of a given nucleic acid molecule.

[0074] Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0075] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of theRU: [P2025-040-01] / VHPM: 08035.154WO1

[0076] degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g, 81%-84%, at least 85%, e.g, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the native (endogenous) nucleotide sequence.

[0077] “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0078] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, anRU: [P2025-040-01] / VHPM: 08035.154WO1

[0079] "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (z.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, culture medium may represent less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0080] A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0081] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0082] Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is providedRU: [P2025-040-01] / VHPM: 08035.154WO1

[0083] with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0084] "Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0085] "Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0086] The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.

[0087] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, butRU: [P2025-040-01] / VHPM: 08035.154WO1

[0088] may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0089] The invention will now be illustrated by the following non-limiting Example.

[0090] EXAMPLE

[0091] The Amyloid Precursor Protein (APP), a genetic cause of Alzheimer’s disease (AD), is a type-I transmembrane protein that is metabolized by proteolysis in the endolysomal system. APP and its metabolites are secreted by cells in extracellular vesicles (EVs). To study the function of APP-containing EVs, App-EVs were isolated from rat primary neuronal conditioned media and proteomic analysis identified the Valosin-containing protein (Vcp) as molecular cargo.

[0092] Pharmacological modulation of Vcp activity was found to alter App processing and global EV secretion in rat primary neurons. AD-associated knock-in App mutations were found to alter the abundance of App-EVs and the trafficking of App metabolites within App-EVs, in a manner related to the epitopes generated by the nonamyl oidogenic processing of App. The presence of Vcp suggests a role for App-EVs in the clearance of protein aggregates.

[0093] This study (1) identifies Vcp as molecular cargo in App-EVs, (2) explores the consequence of amyloidogenic vs nonamyl oidogenic processing of App in App-EV biogenesis, and (3) uncovers a new role of Vcp in EV secretion. There is a significant genetic connection between Vcp and neurodegeneration. Autosomal dominant mutation of VCP causes tau-only frontotemporal dementia50, multisystem proteinopathy51, characterized by frontotemporal dementia with tauopathy plus extra-CNS proteinopathies in muscle and bone, and amyotrophic lateral sclerosis52, characterized by upper motor neuron loss and intracellular proteinopathy. No VCP mutation has been found to cause AD, though some observations support a functional link between VCP and the main pathological features of AD, / .< ., tau and amyloid. The VCP-tau connection has been established by numerous studies which show that VCP can directly bind aggregated tau, disassemble it, and potentially affect the spread of tau tangle pathology50,53,54. Evidence of a relationship between VCP function and amyloid is less established. Patients with inclusion-body myositis, related to the muscle proteinopathy caused by VCP mutations, have amyloid positive rimmed vacuoles within muscle cells55,56. Additionally, in AD patients, VCP is increased in brain-derived EVs, as compared to nondemented controls57. Our study is the first to provide a direct cell biological link between Vcp and amyloid in the form of Vcp as molecular cargo in App-EVs. The ability of Vcp to localize to App-EVs suggests a new cellular function of App related to the clearance of protein aggregates.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0094] The precise cellular function of APP is unknown. Most studies of AD-causing APP mutations focus on the biochemical changes to Ap amount, Ap length, and Ap self-association. These processes affect the extent to which Ap aggregates, and this metric features heavily in how AD is defined. However, Ap is just one metabolite of APP, and the mutations which govern Ap production also affect the numerous non-Ap metabolites as well as the function of the full length protein. Altered APP function may have a pleiotropic effect: one manifestation of which is the production of aggregation-prone Ap species, and another contemporaneous manifestation is a change in APP -EV production. The advantage of expanding our understanding of APP function to include APP-EVs is that it allows for a new connection between amyloid and tau. Vcp, in its capacity to disaggregate tau and localize to App-EVs, may underpin this link between the two canonical AD pathologies.

[0095] The A mutations that cause or prevent AD occur within the juxtamembranous or transmembrane regions of APP3. These regions are also the subject of extensive proteolysis which results in different species of APP-CTFs, with APP a- and P-CTFs most abundant.

[0096] Unexpectedly, it was discovered that this region governs the localization of App-CTFs to ILVs, with App a-CTF localizing predominantly to ILVs while the longer App P-CTF and full length App are only present in ILVs in minor amounts (Fig. 3D). The biophysical cause of this change in ILV localization is unclear but may be related to changes in membrane curvature that are required for the invagination of the limiting endosomal membrane to form ILVs. The juxtamembranous region of App a-CTF re-inserts into the endosomal membrane46. This close apposition may modulate membrane curvature and be lost when longer or mutated forms of App-CTFs are present. This ability may not be limited to membrane-bound forms of App. The effect of soluble forms of App, which contain this neoepitope formed by a-cleavage, such as p3, was considered it was found that exogenous p3 increases small EV biogenesis.

[0097] The study of App-EVs has discovered a new function of Vcp in its ability to cause global secretion of EVs. This new function is relevant to AD and other types of neurodegeneration, as EVs may be involved in cell-to-cell spread of toxic protein aggregates, including tau. It is proposed that the disruption of the autophagy -promoting ability of Vcp, accomplished pharmacologically in this study, mimics the autophagy failure seen in AD patients and animal models. Secretion of EVs may be an alternative route of clearance when normal degradative pathways are impaired. Therefore, in addition to the known effects of Vcp function on the seeding-potential of tau aggregates within a cell53, Vcp function may be relevant for the cell-to-cell spread of tau as well.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0098] To understand the function of App-EVs, it is necessary to isolate App-EVs from other vesicles. This presents several considerations, such as cell-type source, organism source, and purification method. App is expressed in multiple cell types, but given that App is predominantly expressed in neurons25, and that neuronal cell death underlies the progressive cognitive impairment seen in AD, the choice of neurons is most relevant. EV isolation from primary neuronal conditioned media allows for the study of exclusively neuronal EVs and removes the possibility of contamination with intracellular vesicles, which have the same biophysical properties, such as size and density, and protein markers as EVs. For the study of AD, it would be ideal to use a human source of neurons, but given the scale of induced pluripotent stem cell (iPSC)-derived neurons required for App-EV isolation and the nonphysiological features of neuronal cancer cell lines, the ability to study neuronal App-EVs from human sources is limited. Transgenic animals have been engineered to express human APP, with and without AD-associated mutations, though many rodent models rely on overexpression of the transgene or the use of multiple AD-mutations, both of which may alter the physiological function of APP26. Recently, App knock-in rats (Apphand 4 / y ) have been developed which offer advantages over other transgenic animals27'29. Apphrats express a humanized form of rodent App under the control of the endogenous rodent App promoter, therefore, each

[0099]

[0100] rat in this study produces human Ap, human p3, and human App-CTFs. To examine the effect of AD-related amyloidogenic App processing on App-EV function, App rats were engineered to additionally express the Swedish mutation, which drives App metabolism toward amyloidogenic processing in a manner similar to familial AD patients with the Swedish APP mutation. For these technical considerations, conditioned media from primary neurons from Apphand App' rats were used in this study.

[0101] Immunocapture of EVs using antibodies against common EV markers such as Alix, CD9, CD63, or CD81 has been found to enrich EVs from a heterogenous mixture of vesicles23, and, as App is a type-I transmembrane protein with its N-terminus exposed to the extracellular space in EVs, it was predicted that App-EVs could be isolated by similar methods using anti-App antibodies. Neuronal conditioned media was filtered to remove large debris and concentrated 80X for use as input. Concentration of EV-containing media was chosen over the standard use of ultracentrifugation to pellet EVs because ultracentrifugation has been shown to cause aggregation of vesicles30which may result in nonspecific co-immunoprecipitation of non-App-EVs. The anti-App 4G8 antibody was chosen, as it recognizes epitopes at theRU: [P2025-040-01] / VHPM: 08035.154WO1

[0102] juxtamembranous extracellular-facing region of App and does not recognize, and therefore does not compete against, the abundant App-metabolite sAppa present in conditioned media (Fig. IA). The detection of ~110 kDa App with a C-terminal anti -App antibody in anti -App 4G8 eluate indicates the presence of the full length protein, including its transmembrane domain (Fig. IB). As no detergents were used in the immunocapture, the presence of the full length App protein suggests a membranous source, which is herein termed App-EVs. Mass spectrometry sequencing of immunoisolated App-EVs i ovaApp11and App' neuronal conditioned media revealed the presence of Vcp in both samples, with no Vcp peptides detected in IgG controls (Fig. 1C). Vcp is an abundant multifunctional protein that binds and unfolds multiple protein substrates, including polyubiquitinated aggregates, for degradation24. In addition to Vcp, known Vcp interactors, including polyubiquitin31, histone subunits32,33, and ribosome subunits32,34,35, were detected (Fig. 1C). Western analysis of App-EVs confirmed the presence of Vcp m Apphand App' samples (Fig. ID). Direct binding of App and Vcp was investigated by coimmunoprecipitation of App and Vcp in Triton-X solubilized brain lysate (Fig. 4). The absence of co-immunoprecipitation of App by Vcp, and vice-versa, suggests a lack of direct binding and excludes the possibility that Vcp is binding aggregated App. App-EVs were further characterized by western analysis using antibodies against the common EV markers flotillin- 1 , Alix, and CD9 (Fig. IE). These markers were not detected in App-EV samples, in agreement with their absence from the mass spectrometric results. Given that these common EV markers were not present in App-EVs, further confirmation of the App-EV’ s vesicular identity was accomplished using transmission electron microscopic analysis (Fig. IF). Numerous spherical particles less than 40 nm in diameter were detected and are consistent with electron micrographs of small EVs36. The combined immunoprecipitation, mass spectrometric, and electron micrographic results support the conclusion that App-EVs have been isolated.

[0103] Vcp inhibition causes global EV release

[0104] Given the finding that App-EVs contain Vcp cargo, the functional effect of Vcp on App levels and metabolism was investigated. While Vcp knockout is embryonically lethal37, pharmacological inhibition of Vcp can be accomplished by NMS-873, a potent allosteric inhibitor which binds the region between the Vcp DI and D2 ATPase domains38. 8h treatment of primary Apphneurons with 2.5 pM NMS-873 resulted in significantly less App a-CTF in cell lysate, with no concomitant changes to full length App levels (Fig. 2A). This result was mirrored by the increase in App a-CTF caused by the dual activation of DI and D2 domains byRU: [P2025-040-01] / VHPM: 08035.154WO1

[0105] Smer2839and VAI40, respectively (Fig. 5). The effect of Vcp inhibition on App a-CTF independent of full length App suggests that lower App a-CTF levels are not the result of a transcriptional response. Decreased production (by reduced a-secretase activity) or increased clearance (by increased macroautophagy or y-secretase activity) could explain lower App a-CTF levels. sAppa and App a-CTF are produced in equimolar amounts when App is cleaved by a-secretase; therefore, sAppa levels in conditioned media indicate a-secretase activity.

[0106] Paradoxically, increased sAppa levels were observed in NMS-873 -treated samples (Fig. 2B), which rules out a decrease in production. The effect of NMS-873 on major App-CTF degradative pathways, including macroautophagy and y-secretase proteolysis, was investigated. Vcp is required for autophagy and binds Beclin 1 and the PI3K complex41,42. In agreement with this function, rather than increasing autophagy, inhibition of Vcp by NMS-873 lowers autophagy, as indicated by a lower LC3II / I ratio in chloroquine-treated samples (Fig. 2C). y-Secretase activity results in the production of Ap, which can be measured in neuronal conditioned media. No significant difference in the most abundant form of Ap, Ap40, was observed in NMS-873 -treated samples, while a statistically significant decrease in the second most abundant form, Ap42, was detected (Fig. 2D). Together, these data indicate that neither decreased production nor increased clearance is responsible for the NMS-873 -mediated decrease in App a-CTF.

[0107] The significant increase in sAppa (Fig. 2B) suggests an increased amount of App is trafficked to the cell surface, the predominant subcellular localization of a-secretase activity. Therefore, a cell-surface labelling assay was used to determine cell surface levels of App a-CTF in NMS-873 -treated neurons. Despite a decrease in total App a-CTF levels, no such difference was observed at the cell surface (Fig. 2E), implicating a change in App trafficking. One possible change in App trafficking that would result in more App available for a-secretase processing at the cell surface is the fusion of App-containing MVBs to the cell surface. The ILVs within, which are selectively enriched in App-CTFs16'19, would be secreted and detected in total EV preparations. Increased MVB fusion to the cell surface would likewise result in decreased cellular levels of App-CTFs (Fig. 2A). Western analysis of Alix and App a-CTF showed significantly increased levels in conditioned media from NMS-873-treated neurons, consistent with increased EV secretion (Fig. 2F). Increased global EV secretion was confirmed with nanoparticle tracking analysis (Fig. 2G). It was hypothesized that the reduction of autophagy by Vcp inhibitors results in the secretion of EVs, a phenomenon which has been seen with other modulators of autophagy43,44(Fig. 2H).RU: [P2025-040-01] / VHPM: 08035.154WO1

[0108] App8mutation reduces App-EV levels

[0109] App is extensively processed by sequential proteolysis along the nonamyl oidogenic and amyloidogenic pathways. These pathways result in different App membrane-bound CTFs which have been reported to be enriched in App-EVs16'19. The use of genetic AD-associated mutants would allow us to determine the effect of alterations in App processing on App-EV composition and function. One well-characterized App mutation, the Swedish mutation, is located at the two amino acids N-terminal to the P-cleavage site45. Swedish-App preferentially binds P-secretase and commits App to the amyloidogenic pathway, generating App P-CTF and, in turn, Ap. Wild type App is processed primarily along the nonamyloidogenic pathway, which results in App a-CTF, and, in turn, p3. The Swedish mutation-induced shift toward amyloidogenic App processing is recapitulated in App rat primary neurons, which display significantly higher App P-CTF levels than Apphcontrols, where P-CTF is often below the limits of detection (Fig. 3A).

[0110] To determine if the cellular changes in App metabolite abundance caused by the Swedish mutation are reflected in EVs, total EVs were islolated from Apphand App' primary neuronal conditioned media and analyzed App content by western blot. Significant decreases in both full length App and App a-CTF were observed in App' samples (Fig. 3B). App P-CTF, which composed roughly half of total App-CTFs in App' neuronal lysates, was strikingly undetectable in EVs, and therefore omitted from quantifications where relevant. To determine if this decrease in full length App and App-CTFs was the result of a global reduction in EVs, conditioned media from ApphanAApp' primary neuronal cultures was analyzed by nanoparticle tracking, which showed no differences (Fig. 3C). The effect of the Swedish^ / ? / ? mutation appears to be confined to App-EVs.

[0111] App a-cleaved neoepitope determines App-EV sorting

[0112] The absence of App P-CTF in EVs seen in Fig. 3B may be the result of a retention of App P-CTF-containing ILVs within the cell or, alternatively, a failure of App P-CTF to traffic to ILVs. To distinguish between these two possibilities, a proteinase protection assay was performed. Transmembrane proteins retain their original membrane topology when membranous fractions are homogenized without detergents. Cytosolic-facing regions can be mapped by proteinase K digestion of exposed epitopes. Lumen-facing epitopes are protected from proteinase K digestion by the intact membrane. The C-terminus of a type-I transmembrane protein, such as App, faces the cytosol and is therefore susceptible to proteinase K cleavage.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0113] However, if App traffics to a double membranous structure, e.g., to ILVs within MVBs, the C-terminus is protected by the limiting membrane. To eliminate other sources of double membranous vesicles, a post mitochondrial supernatant was prepared from pO brain homogenates from Apphand App' rats, treated with proteinase K, and analyzed by western blot. Intralumenal Grp78 and cytosolic Vamp2 controls displayed the expected digestion pattern (Fig.

[0114] 3D, right middle and bottom). Full length App from both Apphand App' rats displayed the expected proteinase K digestion pattern of a type-I transmembrane protein, with a vast majority of the C-terminal epitopes susceptible to digestion (Fig. 3D, left). The N-terminus of App, which is detected by the 6E10 antibody, was protected from degradation but instead shifted down ~5 kDa in a manner consistent with the digestion of the exposed C-terminus (Fig. 3D, right, top). App P-CTF, seen exclusively in App' homogenates, was also digested in the same pattern as full length App. Interestingly in Apphhomogenates, C-terminal epitopes of App a-CTF were mostly protected, indicating that App a-CTF preferentially trafficked to ILVs (Fig. 3D, left, bottom).

[0115] App a- and P-CTF differ by an additional 16 amino acids present at the N-terminus of App P-CTF, and these N-terminal neoepitopes may underlie the different trafficking pattern of these App-CTFs. The N-terminus of App a-CTF is flexible and loops back into the membrane to terminate at the surface, whereas the additional N-terminal 16 amino acids in App P-CTF re-emerge in the intralumenal / extracellular space46. Interestingly, a significantly higher percentage of App a-CTF is protected in Apphsamples as compared App ' samples (Fig. 3D), suggesting that the N-terminal neoepitope of App a-CTF alone does not determine ILV / EV localization. P3, the soluble product of App a-CTF digestion by y-secretase, contains the same a-cleaved neoepitope present in App a-CTF. As p3 is the product of the a- and y-secretase pathway, the Swedish App mutation results in lower p3 production. To determine the contribution of soluble p3 to the decrease in EVs seen in App samples, recombinant p3-40 (corresponding to AP17-40) was added to App' primary neuronal cultures, and total EVs were analyzed by nanoparticle tracking. Three doses of p3 were used which span the p3 concentrations found in human CSF47. Increasing doses of p3 showed no increase in total EV levels, with a slight but statistically significant decrease found at the 10 pM dose (Fig. 3E). When EVs were analyzed by size, a dose-dependent increase was seen in small EV (<40 nm), corresponding to the size of App-EVs (Fig. IF), reaching statistical significance at the 1 nM dose. The neoepitope formed by a-cleavage of App, present in both the membrane bound App a-CTF and soluble p3, can therefore modulate small EV biogenesis.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0116] METHODS

[0117] Animals

[0118] All animal breedings, maintenance, care, and experimental use was performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Rutgers Institutional Animal Care and Use Committee has approved the experimental use of animals generated in this study. Colony genotyping was performed by Transnetyx (TN, USA).

[0119] Primary Neuronal Culture

[0120] Plates and flasks were coated overnight with poly-L-lysine (Sigma P4707) and washed 3X with deionized water prior to use. Total cortex was dissected, and meninges were removed from p0-l rat pup brains. Dissected cortical tissues were digested with trypsin (Gibco 25200056), triturated, filtered with a 0.70 pm cell strainer, and plated onto coverslips or flasks.

[0121] 12-well plates without coverslips were seeded at 5xl05cells / well for western analysis and nanoparticle tracking analysis. T-75 flasks were seeded at 7.5xl06cells / flask and T-175 flasks were seeded at 1.5xl07cells / flask. Neurons were maintained in Neurobasal (Gibco 21103049) supplemented with 10% B-27 (Gibco 17504044), 1% Pen-Strep (Gibco 15140163), and 2mM glutamine (Gibco 25030081). Cultures were incubated at 37°C and 5% CO2 and given half-feeds twice a week. Neurons were treated with 2.5 pM NMS-873 (Sigma SML1128), 25 pM Smer28 (Sigma S8197), 25 pM VCP Activator 1 (MedChemExpress HY-157508), and 50 pM chloroquine (Cell Signaling Technology 14774). Neurons were treated with 0-1 nM p3 / Api7-40 peptide (Anaspec AS-22813).

[0122] Total EV Isolation

[0123] 14 DIV primary neuronal conditioned media was collected from 1 T-75 flask per biological replicate, and debris was removed by 0.22 pm PVDF syringe filtration. Total EVs were pelleted by ultracentrifugation of the filtrate at 150,000 x g for Ih at 4°C. Total pellet was lysed in IX loading buffer (LDS Thermo 84788 supplemented with 10% P-mercaptoethanol) prior to western analysis.

[0124] Immunocapture of App-EVs

[0125] 21 DIV primary neuronal conditioned media was collected from 4 T-175 flasks per biological replicate, and debris was removed by 0.22 pm PVDF syringe filtration. The filtrate was then concentrated to 80X with a 100 kDa MWCO Vivaspin 20 filter (Sigma Z614661) atRU: [P2025-040-01] / VHPM: 08035.154WO1

[0126] 3000 x g, 4°C. Anti-App 4G8 (BioLegend 800703), which targets amino acids 17-24 of Ap, or Anti -Mouse IgGl kappa Isotype Control (Thermo 14-4714-82) was bound to Protein A / G agarose beads (Thermo 20421) at 4°C for Ih with end-over-end rotation, at a concentration of 20 pg antibody per 100 pL beads. Unbound antibody was washed off 3X with IP buffer (1 mM EDTA, 50 mM Tris, 150 mM NaCl, pH 8) at 500 x g for Im. Primary neuronal conditioned media concentrate was incubated with 4G8 or anti-IgG beads at 4°C overnight with end-over-end rotation. Beads were washed 7X with IP buffer and eluted with 300 ng / mL Api7-24 peptide (Anaspec AS-61978) for Ih at room temperature with gentle agitation. Eluted EVs were used for downstream mass spectrometry, western, or transmission electron microscopic analysis.

[0127] Proteinase K Protection Assay

[0128] Brains from pO pups were homogenized with a glass-glass homogenizer in SEMK buffer (220 mM sucrose, 10 mM MOPS, 1 mM EDTA, 20 mM KC1, pH 7.2), supplemented with 1% protease / phosphatase inhibitor cocktail (Sigma PPC1010), on ice. The homogenate was centrifuged twice at 15,000 x g for 10m to produce a post-mitochondrial supernatant. The supernatant was ultracentrifuged at 150,000 x g for Ih to produce a pellet containing membranous organelles. The pellet was resuspended in SEMK buffer and total protein content was determined by Bradford analysis. In a 50 pL reaction, 50 pg of the membranous organellar fraction in SEMK buffer was digested with 0.5 pL of 2 pg / mL proteinase K (PK) (Sigma P6556) at 37°C for 10m. A negative control without proteinase K and a positive control with proteinase K and 0.1% SDS were performed simultaneously. Digestion was halted with 100 mM phenylmethyl sulfonyl fluoride (Roche 10837091001) and by boiling reaction mixture for 10m at 100°C. Total reaction mixture was analyzed by western analysis.

[0129] Mass Spectrometry

[0130] Mass spectrometry experiments were performed by MSBioworks (MI, USA) as follows: App-EV eluate was processed by SDS-PAGE using a 10% Bis-Tris NuPAGE gel (Invitrogen) with the MES buffer system. The mobility region was excised into 10 equal sized segments and in-gel digestion was performed on each using a robot (DigestPro, CEM) with the following protocol: washed with 25 mM ammonium bicarbonate followed by acetonitrile, reduced with 10 mM dithiothreitol at 60°C followed by alkylation with 50 mM iodoacetamide at RT, digested with sequencing grade trypsin (Promega) at 37°C for 4h, and quenched with formic acid. The supernatants were combined and lyophilized. Samples were dissolved in 0.1% TFA for analysis.RU: [P2025-040-01] / VHPM: 08035.154WO1

[0131] Half of each digested sample was analyzed by nano LC-MS / MS with a Waters M-Class LC system interfaced to a ThermoFisher Exploris 480 mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75 pm analytical column at 350 nL / min; both columns were packed with XSelect CSH C18 resin (Waters); the trapping column contained a 3.5 pm particle, the analytical column contained a 2.4 pm particle. The column was heated to 55°C using a column heater (Sonation). The mass spectrometer was operated in data-dep endent mode, with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS / MS respectively. The instrument was run with a 3s cycle for MS and MS / MS. Advanced Precursor Determination48was enabled. 5h of instrument time was used for the analysis of each sample.

[0132] Data were searched using a local copy of Mascot (Matrix Science) with the following parameters: Enzyme: Trypsin / P; Database: UniProt Rat (concatenated forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q), Deamidation (N,Q); Mass values:

[0133] Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. Mascot DAT files were parsed using Scaffold (Proteome Software) for validation, filtering and to create a non-redundant list per sample. Data were filtered at 1% protein and peptide FDR and requiring at least two unique peptides per protein.

[0134] Cell Surface Labeling

[0135] Total primary neuronal surface membrane proteins were labeled with Sulfo-NHS-SS-biotin and isolated by immunoprecipitation adapted from published protocols49. Briefly, primary neurons grown in 12-well plates were biotinylated with 0.3 mL of 0.5 mg / mL Sulfo-NHS-SS-biotin solution (Thermo 21331) for 30m on ice. Unreacted linker was quenched with 50 mM glycine in PBS 3X for 5m on ice. Neurons were lysed in 120 pL IP buffer supplemented with 1% Triton-X for 10m on ice. Lysate was centrifuged at 17,000 x g for 10m at 4°C and the supernatant was collected. An aliquot of total lysate was stored separately for western analysis. Total surface protein was isolated by immunoprecipitation with Neutravidin beads (Thermo 29200). 50 pL of 50% bead slurry was incubated with 100 pL lysate for 2h at 4°C with endover-end rotation. Beads were washed 7X with IP buffer and total surface protein was eluted by boiling for Im in 50 pL IX loading buffer.

[0136] Ap ELISA

[0137] Primary neuronal conditioned media from 14 DIV neurons was collected and dead cells were removed by 0.22 pm PVDF syringe filtration. Conditioned media levels of Ap40 and Ap42RU: [P2025-040-01] / VHPM: 08035.154WO1

[0138] were determined by Meso Scale Discovery (MSD) multi-array electrochemiluminescence assay kit (K15199G-1). MSD kit was used according to manufacturer’s recommendations and read on a MESO QuickPlex SQ 120 plate reader.

[0139] Western Analysis

[0140] For analysis of conditioned media, total conditioned media was passed through a 0.22 pm PVDF syringe filter. IX loading buffer was added to the filtrate, which was then boiled for Im and loaded. For analysis of cell lysate, primary neurons were lysed in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mMEDTA,l% Triton X-100, 0.1% SDS, 140 mMNaCl). 15 pg of protein was brought to 15 pl with PBS and IX loading buffer and loaded on a 4%-12% BisTris polyacrylamide gel (Bio-Rad 3450125). Proteins were transferred onto nitrocellulose at 25 V for 7m using the Trans-blot Turbo system (Bio-Rad) and visualized by red Ponceau staining.

[0141] Membranes were blocked for Ih in 5% milk (Bio-Rad 1706404) and washed extensively in PBS / Tween 20 (0.05%). Primary antibody was applied overnight at 4°C at 1:1000 dilution in 5% BSA (Fisher BP9703100). The following primary antibodies were used: 6E10 used for sAppa (App AP3-8 epitope, Biolegend 803001), Y188 used for App (App-C-terminus epitope, Abeam AB32136), Gapdh (Cell Signaling Technology 2118), Alix (Cell Signaling Technology 92880), Vcp (Cell Signaling Technology 2649), Lc3b (Cell Signaling Technology 83506), flotillin-1 (Cell Signaling Technology 18634), CD9 (Cell Signaling Technology 98327), Bip / Grp78 (Cell Signaling Technology 3183), and Vamp2 (Synaptic Systems 104202). Primary antibodies were washed off extensively and 1:1000 dilutions of secondary antibodies, either anti-mouse (Southern Biotech 1030-05) or anti-rabbit (Southern Biotech 4030-05) in 5% milk PBS / Tween 20, were applied for Ih at room temperature with shaking. Blots were developed with Clarity and Clarity Max ECL Western Blotting Substrates (Bio-Rad 1705060 and 1705062) and visualized on a ChemiDoc MP Imaging System (Bio-Rad). Signal intensity was quantified with Image Lab software (Bio-Rad).

[0142] Immunoprecipitation

[0143] Total pO brain lysate was diluted in IP buffer supplemented with 1% Triton-XlOO, solubilized for Ih at 4°C with end-over-end rotation. Samples were spun at 17,000 x g for 10m. Solubilized lysate was used as input for immunoprecipitation with anti-App 4G8, anti-Vcp (Invitrogen MA3-004), or control anti -Mouse IgGl kappa Isotype Control and protein A / G beads overnight at 4 °C with end-over-end rotation. Beads were washed 7X with IP buffer, andRU: [P2025-040-01] / VHPM: 08035.154WO1

[0144] bound protein was eluted by Im boiling in IX loading buffer. Input (diluted 1:20 in IX loading buffer) and eluates were analyzed by western blot analysis.

[0145] Statistical Analysis

[0146] Statistical significance was evaluated using ordinary one-way ANOVA followed by post hoc Tukey's multiple comparisons test when applicable (i.e. when the ordinary one-way ANOVA showed statistical significance) or by Student’s / -test. Statistical analysis was performed with GraphPad Prism vlO for Windows. Significant differences were accepted at < 0.05, with error bars representing SEM.

[0147] Nanoparticle Tracking Analysis

[0148] Conditioned primary neuronal culture media was analyzed by Alpha Nano Tech (Morrisville, NC). Briefly, samples were diluted with fresh 0.2 pm filtered (Sarstedt 831826001) deionized water to achieve a concentration of 100-300 particles per screen. The diluted samples were briefly vortexed and loaded into 1 mL syringes for loading into the machine. Zetaview Quatt NTA instrument (Particle Metrix, Meerbusch, Germany) was used for analyzing after alignment with 100 nm polystyrene beads. The following instrument settings were used: Mode at Scatter (488 nm), Sensitivity at 83, Shutter at 100, Cycles / positions at 1 / 11, Frame rate at 30, Maximum Size at 1000, Minimum Size at 20, Track Length at 15, Minimum Brightness at 20. Data in figures are represented after dilution factor adjustments.

[0149] TEM

[0150] Isolated App-EVs were analyzed by Alpha Nano Tech. Briefly, copper carbon Formvar grids were cleaned with glow discharge and floated on a sample drop for 10 minutes for sample adsorption. The grids were then washed twice by floating on a drop of deionized water and stained with 2% uranyl acetate for imaging using JEM-1230 (Jeol).

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[0223] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

RU: [P2025-040-01] / VHPM: 08035.154WO1CLAIMSWhat is claimed is:

1. A method for treating a neurodegenerative disease in a patient, comprising administering to a patient in need thereof a peptide that comprises the following amino acid sequence, or a therapeutically effective variant of the sequence: LVFFAEDVGSNKGAIIGLMVGGVV (SEQ IDNO:1).

2. The method of claim 1, comprising administering to a patient in need thereof a peptide that comprises the following amino acid sequence, or a therapeutically effective variant of the seqquence: LVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:2).

3. The method of any one of claims 1-2, wherein the peptide comprises a therapeutically effective variant of SEQ ID NO: 1 or SEQ ID NO:2 that has at least 90% identity to SEQ ID NO:1 or SEQ ID NO:2.

4. The method of any one of claims 1-3, wherein the peptide comprises a therapeutically effective variant of SEQ ID NO: 1 or SEQ ID NO:2 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1 or SEQ ID NO:2.

5. The method of any one of claims 1-4, wherein the disease is an amyloid beta dependent neurodegenerative disease.

6. The method of claim 5, wherein the disease is Alzheimer's disease (AD) or cerebral amyloid angiopathy (CAA).

7. The method of claim 6, wherein the disease is cerebral amyloid angiopathy (CAA).

8. The method of claim 6, wherein the disease is Alzheimer's disease (AD).

9. The method of claim 5, wherein the disease is early-onset AD.RU: [P2025-040-01] / VHPM: 08035.154WO110. The method of claim 5, wherein the disease is late-onset AD.

11. A peptide as described in any one of any one of claims 1-4 for the prophylactic or therapeutic treatment of an amyloid beta dependent neurodegenerative disease in a subject.

12. The peptide as described in any one of any one of claims 1-4 for the prophylactic or therapeutic treatment of Alzheimer's disease (AD) or cerebral amyloid angiopathy (CAA) in a subject.

13. The peptide as described in any one of any one of claims 1-4 for the prophylactic or therapeutic treatment of early-onset AD.

14. The peptide as described in any one of any one of claims 1-4 for the prophylactic or therapeutic treatment of late-onset AD.

15. The use of a peptide according to any one of claims 1-4 to prepare a medicament for the treatment of an amyloid beta dependent neurodegenerative disease.

16. The use of a peptide according to any one of claims 1-4 to prepare a medicament for the treatment of Alzheimer's disease (AD) or cerebral amyloid angiopathy (CAA) in a subject.

17. The use of a peptide as described in any one of any one of claims 1-4 for the prophylactic or therapeutic treatment of early-onset AD.

18. The use of a peptide as described in any one of any one of claims 1-4 for the prophylactic or therapeutic treatment of late-onset AD.

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