Delivery of Aβ mutants to inhibit aggregation

Abstract

Aspects of the present disclosure relate to compositions and methods for treating a subject having a neurodegenerative disorder, disease or condition. Particular aspects relate to treatment with a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide variant. Further aspects relate to a method of inhibiting aggregation of endogenous Aβ peptide in vivo by contacting at least one endogenous Aβ peptide in vivo with a therapeutically effective amount of an Aβ peptide variant expressed from a vector encoding the Aβ peptide variant, said vector being present in a composition.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 127,815, filed December 18, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention was made with Government support under awards NS092615, AG054160, AG056028 and AG058188 from the National Institutes of Health. The Government has certain rights in this invention.

[0003] Aspects of the present disclosure relate to at least the fields of cell biology, molecular biology, protein biology, neurobiology, and medicine. [Background technology]

[0004] Alzheimer's disease (AD) is a fatal neurodegenerative disorder and the most common cause of dementia. The pathological hallmark of Alzheimer's disease is the presence of neurofibrillary tangles and amyloid deposits in the brains of patients. Beta-amyloid peptide (Aβ) and its aggregated forms are the major components of amyloid plaques. Aβ1-42 is particularly prone to aggregation, forming oligomers, protofibrils, insoluble fibrils and plaques. Engineered antibody therapy is effective in lowering Aβ aggregation; however, the side effect profile and requirement of repeated intravenous delivery pose obstacles to the widespread use of engineered antibodies for the treatment of neurodegenerative diseases such as Alzheimer's disease. Thus, there is a need in the art for novel therapeutic approaches with high specificity, low toxicity and extended in vivo half-life that can prevent Aβ peptide oligomerization and aggregation. Summary of the Invention [Problem to be solved by the invention]

[0005] Aspects of the present disclosure address the need in the art by providing methods and compositions for treating a subject with a neurodegenerative disease, disorder or condition (e.g., Alzheimer's disease). Thus, in some aspects, the present invention provides methods and compositions for treating a subject with a neurodegenerative disease, disorder or condition, or inhibiting aggregation or promoting degradation of amyloid beta (Aβ) peptide in vivo, comprising administering a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof. In some embodiments, the disclosed methods also include diagnosing a subject with a neurodegenerative disease, disorder or condition, diagnosing a subject with symptoms of a neurodegenerative disease, disorder or condition, or diagnosing a subject at risk of having a neurodegenerative disease, disorder or condition. In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease, Parkinson's disease, Parkinson's dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down's syndrome, and / or pathological aging. In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease. [Means for solving the problem]

[0006] Embodiments of the present disclosure include methods and compositions for treating a subject having a neurodegenerative disease, disorder or condition and / or inhibiting aggregation of Aβ peptide in vivo. The methods of the present disclosure may include one, two, three, four, five or more of the following steps: providing an Aβ peptide variant to a subject, providing one or more additional therapies for a neurodegenerative disease, disorder or condition to the subject, diagnosing a subject with a neurodegenerative disease, disorder or condition, diagnosing a subject as having symptoms of a neurodegenerative disease, disorder or condition, and diagnosing a subject at risk for having a neurodegenerative disease, disorder or condition. Certain embodiments of the present disclosure may exclude one or more of the preceding elements and / or steps.

[0007] In some aspects, the present application discloses a method for treating or preventing a neurodegenerative disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof. In some embodiments, administering a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof, prevents or reduces protein misfolding, endogenous Aβ peptide aggregation, amyloid plaque formation, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; reduces tau levels, tau phosphorylation or phosphorylated tau levels; slows / slows tau seeding or endogenous Aβ peptide seeding or promotes cognitive improvement. In some embodiments, administering a vector encoding an Aβ peptide variant prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils or plaques. In some embodiments, administering a vector encoding an Aβ peptide mutant prevents or reduces the cytotoxicity of endogenous Aβ peptide ensembles.

[0008] In some aspects, a method of inhibiting aggregation of endogenous Aβ peptide in vivo is described herein, comprising contacting at least one endogenous Aβ peptide in vivo with a therapeutically effective amount of an Aβ peptide variant expressed from a vector encoding the Aβ peptide variant, said vector being present in a composition. In some embodiments, the method of inhibiting aggregation of endogenous Aβ peptide treats or prevents a neurodegenerative disease, disorder, or condition in a subject. In some embodiments, the method of inhibiting aggregation of endogenous Aβ peptide prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils, or plaques. In some embodiments, the method of inhibiting aggregation of endogenous Aβ peptide prevents or reduces the cytotoxicity of endogenous Aβ peptide aggregates.

[0009] In some embodiments, the method further comprises diagnosing a subject with a neurodegenerative disease, disorder or condition, diagnosing a subject with symptoms of a neurodegenerative disease, disorder or condition, and diagnosing a subject at risk of having a neurodegenerative disease, disorder or condition. In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease, Parkinson's disease, Parkinson's dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down's syndrome, and / or pathological aging. In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease.

[0010] In some aspects, disclosed herein is a pharmaceutical composition comprising a vector encoding an Aβ peptide variant.

[0011] In some embodiments of the methods and compositions disclosed herein, the Aβ peptide variant comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a fragment or functional derivative thereof.

[0012] In some embodiments, the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:3, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant comprises SEQ ID NO:3, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprising SEQ ID NO:3 comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids. In some embodiments, the Aβ peptide variant has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprises SEQ ID NO:3 and comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids. In some embodiments, a fragment or functional derivative of SEQ ID NO:3 comprises an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to KLVDFAE (SEQ ID NO:10).

[0013] In some embodiments, the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:4, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant comprises SEQ ID NO:4, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprising SEQ ID NO:4 comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids. In some embodiments, the Aβ peptide variant has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprises SEQ ID NO:4 and comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids. In some embodiments, a fragment or functional derivative of SEQ ID NO:4 comprises an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to KLVFPAE (SEQ ID NO:11).

[0014] In some embodiments, the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:5, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant comprises SEQ ID NO:5, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprising SEQ ID NO:5 comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids. In some embodiments, the Aβ peptide variant has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprises SEQ ID NO:5 and comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids. In some embodiments, a fragment or functional derivative of SEQ ID NO:5 comprises an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to KLVDFAE (SEQ ID NO:10).

[0015] In some embodiments, the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:6, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant comprises SEQ ID NO:6, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprising SEQ ID NO:6 comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid truncation. In some embodiments, the Aβ peptide variant has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to or comprises SEQ ID NO:6 and comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids. In some embodiments, a fragment or functional derivative of SEQ ID NO:6 comprises an amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to KLVPFAE (SEQ ID NO:12).

[0016] In some embodiments of the methods and compositions disclosed herein, the Aβ peptide variant is a polypeptide or polynucleotide encoding the Aβ peptide variant, hi some embodiments, the vector encodes a polynucleotide encoding the Aβ peptide variant.

[0017] In some embodiments, the vector or polynucleotide encoded by the vector encodes an Aβ peptide variant or a fragment or functional derivative thereof. In some embodiments, the vector or polynucleotide encoded by the vector encodes a minigene encoding an Aβ peptide variant or a fragment or functional derivative thereof. In some embodiments, the minigene encoding the Aβ peptide variant encodes a nucleotide sequence corresponding to an amino acid sequence comprising a truncated beta-carboxyl-terminal fragment (β-CTF) of amyloid precursor protein. In some embodiments, the truncated β-CTF is fused to a signal peptide sequence. In some embodiments, the signal peptide sequence comprises a nucleotide sequence corresponding to an amino acid sequence comprising a Gaussia luciferase signal peptide or a nucleotide sequence corresponding to an amino acid sequence comprising a mouse immunoglobulin heavy chain signal peptide. In some embodiments, the truncated β-CTF comprises an Aβ peptide variant sequence, a transmembrane domain sequence, and a cytoplasmic sequence. In some embodiments, the transmembrane domain sequence comprises a nucleotide sequence corresponding to an amino acid sequence comprising SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:86, or SEQ ID NO:87. In some embodiments, the cytoplasmic sequence comprises a nucleotide sequence corresponding to an amino acid sequence for membrane anchoring, facilitating gamma-secretase cleavage, and / or extracellular release of the Aβ peptide. In some embodiments, the cytoplasmic sequence corresponds to an amino acid sequence comprising two lysine residues. In some embodiments, the cytoplasmic sequence corresponds to an amino acid sequence comprising three lysine residues. In some embodiments, the cytoplasmic sequence corresponds to an amino acid sequence comprising, in the 5' to 3' direction, an arginine residue followed by two lysine residues.

[0018] In some embodiments, the minigene comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:8, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. In some embodiments, the minigene comprises SEQ ID NO:8, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. In some embodiments, the minigene comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70. In some embodiments, the minigene comprises SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70. In some embodiments, the minigene comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In some embodiments, the minigene comprises SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75. In some embodiments, the minigene comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80. In some embodiments, the minigene comprises SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80.

[0019] In some embodiments, minigene expression is regulated by a constitutive promoter. In some embodiments, minigene expression is regulated by a tissue-specific or cell-specific promoter. In some embodiments, the cell-specific promoter is a neuron-specific promoter. In some embodiments, the neuron-specific promoter is a synapsin promoter. In some embodiments, the tissue-specific promoter is a choroid plexus-specific promoter. In some embodiments, the tissue-specific promoter is Prlr, Spint2, or F5.

[0020] In some embodiments, the polynucleotide encoding the Aβ peptide or a fragment or functional derivative thereof is included in a vector. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the vector is an adenoviral, lentiviral, retroviral, or adeno-associated viral vector. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAV-DJ, AAVrhlO.XX, AAVrh.8, AAVrh.10, AAVrh.43, AAVpi.2, AAVhu.11, AAVhu.32, AAVhu.37, or PHP.eB AAV. In some embodiments, the vector is AAV9, PHP.eB, AAVrh.8, AAVrh.10, or AAVrh.43.

[0021] In some embodiments, 1×10 per kg of subject body weight 8 〜1×10 18 A dose of vector genome is administered to the subject. In some embodiments, a dose of about 1×10 per kg of the subject's body weight is administered. 11 to about 1×10 14 A dose of vector genome is administered to the subject. In some embodiments, a dose of about 1×10 per kg of the subject's body weight is administered.12 to about 1×10 15 A dose of the vector genome is administered to the subject. In some embodiments, the vector transduces the cells of the subject, and the cells of the subject express the minigene.

[0022] In some embodiments of the methods and compositions disclosed herein, the composition further comprises a pharma- ceutically acceptable carrier, hi some embodiments, the pharma- ceutically acceptable carrier comprises a liposome, a polymeric micelle, a microsphere, or a nanoparticle.

[0023] In some embodiments, the composition is delivered systemically or locally. In some embodiments, the composition is delivered systemically to the central nervous system through peripheral injection. In some embodiments, the peripheral injection is an intravenous injection. In some embodiments, the composition is delivered to the cerebrospinal fluid (CSF). In some embodiments, the composition is delivered to the cerebrospinal fluid (CSF) by non-surgical injection. In some embodiments, non-surgical injection into the cerebrospinal fluid (CSF) comprises non-surgical intrathecal injection. In some embodiments, the composition is delivered to the cerebrospinal fluid (CSF) by neurosurgical injection. In some embodiments, neurosurgical injection into the cerebrospinal fluid comprises neurosurgical injection into the cisterna magna. In some embodiments, the composition is delivered to the ventricular system. In some embodiments, the composition is delivered to the ventricular system by neurosurgical injection. In some embodiments, the composition crosses the blood-brain barrier.

[0024] In some embodiments, the composition is delivered to the subject once. In some embodiments, the composition is delivered before the onset of Aβ peptide oligomer, protofibril or fibril formation. In some embodiments, the composition is delivered after the onset of Aβ peptide oligomer, protofibril or fibril formation. In some embodiments, the composition is delivered before the onset of amyloid plaque formation. In some embodiments, the composition is delivered after the onset of amyloid plaque formation.

[0025] In some embodiments, a subject is provided with an effective amount of one or more additional therapies for a neurodegenerative disease, disorder, or condition. In some embodiments, the one or more additional therapies include an Alzheimer's disease medication. In some embodiments, the Alzheimer's disease medication includes aducanumab, donepezil, rivastigmine, galantamine, memantine, or tacrine.

[0026] The terms "individual," "subject," and "patient" are used interchangeably and may refer to a human or non-human.

[0027] Throughout this application, the term "approximately" is used to indicate that a value includes the inherent error range for the method of measurement or quantification.

[0028] When used in conjunction with the term "comprising," the use of the word "a" or "an" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0029] The phrase "and / or" means "and" or "or." For example, A, B and / or C includes A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C. That is, "and / or" functions as an inclusive or non-exclusive.

[0030] The words "comprising" (and all inclusive forms such as "including" and "including"), "having" (and all inclusive forms such as "having" and "having"), "including" (and all inclusive forms such as "including" and "including") or "containing" (and all inclusive forms such as "containing" and "containing") are inclusive or open-ended and do not exclude elements or method steps not further stated.

[0031] Compositions and methods for use may "comprise," "consist essentially of," or "consist of" any components or steps disclosed throughout the specification. Compositions and methods "consisting essentially of" any disclosed components or steps limit the claims to particular materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0032] In connection with a therapeutic, diagnostic or physiological purpose or effect, any method may be described as a "use" claim term, such as the "use" of any compound, composition or formulation discussed in this application to achieve or realize the described therapeutic, diagnostic or physiological purpose or effect.

[0033] It is contemplated that any embodiment discussed herein may be implemented with respect to any method or composition of the invention, and vice versa, and a composition of the invention may be used to achieve a method of the invention.

[0034] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0036] [Figure 1]Figures 1A-1E show that mutant Aβ peptides reduce fibrillization and cytotoxicity of wild-type Aβ. Figure 1A shows a thioflavin-T (ThT) assay for self-aggregation of mutant peptides compared to wild-type (WT) Aβ42. None of the five mutants showed self-aggregation during the reaction; for comparison, WT Aβ42 (green) reached a ThT binding plateau within 1 h. Figure 1B shows a competition assay testing inhibition of WT Aβ42 aggregation. Each mutant was mixed 1:1 with WT Aβ42 and incubated with ThT. V18P exacerbated aggregation of the WT peptide; the other four mutants reduced fibrillization. F20P (red) and F19D / L34P (blue) prevented aggregation and were selected for further studies. Figure 1C shows a fibril disassembly assay for F19D / L34P. WT Aβ42 fibrils were exposed to various concentrations of monomeric F19D / L34P peptide. Incubation with F19D / L34P compared to fibrils alone produced a concentration-dependent decrease in ThT fluorescence consistent with disassembly of Aβ fibrils. Figure 1D shows disassembly of fibrils for F20P as described for F19D / L34P. F20P also reduced ThT fluorescence in a concentration-dependent manner, but was significantly less effective than F19D / L34P. Figure 1E shows MTS analysis showing that unlike wild-type Aβ42 oligomers (green), F20P (red) or F19D / L34P (blue) did not induce toxicity in N2a cells per se, with all two mutants similar to untreated controls (black). In contrast, equimolar co-incubation of any mutant with WT Aβ during oligomeric Aβ formation reduced subsequent N2a cytotoxicity. X-axis values ​​indicate the volume (μl) of 100 μL oligomeric Aβ reaction added to N2a medium for testing. This corresponds to 0.1 or 10 μM of monomeric WT Aβ42 starting material and identical amounts of mutant peptide (where indicated). ANOVA, *p<0.05, **p<0.01 with Bonferroni post-hoc test for comparison with WT Aβ42 alone. All data are presented as mean ± SEM. [Diagram 2]Figures 2A-2D show expression constructs optimized for efficient secretion of mutant Aβ. Figure 2A shows the sequential deletion strategy used to identify the shortest CTF fragment sufficient for γ-secretase cleavage. Constructs were transfected into N2a cells and secretion was measured by recovering Aβ from the medium. The optimal construct contained the entire transmembrane domain and two intracellular lysines (KK). This minigene achieved Aβ secretion equivalent to the full-length CTF (left blot, arrows indicate secreted Aβ vs. uncleaved Aβ+ remaining transmembrane (TM) domain for each construct labeled with terminal residues). Secretion of wild-type Aβ42 using the 'KK' minimal construct was blocked in a dose-dependent manner by the β-secretase inhibitor LY411575 (right blot, arrows again indicate secreted Aβ vs. uncleaved Aβ+TM domain, GSI concentration in nM). Figure 2B shows the design of AAV vectors for delivery of mutant Aβ peptides in vivo. The expression cassette contains a Gaussia luciferase signal peptide at the N-terminus of Aβ followed by Aβ42 mutant and minimal APP C-terminal transmembrane sequences, both controlled by the CAG promoter. Figure 2C shows the extracellular release of full-length mutant Aβ using mass spectrometry analysis of immunoprecipitated Aβ. N2a cells expressing Aβ F20P were used to isolate secreted peptides from the medium by 6E10 immunoprecipitation. MS1 spectra of eluted peptides display the expected masses for Aβ40 F20P and Aβ42 F20P (data not shown). The peak for intact Aβ40 F20P at m / z = 856.4441 in the +5 charge state exhibits a monoisotopic mass of 4,277.1841 Da, while the most abundant isotope exhibits a mass of 4,279.1851 Da, within 10 ppm error of the expected mass. FIG. 2D shows MS2 fragmentation of a peptide matching the mass of Aβ40 F20P, confirming sequence identity with a fragment ion tolerance of less than 20 ppm. [Diagram 3]Figures 3A-3G demonstrate that neonatal AAV injection produces neural expression of mutant Aβ. AAV encoding F19D / L34P or F20P Aβ was injected into the lateral ventricle of wild-type neonatal mice. After 3 weeks or 7 months, mice were harvested for immunostaining and / or ELISA analysis. Figure 3A shows that anti-human Aβ immunostaining (6E10, green) in mice harvested 3 weeks after P0 injection of AAV-F20P demonstrates widespread viral expression in the cortex of sagittal sections. Figure 3B shows that co-staining for virus-transmitted mutant Aβ (F20P; 6E10, green) and endogenous mouse APP (Y188, red) demonstrates excellent concordance, suggesting membrane delivery of the mutant peptide in cortical neurons. Figure 3C shows mass spectrometry analysis of immunoprecipitated Aβ to confirm the generation of full-length mutant Aβ in vivo. Peptides were isolated from brain homogenates by 6E10 immunoprecipitation using mice expressing Aβ F20P. MS1 spectra of eluted peptides display the expected masses for Aβ42 F20P (shown) and Aβ40 F20P. The peak for intact Aβ42 F20P at m / z = 893.2675 with a +5 charge state (d = 0.2) exhibits a monoisotopic mass of 4,461.3011 Da, whereas the most abundant isotopic configuration is 4,463.3136 Da. Da, within 15 ppm error of the expected mass. Figure 3D shows MS2 fragmentation of the peptide to confirm the sequence identity of Aβ42 F20P, with a fragment ion tolerance of 20 ppm. Figure 3E shows human Aβ detected by ELISA in the soluble fraction of frontal lobe homogenates from wild-type mice euthanized at 3 weeks of age. All two mutants produced human Aβ42, while Aβ40 was only detected in mice transduced with F20P. F20P produced high levels of Aβ40 compared to uninjected mice, whereas F19D / L34P showed negligible Aβ40 production, speculated to be due to poor detection of the mutant peptide (abbreviated as F19D in the graph). Aβ42 production was lower than Aβ40 as expected for a construct encoding a wild-type γ-secretase site.In Fig. 3F, expression of mutant human Aβ can still be detected by ELISA 7.5 months after P0 virus injection. Values ​​in the figure refer to the soluble fraction of frontal lobe homogenates of non-transgenic animals. Absolute values ​​in panels C and D cannot be directly compared because assays were performed at different times with different production lots of kits. In Fig. 3G, the ratio of Aβ40:42 produced by each mutant remains constant between 3 weeks (upper panel) and 7.5 months (lower panel). ANOVA, *p<0.05, ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. n=2-10 / treatment. [Figure 4] Figures 4A-4C demonstrate that lifelong expression of mutant Aβ reduces plaque burden and Aβ accumulation in APP / PS1 mice. APP / PS1 mice were injected with AAV encoding Aβ F19D / L34P or F20P at P0 and harvested 7.5 months later. Figure 4A shows that Aβ immunostaining indicates reduced plaque accumulation in mice treated with mutant Aβ peptides. Figure 4B shows that cortical plaque burden, measured by %Aβ area, confirms that F20P mice have less amyloid than untreated mice. n=5 uninjected, n=4 F19D / L34P, n=8 F20P. Figure 4C shows that MSD ELISA for human Aβ peptides in guanidine extracts of cortical tissue reflects plaque histology. Levels of Aβ40 were reduced in all two mutants, whereas F20P also reduced levels of Aβ42. Reduction of Aβ reached significant levels in F20P and showed a trend in F19D / L34P. n=8 uninjected, n=5 F19D / L34P, n=12 F20P. ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as mean ± SEM. [Diagram 5]Figures 5A-5B demonstrate the use of viral serotypes to control the spatial distribution and timing of transgene expression. Figure 5A shows the PHP.eB virus encoding red fluorescent protein used to demonstrate CNS expression after peripheral injection. 1.6 x 1011 particles of PHP.eB encoding CAG-tdTomato were injected into the retro-orbital sinus of 3-month-old mice; mice were harvested 2 weeks later. Figure 5B shows that, unlike AAV8 used in Figure 3, low titer AAV1 preferentially transduces ependymal cells when injected into the lateral ventricles of neonatal mice. Note the spread of virus to the fourth ventricle. The insert in the figure shows the lateral ventricle (boxed) at higher magnification. Alternative serotypes such as AAV4 or AAV5 may be used to provide specificity for ependymal cells when injected into the lateral ventricles. [Figure 6]Figures 6A-6D show that mutant Aβ peptides reduce reactive gliosis commensurate with plaque burden. All APP / PS1 and wild-type (non-transgenic, NTG) mice were harvested 7.5 months after icv P0 virus injection to deliver F20P mutant Aβ. Non-injected siblings were used for comparison. Figures 6A, 6B show GFAP immunostaining used to detect astrocytes; Iba1 to detect microglial cells. Fluorescent immunostaining shown in the bottom row was counterstained with thioflavin-S to detect amyloid plaques. F20P treatment in APP / PS1 mice all reduced the size of neuroglial foci and the number of surrounding cells compared to uninjected animals (top and bottom rows). In contrast, virus injection did not affect microglial cell morphology or density in NTG mice, but increased the number of GFAP+ cells along the corpus callosum (middle row). Figure 6C shows quantification of colorimetric immunostaining for GFAP and Iba1, confirming the qualitative finding that F20P treatment reduced the corresponding glial stained areas in APP / PS1 mice, but increased the levels of GFAP in NTG mice compared to uninjected controls. Figure 6D shows that viral expression of human Aβ was detected in a subset of forebrain astrocytes. Co-immunofluorescence for human Aβ (6E10, green) and GFAP (red) detected sparse co-labeled cells (arrows) in NTG mice injected with F20P. APP / PS1 n=5 uninjected, n=7-8 F20P; NTG n=5-6 uninjected, n=5 F20P. A small proportion of GFAP+ astrocytes (red) co-labeled with anti-human Aβ antibody 6E10 (green) suggests that they were transduced at P0 by AAV injection. Two-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001. Data are presented as mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention is based at least in part on the surprising discovery that certain amyloid beta (Aβ) peptide mutants, vectors encoding the mutants, or vectors encoding minigenes enabling protein expression of mutant Aβ peptides, can inhibit aggregation of endogenous Aβ peptides. It has also been surprisingly found that administering vectors encoding said mutants or vectors encoding minigenes enabling protein expression of mutant Aβ peptides prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils or plaques and the cytotoxicity of endogenous formation of endogenous Aβ peptide aggregates. As disclosed herein, administration of a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide mutant or a vector encoding a minigene enabling protein expression of the mutant Aβ peptide can prevent or reduce protein misfolding, aggregation of endogenous Aβ peptide, formation of amyloid plaques, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; can reduce tau levels, tau phosphorylation or phosphorylated tau levels; can / can blunt tau seeding or endogenous Aβ peptide seeding, or can promote cognitive improvement.

[0038] Thus, in some embodiments, methods and compositions are disclosed for treating or preventing a neurodegenerative disease, disorder or condition from a subject, or inhibiting the aggregation of endogenous Aβ peptide in vivo, comprising administering a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide variant or a vector encoding a minigene enabling protein expression of the variant Aβ peptide. In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease, Parkinson's disease, Parkinson's dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down's syndrome, and / or pathological aging. In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease. In some embodiments, the subject has previously been diagnosed with a neurodegenerative disease, disorder or condition, symptoms of a neurodegenerative disease, disorder or condition, or is at risk of having a neurodegenerative disease, disorder or condition.

[0039] I. Neurodegenerative Diseases, Disorders and Conditions In some embodiments, a method for treating a neurodegenerative disease is disclosed. The term "neurodegenerative disease, disorder or condition" or "neurodegenerative disease" as used herein refers to a condition that primarily affects neurons in the human brain, resulting in the gradual loss of neuronal structure or function, including neuronal death. The neurodegenerative disease may be specifically of the following types, but is not limited to: Alzheimer's disease, Parkinson's disease, Parkinson's disease dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down's syndrome, and / or pathological aging. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0040] Neurodegenerative diseases are incurable, debilitating conditions that result in the progressive degeneration and / or death of nerve cells in the brain or peripheral nervous system. This causes problems with movement or mental function (called dementia). Dementia is the loss of cognitive functions, such as the ability to think, remember, reason and act, to a degree that interferes with daily life and activities. Dementia ranges in severity from the mildest stages, where it only just begins to affect a person's functioning, to the most severe stages, where a person must become completely dependent on others for the basic activities of daily life.

[0041] The causes of dementia can be diverse depending on the type of brain changes that may occur. Dementia is responsible for the largest burden of neurodegenerative diseases, with Alzheimer's disease representing approximately 60-70% of dementia cases. In 2016, approximately 5.4 million Americans were affected by Alzheimer's disease. Other dementias include Lewy body dementia, anterior temporal lobe disorder, and vascular dementia. In some cases, patients may have mixed dementia, where two or more types of dementia are combined. For example, some patients have both Alzheimer's disease and vascular dementia.

[0042] Alzheimer's disease (AD) is a chronic neurodegenerative disorder that results in the loss of neurons and synapses in the cerebral cortex and certain cortical substructures, including the general atrophy and degeneration of the temporal, parietal, and frontal cortices, as well as parts of the cingulate gyrus. Degeneration may also occur in brainstem nuclei such as the locus ceruleus. MRI and PET studies have documented a decrease in the size of certain brain regions in Alzheimer's patients compared to similar images of healthy elderly subjects as they progress from mild cognitive impairment to Alzheimer's disease. Alzheimer's disease pathology is primarily characterized by the presence of senile plaques and neurofibrillary tangles that disrupt normal brain function and chemistry, resulting in a significant deficiency of neurotransmitters and a gradual loss of brain function. Abnormal amounts of beta-amyloid and tau proteins form in the brain and begin to eat away at brain cells, forming plaques and neurofibrillary tangles. Both amyloid plaques and neurofibrillary tangles are clearly visible under a microscope in Alzheimer's disease brains, particularly in the hippocampus. While many older people develop some plaques and tangles as a result of aging, the brains of Alzheimer's disease patients have higher amounts of plaques and tangles in certain brain regions, such as the temporal lobe.

[0043] The most common early symptom is difficulty experienced in remembering recent events. As Alzheimer's disease progresses, symptoms may include language problems, disorientation, mood changes, loss of motivation, mismanagement of self-care, and behavioral problems. Patients with more severe Alzheimer's disease often withdraw from their families and society, leading to gradual loss of physical function and ultimately death. Although the rate of progression can vary, the typical life expectancy after an Alzheimer's disease diagnosis is 3 to 9 years.

[0044] A. The Alzheimer's Disease Process The disease process is divided into distinct stages with a gradual pattern of cognitive and functional impairment. Preclinical Alzheimer's disease is characterized by the presence of biomarkers for Alzheimer's disease, such as amyloid detected by PET or CSF examination. Mild cognitive impairment (MCI) is characterized by the onset of Alzheimer's disease symptoms. It has often been found to be a transitional stage between normal aging and dementia. MCI can present with a variety of symptoms, and when memory loss is the predominant symptom, it is referred to as "amnesic MCI" and is frequently considered as a prodromal stage of Alzheimer's disease. Although the first symptoms are often mistakenly attributed to aging or stress, through detailed neuropsychological testing, subtle cognitive impairment can appear up to eight years before clinical criteria for the diagnosis of Alzheimer's disease are met. The most significant deficit is short-term memory loss, which manifests as difficulty remembering recently learned facts and an inability to acquire new information. Subtle problems with executive functions such as attention, planning, flexibility and abstract thinking or impairment of semantic memory, meaning memory and conceptual relations can also be symptoms of early Alzheimer's disease. Apathy may also be observed and remains the most persistent neuropsychiatric symptom throughout the course of the disease. Depressive symptoms, irritability, and decreased awareness of subtle memory impairment are also common.

[0045] Increasing learning and memory impairments in Alzheimer's patients eventually lead to the final diagnosis. In a small percentage of patients, difficulties with language, executive function, perception (akinesia) or execution of movements (akinesia) are more pronounced than memory problems. Alzheimer's does not affect all memory capacities equally. Old memories (episodic memory), learned facts (semantic memory) and implicit memory (physical memory for how to do things) about an individual's life are less affected than new facts and memories. Language problems are primarily characterized by a decrease in vocabulary and word fluency, leading to a generalized deficit in oral and written language. At this stage, basic thoughts can generally be communicated adequately. Difficulties in coordinating and planning specific movements or akinesia may be present while performing fine motor tasks, but may not be noticeable. As Alzheimer's progresses, patients can continue to perform many tasks independently, but may need help or supervision for the most cognitively demanding activities.

[0046] Gradual deterioration eventually leads to the patient being unable to carry out the most common activities of daily living, hindering independence. Language impairment may manifest as an inability to memorize vocabulary, leading to frequent incorrect word substitutions or paraphrases. Reading and writing skills may be gradually lost. As Alzheimer's disease progresses, complex motor sequences may become poorly coordinated, increasing the risk of falls. Memory problems may worsen and long-term memory may be damaged. Behavioral and neuropsychiatric changes may become more widespread and may include wandering, irritability, labile affect leading to crying, unplanned outbursts of aggression, or resistance to caregiving. Sundowning may also be present. Approximately 30% of patients with Alzheimer's disease have optical illusions and other delusional symptoms. Patients may also lose insight into the disease process and limitations, known as anosognosia.

[0047] In the final stages of Alzheimer's disease, patients may become totally dependent on caregivers. Speech may be reduced to mere phrases or single words, eventually resulting in complete loss of speech. Aggression may still be present, as well as extreme apathy and weakness. Muscle mass and mobility may deteriorate to the point where patients become bedridden and unable to feed themselves. The cause of death is generally external factors such as infection or pneumonia rather than the disease itself.

[0048] B. Causes of Alzheimer's Disease The genetic heritability of Alzheimer's disease, based on examination of twin and family studies, ranges from 49% to 79%. Approximately 0.1% of cases are autosomal dominant with familial forms of onset before age 65, known as early-onset familial Alzheimer's disease. Most autosomal dominant familial Alzheimer's disease cases may be due to mutations in one of three genes: the genes encoding amyloid precursor protein (APP) and presenilin 1 and 2. Most mutations in the APP and presenilin genes increase production of beta-amyloid protein (A-beta protein or Aβ protein), the main component of senile plaques, or increase the ratio of Aβ42 protein (made of 42 amino acids) to Aβ40 protein (made of 40 amino acids). Some mutations remodel the ratio between Aβ42 and the other major forms of amyloid beta protein, such as Aβ40, without increasing total Aβ levels.

[0049] Most cases of Alzheimer's disease do not show autosomal dominant inheritance, but are referred to as sporadic Alzheimer's disease, where environmental and genetic variations may act as risk factors. The best-known genetic risk factor is the inheritance of the ε4 allele of apolipoprotein E (APOE). Between 40 and 80% of Alzheimer's disease patients carry at least one APOE ε4 allele. The APOE ε4 allele increases the risk of disease by up to three-fold in heterozygotes and 15-fold in homozygotes. Genome-wide association studies (GWAS) have found more than 30 regions in additional genes that appear to affect the risk of Alzheimer's disease. These genes include, but are not limited to, ABCA7, SORL1, CASS4, CELF1, FERMT2, HLA-DRB5, INPP5D, MEF2C, NME8, PTK2B, SORL1, ZCWPW1, SLC24A4, CLU, PICALM, CR1, BIN1, MS4A, ABCA7, EPHA1, and CD2AP. Additional genes that are associated with risk for Alzheimer's disease can be found in the literature [see BW Kunkle et al., Nature Genetics 51, 414-430 (2019)], the entire text of which is incorporated by reference herein. Alleles of the TREM2 gene are also associated with a 3-5 times higher risk of developing Alzheimer's. Microglial cells in the brain in some TREM2 mutants are no longer able to regulate the amount of beta-amyloid present. According to a 2018 study by Mukherjee et al. (Genetic and cognitively defined subgroups of late-onset Alzheimer's disease; Mol Psychiatry (2018), specifically incorporated herein by reference in its entirety, which adds 30 SNPs by dividing Alzheimer's disease into six categories including memory, language, spatiotemporal, and executive function), many single nucleotide polymorphisms (SNPs) are associated with Alzheimer's disease.

[0050] Familial Alzheimer's disease in Japanese pedigree has been shown to be associated with a deletion mutation at codon 693 of APP, known as the Osaka mutation. Homozygotes for this mutation develop Alzheimer's disease. The mutation accelerates Aβ oligomerization, but the protein does not form amyloid fibrils. Mice expressing this mutation have all the common pathology of Alzheimer's disease due to the Osaka mutation.

[0051] An alanine to valine substitution mutation at codon 673 of the APP gene increases the risk of Alzheimer's disease in homozygous carriers along with the development of amyloid fibrils, possibly by promoting the formation of Aβ, whereas heterozygous carriers of A673V may be protected from Alzheimer's disease. This substitution is adjacent to the beta-secretase cleavage site and may result in increased APP cleavage by beta-secretase through the amyloidogenic pathway. In contrast, substitution of the same alanine at position 673 with threonine (A673T) can prevent Alzheimer's disease. This substitution is adjacent to the beta-secretase cleavage site and can reduce the formation of amyloid beta by 40% in vitro.

[0052] It has also been proposed that extracellular Aβ deposits are the underlying cause of Alzheimer's disease. Support for this theory is the location of the APP gene on chromosome 21 as well as the fact that many trisomy 21 (Down's syndrome) patients with an additional gene copy show the earliest symptoms of AD by age 40. APOE4 is also a major genetic risk factor for Alzheimer's disease (see, e.g., M. Safieh et al., BMC Medicine 17:64 (2019); and C.G. Fernandez et al., Front. Aging Neuroscience 11:14 (2019); specifically, these references are incorporated herein by reference in their entirety). Transgenic mice expressing a mutant form of the human APP gene develop Alzheimer's-like brain pathology with fibrillar amyloid plaques and spatial learning defects.

[0053] Aβ deposits are formed from Aβ (also called A-beta peptide or beta-amyloid), which are typically 39-43 amino acids in length. Aβ peptides are fragments of a larger protein called transmembrane protein (APP) that spans cell membranes. APP appears to play a role in normal neuronal growth, survival, and recovery after injury. In Alzheimer's disease, gamma-secretase and beta-secretase work together in a proteolytic process to split APP into smaller fragments. These fragments generate beta-amyloid fibrils that can self-assemble into dense extracellular deposits known as senile or amyloid plaques. Plaques are dense and largely insoluble deposits composed of beta-amyloid peptide and cellular material outside and around neurons. Experimental vaccines have been shown to clear amyloid plaques in early human trials but have not had a significant effect on dementia, which may indicate that non-plaque Aβ oligomers are the predominant pathogenic form of Aβ. These toxic oligomers, also known as amyloid-derived diffusible ligands, disrupt neuronal communication by binding to neuronal surface receptors and altering synaptic structure.

[0054] Other studies have proposed that tau protein abnormalities initiate the AD cascade. Every neuron has a cytoskeleton that is partially composed of microtubules. When tau protein is phosphorylated, it stabilizes the microtubules. Tau in Alzheimer's disease models undergoes chemical changes and becomes hyperphosphorylated. Hyperphosphorylated tau begins to pair with other threads of tau. Eventually, they form neurofibrillary tangles inside the neuronal cell body. Tangles (neurofibrillary tangles) are aggregates of the microtubule-associated protein tau that are hyperphosphorylated and accumulate inside the cell. When tangles occur, the microtubules are disassembled, the cytoskeleton structure of the cell is destroyed, and the neuronal transport system is disrupted. Initially, it can cause a malfunction of biochemical communication between neurons and later cell death. Pathogenic tau can also induce neuronal death through translocation factor dysregulation.

[0055] Age is an important risk factor for Alzheimer's disease. Age-related changes in the brain can harm neurons and affect other types of brain cells, contributing to Alzheimer's damage. Age-related changes include atrophy (shrinkage) of certain parts of the brain, inflammation, blood vessel damage, production of unstable molecules such as free radicals, and mitochondrial dysfunction (disruption of intracellular energy production). Thus, older age (over 60 years of age) is a risk factor for Alzheimer's disease.

[0056] There are several additional theories that have been presented to explain the causes of Alzheimer's disease. For example, the inflammatory hypothesis is that Alzheimer's disease is triggered by a self-sustaining progressive inflammation in the brain that culminates in neurodegeneration. In some models, infectious viral and microbial agents appear to act together with Aβ to generate a positive feedback mechanism for neuroinflammation. See AL Komaroff, JAMA 324(3):239-240(2020)]. A neurovascular hypothesis has also been proposed in which impaired function of the blood-brain barrier (BBB) ​​may be involved. Cellular homeostasis of biometals such as ionic copper, iron and zinc is also disrupted in Alzheimer's disease; these ions affect and are affected by tau, APP and APOE, and their dysregulation can induce oxidative stress that can contribute to the pathology. Another hypothesis is that during aging, dysfunction of oligodendrocytes and associated myelin contributes to axonal injury, inducing amyloid production and tau hyperphosphorylation as a side effect.

[0057] C. Diagnosis of Alzheimer's Disease Alzheimer's disease is generally diagnosed based on the patient's medical history, the medical history of relatives, and behavioral observations. It is supported by the presence of characteristic neurological and neuropsychological features and the absence of alternative conditions. Advanced medical imaging with computed tomography or magnetic resonance imaging and single-photon emission computed tomography or positron emission tomography can be used to exclude other brain pathologies or subtypes of dementia. Also, the prodromal stage, represented by mild cognitive impairment, can predict the conversion to Alzheimer's disease.

[0058] The National Institute on Aging and Alzheimer's Association Workgroup Research Framework for Diagnosis of Alzheimer's Disease currently uses a biomarker classification system that divides the major Alzheimer's disease biomarkers into three categories based on pathological change typology: β-amyloid (A), pathological tau (T) and neurodegenerative (N). The ATN nomenclature represents a conceptual framework based on empirical observations over the past decade of the relationship between amyloid, tau and neurodegenerative markers. "A" refers to amyloid β (Aβ) measured by amyloid positron emission tomography (PET) imaging of amyloid plaques or in cerebrospinal fluid (CSF) as Aβ42 or the ratio of Aβ42 to Aβ40. "T" refers to tau pathology measured by CSF phosphorylated tau or tau PET imaging of parenchymal neurofibrillary tangles. "N" refers to neurodegeneration or neuronal damage and dysfunction as measured, for example, by hippocampal or cortical volume or thickness. "A"+"T" is considered to have diagnostic specificity for Alzheimer's disease, whereas "N" is not specific for the diagnosis of Alzheimer's disease, as it can reflect any number of etiologies besides Alzheimer's disease. The ATN system has been described in detail in the literature (DS Knopman et al., Alzheimer's & Dement. 14(4):563-575 (2018), specifically, the entire text of which is incorporated herein by reference).

[0059] Assessment of intellectual function, including memory testing, can further characterize the disease state. Medical organizations have constructed diagnostic criteria to standardize the diagnostic process. In 1984, the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA, now known as the Alzheimer's Association) established the most commonly used NINCDS-ADRDA Alzheimer's criteria, which require that cognitive impairment and suspected dementia syndromes be confirmed by neuropsychological testing for a possible or probable clinical diagnosis of Alzheimer's disease. Eight intellectual domains, such as memory, language, perceptual abilities, attention, motor skills, orientation, problem solving, and executive function abilities, are most commonly impaired in Alzheimer's disease. These domains are identical to the NINCDS-ADRDA Alzheimer's criteria aligned in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) published by the American Psychiatric Association. Excellent statistical reliability and validity have been demonstrated between the diagnostic criteria and unequivocal histopathological confirmation.

[0060] In 2011, the National Institute on Aging / Alzheimer's Association published an updated approach for clinicians and scientists to provide high-level guidelines for advancing research into diagnosis and treatment. The 2011 guidelines differ from the 1984 diagnostic criteria in several important ways. The organizations recognize that Alzheimer's disease progresses into a spectrum of three stages: an early preclinical stage with no symptoms; an intermediate stage of mild cognitive impairment; and a final stage in which dementia symptoms appear. The 1984 criteria address only one stage of the disease, the final stage of dementia; and expand the criteria for Alzheimer's dementia beyond memory loss being the first or only presenting symptom. The organizations recognize that other aspects of cognition, such as word-finding ability and judgment, may be impaired first. The 1984 criteria focus on memory loss as the core feature of Alzheimer's dementia; and reflect a greater understanding of the differences and associations between Alzheimer's and non-Alzheimer's dementia, as well as between disorders that may affect pathogenesis, such as Alzheimer's and vascular disease. In 1984, these relationships were not well recognized or understood, and the guidelines recognize the potential use of biomarkers that are indicators of underlying brain disease to diagnose Alzheimer's disease. However, the guidelines state explicitly that biomarkers would be used almost exclusively in research rather than clinical settings. These biomarkers did not exist when the original criteria were developed in 1984, and diagnosis was generally confirmed through autopsy after death.

[0061] Neuropsychological tests such as the Mini-Mental State Examination (MMSE) are widely used to assess cognitive impairment necessary for diagnosis. Neurological examinations in early Alzheimer's disease generally provide normal results, except for overt cognitive impairment that may not differ from the outcome of other disease processes, including other causes of dementia. Additional neurological testing is important for the differential diagnosis of Alzheimer's disease and other diseases. Interviews with family members are also utilized in the evaluation of the disease. Caregivers can provide important information regarding the patient's decline in mental functioning over time, as well as daily living abilities. The caregiver's perspective may be particularly important, as patients with Alzheimer's disease are generally unable to recognize their own deficits.

[0062] Supplemental testing can provide additional information about disease characteristics or be used to rule out other diagnoses. Blood tests can identify alternative causes of dementia other than Alzheimer's disease. Also common are thyroid function tests, B12 evaluation, syphilis exclusion, metabolic problem exclusion (including kidney function, electrolyte levels, and diabetes testing), heavy metal (e.g., lead, mercury) levels, and anemia evaluation. Because depression may be a cause in addition to or in conjunction with early signs of Alzheimer's disease and cognitive impairment, forensic testing for depression can also be used.

[0063] II. Aβ peptide variants In some embodiments, the disclosed compositions comprise at least one proteinaceous molecule. In some embodiments, the proteinaceous molecule comprises an amyloid beta (Aβ) peptide or a variant thereof. In some embodiments, the proteinaceous molecule comprising a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof is encoded by a polynucleotide. In some embodiments, the polynucleotide is encoded by a vector. In some embodiments, the polynucleotide is encoded by a vector encoding a minigene that allows for the protein expression of the Aβ peptide. Thus, in some embodiments, the wild-type or variant Aβ peptide or a fragment or functional derivative thereof is encoded by a vector. In some embodiments, the Aβ peptide or a fragment or functional derivative thereof is encoded by a vector encoding a minigene that allows for the protein expression of the Aβ peptide.

[0064] The Aβ peptide is a fragment of a larger protein called transmembrane protein (APP) that penetrates across neuronal membranes. A representative mRNA APP sequence can be found in GenBank® Accession No. NM_000484 and comprises the following amino acid sequence:

[0065] (SEQ ID NO:1)

[0066] Gamma-secretase and beta-secretase act together in the proteolytic process to split APP into smaller amino acid fragments known as Aβ (amyloid beta or A-beta) peptides. Gamma-secretase, which generates the C-terminus of the Aβ peptide, can cleave within the transmembrane region of APP to generate multiple C-terminal fragment isoforms ranging from 30 to 51 amino acid residues in length. In addition to these C-terminal fragments, a 42 amino acid Aβ peptide fragment containing the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:2) is also generated by cleavage of APP. These fragments generate beta-amyloid fibrils that can self-assemble into dense extracellular deposits known as plaques. Aβ42 is the most amyloidogenic form of the peptide because it is more hydrophobic. However, the central amino acid sequence KLVFFAE (SEQ ID NO:39) is known to form amyloid by itself and can form the core of amyloid fibrils.

[0067] Thus, in some embodiments of the present disclosure, the disclosed compositions include wild-type Aβ peptide. In some embodiments of the present disclosure, the disclosed compositions include mutants of wild-type Aβ peptide. In some embodiments, the wild-type Aβ peptide or mutant Aβ peptide promotes aggregation of endogenous Aβ peptide. In some embodiments, the wild-type Aβ peptide or mutant Aβ peptide self-assembles into oligomers, protofibrils, fibrils, or plaques. In some embodiments, the mutant Aβ peptide prevents aggregation of endogenous Aβ peptide. In some embodiments, the mutant Aβ peptide does not self-assemble into oligomers, protofibrils, fibrils, or plaques. In some embodiments, the mutant Aβ peptide reduces the cytotoxicity of remaining Aβ peptide aggregates. In some embodiments, the mutants of full-length Aβ disclosed herein simultaneously allow targeting of multiple aggregation domains to increase affinity and specificity for Aβ.

[0068] In some embodiments, a wild-type Aβ peptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:2), or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, a wild-type Aβ peptide of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:2, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:2 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVFFAE (SEQ ID NO:39), or any value derivable within these ranges.

[0069] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000002.tif12168, or a fragment or functional derivative thereof, or any value derivable therein, wherein bolded residues indicate substitutions from a wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of the present disclosure comprise, consist of, or consist essentially of SEQ ID NO:3, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:3 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVDFAE (SEQ ID NO:10), or any value derivable within these ranges.

[0070] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000003.tif12168, or a fragment or functional derivative thereof, or any value derivable therein, wherein bolded residues indicate substitutions from a wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of the present disclosure comprise, consist of, or consist essentially of SEQ ID NO:4, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:4 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVFPAE (SEQ ID NO:11), or any value derivable within these ranges.

[0071] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000004.tif10168, or a fragment or functional derivative thereof, or any value derivable therein, wherein bolded residues indicate substitutions from a wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of the present disclosure comprise, consist of, or consist essentially of SEQ ID NO:5, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:5 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVDFAE (SEQ ID NO:10), or any value derivable within these ranges.

[0072] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000005.tif10168, or a fragment or functional derivative thereof, or any value derivable therein, wherein bolded residues indicate substitutions from a wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of the present disclosure comprise, consist of, or consist essentially of SEQ ID NO:6, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:6 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVPFAE (SEQ ID NO:12), or any value derivable within these ranges.

[0073] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000006.tif11168, or a fragment or functional derivative thereof, or any value derivable therein, wherein bolded residues indicate substitutions from a wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of the present disclosure comprise, consist of, or consist essentially of SEQ ID NO:7, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:7 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLPFFAE (SEQ ID NO:65), or any value derivable within these ranges. In some embodiments, the Aβ peptide variant comprises, consists of, or does not essentially consist of SEQ ID NO:7.

[0074] In some embodiments, a wild-type Aβ peptide or an Aβ peptide variant comprising, consisting of, or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a 1 to 22 amino acid truncation. In some embodiments, the N-terminal truncation comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid truncation.

[0075] In some embodiments, a wild-type Aβ peptide or an Aβ peptide variant comprising, consisting of, or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a 1 to 27 amino acid truncation. In some embodiments, the C-terminal truncation comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acid truncation.

[0076] In some embodiments, a wild-type Aβ peptide or an Aβ peptide variant comprising, consisting of, or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises an N-terminal truncation and a C-terminal truncation, in some embodiments, the N-terminal truncation comprises a 1-22 amino acid truncation and the C-terminal truncation comprises a 1-27 amino acid truncation. In some embodiments, N-terminal truncations include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid truncations and C-terminal truncations include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acid truncations.

[0077] In some embodiments, the wild-type Aβ peptide or Aβ peptide variant comprises a polypeptide. In some embodiments, the wild-type Aβ peptide or Aβ peptide variant comprises a polynucleotide encoding the wild-type Aβ peptide or Aβ peptide variant, or a fragment or functional derivative thereof. In some embodiments, the vector encodes a polynucleotide encoding the wild-type Aβ peptide or Aβ peptide variant, or a fragment or functional derivative thereof. In some embodiments, the vector or polynucleotide encoded by the vector encodes the wild-type Aβ peptide or Aβ peptide variant, or a fragment or functional derivative thereof fused to a signal peptide. In some embodiments, the vector or polynucleotide encoded by the vector encodes the entire APP beta-carboxyl-terminal fragment (β-CTF), comprising the 99 C-terminal amino acids of amyloid precursor protein comprising the wild-type Aβ peptide or Aβ peptide variant amino acid sequence fused to a signal peptide at the N-terminus of the β-CTF.

[0078] In some embodiments, the vector or polynucleotide encoded by the vector encodes a minigene encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof. In some embodiments, the minigene encodes a nucleotide sequence corresponding to an amino acid sequence comprising a truncated β-CTF fused to a signal peptide at the N-terminus of the β-CTF. In some embodiments, the truncated β-CTF comprises a wild-type Aβ peptide or an Aβ peptide variant amino acid sequence (or a fragment or functional derivative thereof) including all of the extracellular and transmembrane amino acids, the transmembrane domain amino acid sequence, and the cytoplasmic amino acid sequence.

[0079] Thus, in some embodiments, the minigene can encode any signal peptide disclosed herein, a wild-type Aβ peptide or any Aβ peptide variant or a fragment or functional derivative thereof disclosed herein, any transmembrane domain amino acid sequence disclosed herein, and any cytoplasmic amino acid sequence disclosed herein. In some embodiments, the minigene encodes, from 5' to 3', a signal peptide disclosed herein, a wild-type Aβ peptide or any Aβ peptide variant or a fragment or functional derivative thereof disclosed herein, a transmembrane domain amino acid sequence disclosed herein, and a cytoplasmic amino acid sequence disclosed herein.

[0080] In some embodiments, the minigene comprises: TIFF2024500768000007.tif55168, or a fragment or functional derivative thereof, wherein the bolded text corresponds to the Aβ peptide variant amino acid sequence, the underlined text corresponds to the transmembrane domain amino acid sequence, and the unaltered text corresponds to the cytoplasmic amino acid sequence. In some embodiments, the minigene comprises, consists of, or consists essentially of a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0081] In some embodiments, the minigene comprises: TIFF2024500768000008.tif55168, or a fragment or functional derivative thereof, wherein the bolded text corresponds to the Aβ peptide variant amino acid sequence, the underlined text corresponds to the transmembrane domain amino acid sequence, and the unaltered text corresponds to the cytoplasmic amino acid sequence. In some embodiments, the minigene comprises, consists of, or consists essentially of a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.

[0082] In some embodiments, the minigene comprises: TIFF2024500768000009.tif54168, or a fragment or functional derivative thereof, wherein the bolded text corresponds to the Aβ peptide variant amino acid sequence, the underlined text corresponds to the transmembrane domain amino acid sequence, and the unaltered text corresponds to the cytoplasmic amino acid sequence. In some embodiments, the minigene comprises, consists of, or consists essentially of a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.

[0083] In some embodiments, the minigene comprises: TIFF2024500768000010.tif55168, or a fragment or functional derivative thereof, wherein the bolded text corresponds to the Aβ peptide variant amino acid sequence, the underlined text corresponds to the transmembrane domain amino acid sequence, and the unaltered text corresponds to the cytoplasmic amino acid sequence. In some embodiments, the minigene comprises, consists of, or consists essentially of a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80.

[0084] In some embodiments, the minigene comprises: TIFF2024500768000011.tif56168, or a fragment or functional derivative thereof, wherein the bolded text corresponds to the Aβ peptide variant amino acid sequence, the underlined text corresponds to the transmembrane domain amino acid sequence, and the unaltered text corresponds to the cytoplasmic amino acid sequence. In some embodiments, the minigene comprises, consists of, or consists essentially of a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:85.

[0085] In some embodiments, the minigene comprises: TIFF2024500768000012.tif55168, or a fragment or functional derivative thereof, wherein the bolded text corresponds to the wild type Aβ42 peptide amino acid sequence, the underlined text corresponds to the transmembrane domain amino acid sequence, and the unaltered text corresponds to the cytoplasmic amino acid sequence. In some embodiments, the minigene comprises, consists of, or consists essentially of a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0086] In some embodiments, the transmembrane domain comprises a nucleotide sequence corresponding to the 13 C-terminal amino acids and an additional 10 amino acids of the Aβ peptide variant. Thus, one of skill in the art will recognize that the amino acid sequence of the Aβ peptide variant includes or overlaps with a portion of the amino acid sequence of the transmembrane domain. In some embodiments, the amino acid sequence of the transmembrane portion of the minigene encoding the Aβ peptide variant is: TIFF2024500768000013.tif22168, where bold indicates a portion of the transmembrane domain amino acid sequence that overlaps with the amino acid sequence of the Aβ peptide variant. In some embodiments, the minigene comprises a transmembrane sequence corresponding to an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:86, or SEQ ID NO:87, or any value derivable within these ranges.

[0087] In some embodiments, the cytoplasmic sequence comprises a nucleotide sequence corresponding to an amino acid sequence for membrane anchoring, facilitating gamma-secretase cleavage, and / or extracellular release of Aβ peptide. In some embodiments, the cytoplasmic sequence comprises a nucleotide sequence corresponding to an amino acid sequence that includes two lysine residues (e.g., KK). In some embodiments, the cytoplasmic sequence comprises a nucleotide sequence corresponding to an amino acid sequence that includes three lysine residues (e.g., KKK). In some embodiments where the cytoplasmic amino acid sequence includes three lysine residues, the first or 5' lysine may be replaced with an arginine residue (e.g., RKK). In some embodiments, the signal peptide sequence comprises an amino acid sequence as described in the literature [see L. Kober et al., Biotechnology and Bioengineering 110(4):1164-1173 (2013), specifically incorporated herein by reference in its entirety]. Any signal sequence sufficient to ensure trafficking of the Aβ-containing propeptide to the cell surface, which may be cleaved by endogenous proteases to expose the Aβ peptide N-terminus, is contemplated for use in the polynucleotide constructs of the present disclosure.

[0088] As a non-limiting example, in some embodiments, the signal peptide sequence comprises a nucleotide sequence corresponding to an amino acid sequence comprising a Gaussia luciferase signal peptide, where the Gaussia luciferase signal peptide can be codon-optimized for mammalian gene expression as described in the literature (see, for example, BA Tannous et al., Molecular Therapy 11(3):435-443 (2005), and S. Knappskog et al., J. of Biotechnology 128:705-715 (2007), the entireties of which are incorporated herein by reference). In some embodiments, the amino acid sequence of the Gaussia luciferase signal peptide comprises MGVKVLFALICIAVAEA (SEQ ID NO: 19). A corresponding nucleotide sequence encoding the Gaussia luciferase signal peptide of SEQ ID NO:19 may comprise ATGGGCGTGAAGGTCCTGTTCGCCCTGATTTGCATCGCCGTCGCAGAGGCA (SEQ ID NO:20).

[0089] As a second non-limiting example, in some embodiments, the signal peptide sequence comprises a nucleotide sequence corresponding to an amino acid sequence comprising a mouse immunoglobulin heavy chain (MoIgH) signal peptide, where the MoIgH signal peptide can be codon-optimized for mammalian gene expression as described in the literature (see, for example, BA Tannous et al., Molecular Therapy 11(3):435-443 (2005), and S. Knappskog et al., J. of Biotechnology 128:705-715 (2007), the entire contents of which are incorporated herein by reference). In some embodiments, the amino acid sequence of the MoIgH signal peptide comprises MGWSCIILFLVATATGVHS (SEQ ID NO:21). The corresponding nucleotide sequence encoding the MoIgH signal peptide of SEQ ID NO:21 is TIFF2024500768000014.tif22168, where the bolded portion of SEQ ID NO:22 corresponds to intron non-coding sequence of the MoIgH signal peptide.

[0090] In some embodiments, the minigene comprises a signal peptide sequence corresponding to an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO:19 or SEQ ID NO:21, or any derivable value within these ranges.

[0091] In some embodiments, the polynucleotide is comprised in a vector that stably expresses wild-type or mutant Aβ peptide, or a fragment or functional derivative thereof, at the cell membrane where release of Aβ peptide into the extracellular space is regulated by endogenous γ-secretase.

[0092] A. Proteinaceous Compositions As used herein, the terms "proteinaceous molecule," "proteinaceous composition," "proteinaceous compound," "proteinaceous chain," or "proteinaceous substance" generally refer to, but are not limited to, proteins of about 200 or more amino acids or full-length endogenous sequences translated from a gene; polypeptides of about 100 or more amino acids; and / or peptides of about 3 to about 100 amino acids. All of the above-mentioned terms "proteinaceous" may be used interchangeably in this application.

[0093] As used herein, a "protein," "peptide," or "polypeptide" refers to a molecule that contains at least three amino acid residues. As used herein, the term "endogenous" refers to a version of a molecule that occurs naturally in an organism. In some embodiments, an endogenous peptide includes a wild-type peptide. In some embodiments, an endogenous peptide includes amino acid modifications from the wild-type peptide. In some embodiments, a wild-type version of a protein or peptide is used, but in many embodiments of the present disclosure, a modified protein or peptide is used. The terms can be used interchangeably. A "modified protein" or "modified polypeptide" or "mutant" refers to a protein or peptide whose chemical structure, particularly its amino acid sequence, is modified relative to the wild-type protein or peptide. In some embodiments, a modified / mutant protein or peptide has at least one modified activity or function (recognizing that a protein or polypeptide can have multiple activities or functions). It is specifically contemplated that a modified / mutant protein or peptide can be modified with respect to one activity or function, but in other aspects can retain a wild-type activity or function.

[0094] When a protein is specifically mentioned herein, it is generally a reference to a natural (endogenous) or recombinant (modified or mutant) protein or peptide. Proteins can be directly isolated from native organisms, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. A variety of automated synthesizers are commercially available and may be used according to known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), the entire text of each of which is specifically incorporated herein by reference. Alternatively, recombinant DNA techniques may be used in which a nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and incubated under conditions compatible with expression. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that include a nucleic acid sequence encoding the peptide. The term "recombinant" may be used in conjunction with a peptide or the name of a particular peptide, and generally refers to a peptide produced from a nucleic acid molecule engineered in vitro or the product of replication of such a molecule.

[0095] In certain embodiments, the size of the at least one proteinaceous molecule is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 65 The amino acid sequence may include, but is not limited to, 0, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues, and any range derivable therein, or corresponding derivatives of amino acid sequences described or referenced herein.

[0096] The peptides described herein are at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 109, 110, 111, 112, 113, 1 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113 , 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 4, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 , 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more amino acids (or any derivable range within these ranges).

[0097] In certain embodiments, the proteinaceous composition comprises at least one protein, polypeptide, or peptide. It is contemplated that virtually any protein, polypeptide, or peptide-containing component described herein may be used in the compositions and methods disclosed herein. In additional embodiments, the proteinaceous composition comprises a biocompatible protein, polypeptide, or peptide. The term "biocompatible" as used herein refers to a material that does not cause significant side effects when applied or administered to a given organism according to the methods and amounts described herein. Such undesirable and undesirable effects are similar to significant toxicity or adverse immunological responses. In preferred embodiments, the biocompatible protein-, polypeptide-, or peptide-containing composition will generally be a mammalian protein or peptide or a synthetic protein or peptide that is essentially free of toxins, pathogens, and harmful immunogens, respectively.

[0098] Proteinaceous compositions may be produced by any technique known to those of skill in the art, including expression of proteins, polypeptides or peptides through standard molecular biology techniques, isolation of proteinaceous compounds from natural sources, or chemical synthesis of proteinaceous substances. Nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The coding regions for these known genes may be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art. Alternatively, various commercial formulations of proteins, polypeptides and peptides are known to those of skill in the art.

[0099] In certain embodiments, a proteinaceous compound may be purified. "Purified" generally refers to a particular protein, polypeptide or peptide composition that has been fractionated to remove various other proteins, polypeptides or peptides, and wherein the composition may be assessed as substantially retaining its activity (e.g., as assessed by protein assays as known to those of skill in the art for the particular protein, polypeptide or peptide of interest).

[0100] The proteins and peptides suitable for use in the present invention may be self-proteins or peptides, although the present invention does not expressly limit the use of such self-proteins. The term "self-protein, polypeptide or peptide" as used in this application means a protein, polypeptide or peptide derived or obtained from an organism. Organisms that may be used include, but are not limited to, bovine, reptile, amphibian, fish, rodent, bird, canine, feline, fungus, plant or prokaryotic organisms, with the selected animal or human subject being preferred. The "self-protein, polypeptide or peptide" may be used as a component of a composition for application to the selected animal or human subject.

[0101] In that the compositions of the present disclosure are particularly suitable for use in treating or preventing neurodegenerative diseases, preferred proteins including wild-type or mutant Aβ peptides are contemplated.

[0102] In selecting other proteins, polypeptides, peptides, etc. for use in the methods and compositions of the present disclosure, one of skill in the art would preferably select a proteinaceous material that possesses one or more of the following characteristics: forms a solution with a high percentage of solubilized proteinaceous material; has a high viscosity (i.e., about 40 to about 100 poise); has the correct molecular charge to bind the dye if it is a non-covalent mixture (i.e., anionic proteins and cationic dyes, or cationic proteins and anionic dyes); has the correct amino acids present to form covalent cross-links (i.e., one or more tyrosines, histidines, tryptophans, and / or methionines); and / or is biocompatible (i.e., if mammalian, of mammalian origin, preferably if human, of human origin, if canine, of canine origin, etc.; is autologous, non-allergenic and / or non-immunogenic).

[0103] It is contemplated that compositions of the present disclosure have from about 0.001 mg to about 10 mg of total polypeptide, peptide and / or protein per ml. The concentration of protein in the composition may be about, at least about, or up to about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0104] B. Biologically Functional Equivalents Modifications and / or changes may be made to the structure of the polynucleotides and / or proteins according to the invention, obtaining molecules with similar or improved properties, while such biologically functional equivalents are also included in the present invention.

[0105] 1. Modified Polynucleotides and Peptides Biologically functional equivalents may include polynucleotides that have been engineered to contain distinct sequences while retaining the ability to code for a "wild-type" or standard protein or peptide or a "mutant" protein or peptide. This can be accomplished due to the degeneracy of the genetic code, i.e., the presence of multiple codons that code for the same amino acid. The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to codons or changes in codons that code for biologically equivalent amino acids. In one example, one skilled in the art may wish to incorporate a restriction enzyme recognition sequence into a polynucleotide without interfering with the ability of the polynucleotide to code for a protein.

[0106] In relation to functional equivalents, the skilled artisan will appreciate that inherent in the definition of a "biologically functional equivalent" protein and / or polynucleotide is the concept that there are limited changes that can be made within a defined portion of a molecule while maintaining the molecule with an acceptable level of equivalent biological activity. Thus, biologically functional equivalents are defined herein as proteins (and polynucleotides) with substitutions or mutations at selected amino acids (or codons) that maintain the ability to disrupt / disrupt endogenous Aβ peptide aggregation or promote disassembly of endogenous Aβ peptide fibrils or other endogenous Aβ peptide multimeric structures and / or other endogenous Aβ peptide multimeric structures and / or proteins (and polynucleotides) with substitutions or mutations at selected amino acids (or codons) that do not self-aggregate, resulting in, for example, reduced toxicity and / or aggregation of endogenous Aβ peptide. Biologically functional equivalents may also include proteins (and polynucleotides) with substitutions or mutations at selected amino acids (or codons) that maintain the ability to promote aggregation of endogenous Aβ peptide and / or multimeric structures of endogenous Aβ peptide or other endogenous Aβ peptides and / or aggregation of proteins (and polynucleotides) and / or proteins (and polynucleotides) with substitutions or mutations at selected amino acids (or codons) that, for example, self-aggregate.

[0107] Generally, the shorter the length of a molecule, the fewer changes that can be made in the molecule while still maintaining function. Longer domains may have intermediate degrees of variation. Full-length proteins will have the greatest tolerance to greater degrees of variation. However, it should be understood that certain molecules or domains that are highly structurally dependent may tolerate little or no variation at all.

[0108] In one example, a polynucleotide may furthermore be (and encode) a biologically functional equivalent having significant changes: certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, etc.

[0109] Substitutional variants typically involve exchanging one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions may be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. So-called "conservative" changes do not interfere with the biological activity of the protein, since the structural changes do not affect the ability of the protein to perform the designed function. Thus, it is contemplated by the inventors that a variety of changes may be made to the gene and protein sequences disclosed herein while still meeting the objectives of the invention. Alternatively, the substitution may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve chemically replacing one residue with another, such as a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0110] In other embodiments, modification of the polypeptide function is contemplated by introducing one or more substitutions. For example, certain amino acids can be substituted for other amino acids in a protein structure without significant loss of interactive binding capacity. For example, structures such as enzyme catalytic domains or interacting moieties may have substituted amino acids to maintain such function. Since it is the interacting capacity and properties of a protein that define the biological functional activity of a protein, certain amino acid substitutions may be made in the protein sequence and in the basic DNA coding sequence to generate a protein that nevertheless has similar properties. Thus, the inventors contemplate that a variety of changes may be made in the DNA sequence of a gene without significant loss of biological utility or activity.

[0111] Deletion mutants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or multiple adjacent amino acids may be deleted. Stop codons may be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate truncated proteins. For example, it is contemplated that a peptide may be mutated by truncation or deletion of multiple adjacent amino acids to make it shorter than the corresponding endogenous form.

[0112] Insertional mutagenesis generally involves the addition of an amino acid residue to a non-terminal point of a polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated, including fusion proteins, which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein. For example, it is contemplated that peptides may be engineered by fusing or joining heterologous protein or polypeptide sequences having specific functions (e.g., for targeting or localization, for enhanced activity, for purification purposes, etc.).

[0113] Additionally, the polypeptides of the present invention may be chemically modified. The glycosylation of the polypeptide may be altered by altering one or more glycosylation sites within the polypeptide sequence, for example, to increase the affinity of the polypeptide for an antigen (U.S. Patent Nos. 5,714,350 and 6,350,861).

[0114] Also, amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still remain essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including maintenance of biological protein activity upon expression of the protein. The addition of terminal sequences applies particularly to nucleic acid sequences, which may contain a variety of non-coding sequences, for example flanking the 5' or 3' portions of the coding region.

[0115] Amino acid sequence variants of the disclosure may be, for example, substitution, insertion or deletion variants. Regions or fragments of the polypeptides of the disclosure may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 110, 111, 112, 1 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 , 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 or more amino acid substitutions, contiguous amino acid additions or contiguous amino acid deletions.Alternatively, a region or fragment of a polypeptide of the present disclosure may have an amino acid sequence that comprises, or consists of, an amino acid sequence that is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% identical, at least identical, or at most identical to any of SEQ ID NOs:1-19, SEQ ID NOs:21, 23-39, and 65-87.

[0116] In some embodiments, the region or fragment may also be located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 1 31, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 1 62, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 3, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224 , 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 2 87, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 31 8, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349 , 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 4 12, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 44 3, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474 , 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 starting with 4, 5, 6, 7, 8, 9, 10, 11,12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 33 9, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 3 95, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or more contiguous amino acids.

[0117] Polypeptides of the present disclosure may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more variant amino acids of any of SEQ ID NOs: 1-19, SEQ ID NOs: 21, 23-39, and 65-87, or at least or up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 28, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 9, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190 , 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 600 or more (or any derivable range within these ranges) contiguous amino acids and at least 60%, 60%, or more. It may be 1%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similar, identical or homologous.

[0118] The polypeptides of the present disclosure may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 , 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 1 77, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 20 8, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 , 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614 or 615 substitutions (or any range derivable therein).

[0119] The substitutions may be made at amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 , 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 1 67, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 8, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564 , 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614 or 650.

[0120] Amino acid substitutions are generally based on the relative similarity of the amino acid side chain substitutes, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and / or type of amino acid side chain substitutes indicates that arginine, lysine, and / or histidine are all positively charged residues; alanine, glycine, and / or serine are all similar in size; and / or phenylalanine, tryptophan, and / or tyrosine are all generally similar in shape. Thus, depending on such considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents.

[0121] When making such changes to produce biologically functional equivalents, the hydrophobicity index of amino acids may be taken into consideration. The hydrophobicity profile of a protein is calculated by assigning a numerical value (the "hydrophobicity index") to each amino acid and then averaging these values ​​repeatedly along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge properties. These amino acids are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydrophobic amino acid index in conferring interactive biological function to a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophobicity properties of amino acids contribute to the secondary structure of a resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that certain amino acids may be substituted with other amino acids having similar hydrophobicity indices or scores and still maintain similar biological activity. When making changes based on hydrophobicity index, in certain embodiments, substitution of amino acids with hydrophobicity indices within ±2 are included. In some aspects of the present disclosure, hydrophobicity indices within ±1 are included, and in other aspects of the present disclosure, hydrophobicity indices within ±0.5 are included.

[0122] It is also understood in the art that substitution of similar amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference in its entirety, describes that the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values ​​were assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain embodiments substitutions of amino acids with hydrophilicity values ​​within ±2 are included, in other embodiments hydrophilicity values ​​within ±1 are included, and in other embodiments ±0.5 are included. In some cases, epitopes can also be distinguished from a primary amino acid sequence on the basis of hydrophilicity, it being understood that amino acids may be substituted for other amino acids having similar hydrophilicity values ​​and still produce a biologically equivalent and immunologically equivalent protein.

[0123] Additionally, one of skill in the art can consider structure-function studies to identify residues in similar polypeptides or proteins that are important for activity or structure. In view of such comparisons, one can predict the importance of amino acid residues in the protein that correspond to amino acid residues important for the activity or structure of the similar protein. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0124] The skilled artisan can also analyze the three-dimensional structure and the amino acid sequence that spans this structure from similar proteins or polypeptides. In view of such information, the skilled artisan can predict the alignment of amino acid residues relative to the three-dimensional structure. The skilled artisan can choose not to alter amino acid residues that are predicted to be on the protein surface, because such residues can be involved in important interactions with other molecules. In addition, the skilled artisan can generate test mutants that contain single amino acid substitutions at each targeted amino acid residue. These mutants can then be screened using standard assays for binding and / or activity, thus obtaining information gleaned from such routine experiments that allows the skilled artisan to determine amino acid positions where additional substitutions should be avoided, either alone or in combination with other mutations. A variety of tools that may be used to determine secondary structure can be found on the World Wide Web (expasy.org / proteomics / protein_structure).

[0125] In some embodiments of the present disclosure, amino acid substitutions are made to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) modify binding affinity for the formation of protein complexes, (4) modify ligand binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in naturally occurring sequences. Substitutions may be made in portions of proteins that are outside of the domain(s) that form intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the primary, secondary, or tertiary structure that characterizes the native protein) may be used.

[0126] As discussed above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substitutes, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various properties discussed above are well known and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0127] 2. Modified Amino Acids As used herein, "amino moiety" refers to any amino acid, amino acid derivative, or amino acid mimetic known to those of skill in the art. In certain embodiments, the residues of a proteinaceous molecule are sequential without any non-amino moieties interrupting the sequence of amino moiety residues. In other embodiments, the sequence may include one or more non-amino moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule may be interrupted by one or more non-amino moieties. Peptides and polypeptides include the 20 "natural" amino acids and post-translational modifications thereof. However, in vitro peptide synthesis allows for the use of modified and / or unusual amino acids.

[0128] Thus, the term "proteinaceous composition" includes amino acid sequences that contain at least one of the 20 common amino acids from naturally occurring proteins, or at least one modified or unusual amino acid, including, but not limited to, those shown in the table below.

[0129] TIFF2024500768000015.tif116168

[0130] 3. Mimetic In addition to the biologically functional equivalents discussed above, the inventors also contemplate that structurally similar compounds can be formulated to mimic the core portions of the peptides or polypeptides of the invention. Such compounds, which may be referred to as peptidomimetics, are also functional equivalents, since they may be used in the same manner as the peptides of the invention.

[0131] Specific mimetics that mimic elements of protein secondary and tertiary structure have been described in the literature [see Johnson et al. (1993)]. The underlying rationale for using peptidomimetics is that the peptide backbone of proteins exists primarily to orient amino acid side chains in a manner that promotes molecular interactions such as antibody and / or antigen. Thus, peptidomimetics are designed to permit molecular interactions similar to those of the natural molecule.

[0132] Some successful applications of the peptidomimetic concept have focused on mimicking β-turns in proteins known to be highly antigenic. The structure of analogous β-turns in polypeptides can be predicted by computer-based algorithms as discussed herein. Once the component amino acids of the turn have been determined, mimetics can be constructed to achieve a similar spatial orientation of the essential amino acid side chains.

[0133] Other approaches focus on using small, multiple disulfide-containing proteins as attractive structural templates to generate biologically active forms that mimic the binding sites of larger proteins. See Vita et al. (1998). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and highly tolerant to mutations. This motif consists of a beta sheet and an alpha helix connected at the inner core by three disulfides.

[0134] A beta II turn has been successfully mimicked using a cyclic L-pentapeptide with D-amino acids by Weisshoff et al. (1999). Also, the literature [Johannesson et al. (1999)] reports a bicyclic tripeptide with reverse turn inducing properties.

[0135] Methods for generating specific structures are disclosed in the art. For example, alpha-helix mimetics are described in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures make peptides or proteins more thermally stable and more resistant to proteolysis. 6-, 7-, 11-, 12-, 13-, and 14-membered ring structures have been disclosed.

[0136] Methods for generating conformationally restricted beta-turns and beta bulges are described, for example, in U.S. Patent Nos. 5,440,013; 5,618,914; and 5,670,155. Beta-turns tolerate altered lateral substitutions without corresponding changes in backbone morphology and have suitable termini for incorporation into peptides by standard synthetic procedures. Other types of mimetic turns include reverse turns and gamma turns. Reverse turn mimetics are disclosed in U.S. Patent Nos. 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Patent Nos. 5,672,681 and 5,674,976.

[0137] C. Exogenous Delivery of Peptides In certain embodiments, modifications may be made to the proteinaceous compositions and peptide variants disclosed herein to improve exogenous delivery of the peptides to cells.

[0138] In certain embodiments, vectors may be constructed to contain exogenous nucleic acid sequences that cause cells to express the proteinaceous compositions and peptide variants disclosed herein. Details regarding the components and delivery methods of these vectors are disclosed below.

[0139] In some embodiments, the compositions disclosed herein can be administered to cause a cell to contain one or more genetic modifications by genetic engineering of the cell. A cell is "genetically modified," "genetically altered," or "transgenic" when an exogenous nucleic acid or polynucleotide has been delivered into the cell by any suitable means of artificial manipulation, or when the cell is the progeny of an originally altered cell that inherited the polynucleotide.

[0140] In various embodiments, a DNA construct or vector is provided that encodes the peptide sequence of the wild-type or mutant Aβ peptide or a fragment or functional derivative thereof disclosed herein. Genetic modifications may also be introduced into cells. Such modifications include, for example, transducing cells with a vector encoding the wild-type or mutant Aβ peptide or a fragment or functional derivative thereof to generate cells expressing the wild-type or mutant Aβ peptide or a fragment or functional derivative thereof. Viral vectors encoding the peptide sequence of the wild-type or mutant Aβ peptide or a fragment or functional derivative thereof disclosed herein allow for continuous gene expression and release of Aβ peptide.

[0141] A cell according to the present disclosure includes any cell into which a DNA construct or vector made to contain an exogenous nucleic acid sequence such that the cell expresses the proteinaceous compositions and peptide variants disclosed herein, or the proteinaceous compositions and peptide variants disclosed herein, can be introduced and expressed as described herein. It should be understood that the basic concept of the invention described herein is not limited by cell type. Cells according to the present disclosure include eukaryotic cells, mammalian cells, animal cells, human cells, etc. Also included are all cells in which it is beneficial or desirable to regulate the production of functional proteins.

[0142] 1. Modification of peptides to improve exogenous delivery In some embodiments, the wild-type or mutant Aβ peptides disclosed herein may be modified to improve uptake or absorption of the peptide by cells. For example, in some embodiments, cell-penetrating peptides (CPPs) are attached to wild-type or mutant Aβ peptides to generate cell-permeable and / or brain-permeable inhibitors of Aβ oligomer and fibril formation. CPPs are short peptides that facilitate cellular uptake and absorption of molecules ranging from nano-sized particles to small compounds to large pieces of DNA. Cargos are linked to the peptides through chemical linkages or non-covalent interactions via covalent bonds. CPPs generally have amino acid compositions that contain a relative abundance of positively charged amino acids such as lysine or arginine, or sequences that contain alternating patterns of polar, charged and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are hydrophobic peptides that contain only non-polar residues with low net charge or hydrophobic amino acid groups that are important for cellular uptake.

[0143] In some embodiments, the cell-penetrating peptide is a retro-inverted version of the HIV protein transduction domain "TAT"; poly-arginine (e.g., R8-R12); polyamine; MAP; MTS; MPG; Penetratin; Pep-1; Transportan; or VP22. In some embodiments, the cell-penetrating peptide is a retro-inverted version of the HIV protein transduction domain "TAT". In some embodiments, the cell-penetrating peptide is poly-arginine. In some embodiments, the cell-penetrating peptide is a polyamine. In some embodiments, attaching the cell-penetrating peptide to a wild-type or mutant Aβ peptide allows the wild-type or mutant Aβ peptide to become cell-penetrating without vectorization.

[0144] The transactivating transcriptional activator (TAT) of human immunodeficiency virus 1 (HIV-1) penetrates the impermeable phospholipid bilayer of cell membranes, allowing it to cross biological barriers. See M. Green et al., Biochim Biophys Acta 1414:127-139 (1998), the entire text of which is specifically incorporated herein by reference. After being secreted by HIV-infected cells, TAT travels to neighboring cells to alter gene transcription and spread the disease. The first report demonstrating that Tat-derived peptides can deliver large proteins to other cell types and mammalian organelles was published in 1994, which demonstrated the chemical cross-linking and identification of a 36 amino acid region of HIV-1 that can facilitate the uptake of β-galactosidase into living cells as a chimera. See literature [see S. Fawell et al., Proc Natl Acad Sci USA 91:664-668 (1994)]. The HIV TAT protein transduction domain (PTD) contains a cluster of basic amino acid residues and a sequence that is predicted to adopt an α-helical configuration. Numerous studies have aimed to describe whether shorter domains of the TAT peptide are sufficient for cellular internalization. The main determinants required for translocation have been identified as the cluster of basic amino acids, and peptides with α-helical regions are able to enter cells more efficiently, while the predicted α-helical domain has been shown to be dispensable. A truncated polycationic peptide GRKKRRQRRR containing RNA binding and nuclear localization signal (NLS) motifs has been identified as suitable for efficient translocation into cells and tissues and is included in TAT. See literature [see E. Vives E et al., J Biol Chem 272:16010-16017 (1997)]. Thus, in some embodiments, a wild-type or mutant Aβ peptide is attached to a TAT sequence.The TAT sequence may comprise a peptide having an amino acid sequence comprising GRKKRRQRRR (SEQ ID NO:23), YGRKKRRQRRR (SEQ ID NO:24), GRKKRRQRRRPQ (SEQ ID NO:25), which amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs:23-25, or a fragment or derivative thereof, or any value derivable within these ranges.

[0145] Structurally, oligo- and poly-arginines are the simplest CPPs, with Arg as the only building block, and are easy to prepare. Poly-arginines adopt a random coil conformation in aqueous solution or when bound to phospholipid membranes, due to the strong side chain charge repulsion and lack of hydrophobic or amphipathic structures, and membrane permeability depends mainly on electrostatic interactions with lipid membranes mediated by guanidinium charge groups. Thus, in some embodiments, wild-type or mutant Aβ peptides are attached to poly-arginines. In some embodiments, poly-arginines include polymers of L- or D-arginines containing six or more arginine amino acids. In some embodiments, the poly-arginine comprises R8-R12 corresponding to the amino acid sequence comprising RRRRRRRR-RRRRRRRRRRRR (SEQ ID NO: 26) (see G. Tunnemann et al., J. Pept. Sci. 14:469-476 (2008), the entire contents of which are hereby incorporated by reference).

[0146] Polyamines, such as putrescine, spermidine and spermine, have been shown to increase protein permeability at the blood-nerve and blood-brain barriers. Polyamine transporters may be responsible for the transport of polyamine-modified proteins. See, for example, J.F. Poduslo & G.L. Curran, J. Neurochem 66:5705-5709 (1996); J.F. Poduslo & G.L. Curran, J. Neurochem 67:734-741 (1996); and J.F. Poduslo et al., J. Neurobio 39(3):371-82 (1999), specifically incorporated herein by reference in their entirety. Thus, systemic administration of polyamine-modified wild-type or mutant Aβ peptides may be an efficient approach to deliver these therapeutic agents to the CNS for the treatment of various neurological disorders, including Alzheimer's disease, and in some embodiments, the wild-type or mutant Aβ peptides are covalently linked to polyamines. In some embodiments, the polyamine is putrescine. In some embodiments, the polyamine is spermidine. In some embodiments, the polyamine is spermine.

[0147] Other cell-permeable peptides contemplated for attachment to the wild-type or mutant Aβ peptides disclosed herein include, but are not limited to, the following cell-permeable peptides: MAP, which corresponds to an amino acid sequence comprising KLALKLALKALKAALKLA (SEQ ID NO:27) (see J. Oehlke et al., Cell 58:215-223 (1989), specifically incorporated herein by reference in its entirety); MTS, which corresponds to an amino acid sequence comprising AAVALLPAVLLALLAP (SEQ ID NO:28) (see M. Rojas, Nat. Biotechnol. 16:370-375 (1998), specifically incorporated herein by reference in its entirety); MPG, which corresponds to an amino acid sequence comprising GLAFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:29) (see M. Morris et al., Nucleic Acids 58:111-112 (1998)), specifically incorporated herein by reference in its entirety); Res. 25:2730-2736 (1997), specifically, the entire text of which is incorporated herein by reference; penetratin, which corresponds to an amino acid sequence comprising RQIKIWFQNRRMKWKK (SEQ ID NO: 30) (see D. Derossi et al., J. Biol. Chem. 269:10444-10450 (1994), specifically, the entire text of which is incorporated herein by reference); Pep-1, which corresponds to an amino acid sequence comprising KETWWETWWTEWSQPKKRKV (SEQ ID NO: 31) (see M. Morris et al., Nat. Biotechnol. 19:1173-1176 (2001), specifically, the entire text of which is incorporated herein by reference); transportan, which corresponds to an amino acid sequence comprising GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 32) (see M. Pooga et al., FASEB J. 12:67-77 (1998), the entirety of which is incorporated herein by reference); or VP22 corresponding to an amino acid sequence including DAATATRGRSAASRPTQRPRAPARSASRPRRPVQ (SEQ ID NO:33) (see G. Elliott et al., Cell 88:223-233 (1997), the entirety of which is incorporated herein by reference).Additional cell-penetrating peptides contemplated for attachment to the wild-type or mutant Aβ peptides disclosed herein are described, for example, in A. Borrelli et al., Molecules 23:295-318 (2018), the entire contents of which are incorporated herein by reference.

[0148] In some embodiments, the cell-permeable and / or brain-permeable Aβ peptide variants include Aβ peptide variants in which naturally occurring L-amino acids of the Aβ peptide variant are replaced by D-enantiomers of the amino acids. See JF Poduslo et al., J. Neurobio 39(3):371-82 (1999). Peptides containing D-residues are more resistant to proteolysis. See Robson, Nat Biotechnol 14:893-895 (1996), and Schumacher et al., Science 271:1854-1857 (1996). Such modifications can also render the Aβ peptide variants less immunogenic, since L-amino acid peptides are efficiently processed for major histocompatibility complex class II-restricted presentation to T helper cells, thus generating vigorous humoral immune responses that impair the bioactivity of the drug, whereas D-residue peptides are much less antigenic. See Herve et al., J. Immunol. 156:157-163 (1997). Accordingly, in some embodiments, the naturally occurring L-amino acids of the Aβ peptide variants disclosed herein are replaced with D-enantiomers of the amino acids to improve the cell and / or brain permeability of the Aβ peptide variants.

[0149] 2. Vector In some embodiments, the wild-type or mutant Aβ peptides disclosed herein are achieved by operably linking a nucleic acid encoding a wild-type or mutant Aβ peptide or a portion thereof to a promoter and incorporating the construct into an expression vector that is taken up and expressed by a cell. The vector may be suitable for replication and, in some cases, integration in a eukaryotic organism. A typical cloning vector includes transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid sequence of interest. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, a suitable vector includes an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193). In some embodiments, a suitable vector is capable of crossing the blood-brain barrier.

[0150] In certain embodiments, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York and other virology and molecular biology manuals.

[0151] A number of virus-based systems have been developed for gene transfer into mammalian cells. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including self-inactivating lentiviral vectors). For example, adenoviruses provide a convenient platform for gene transfer systems. A selected gene may be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of an in vivo or ex vivo subject. Thus, in some embodiments, a nucleic acid encoding an Aβ peptide variant is introduced into a cell using a recombinant vector, such as a viral vector including, for example, a lentivirus, a retrovirus, a gamma-retrovirus, an adeno-associated virus (AAV), a herpes virus, or an adenovirus.

[0152] In certain embodiments, the vector is an AAV vector. "AAV vector" refers to a recombinant vector derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAV-DJ, AAVrhlO.XX, AAVrh.8, AAVrh.10, AAVrh.43, AAVpi.2, AAVhu.11, AAVhu.32, AAVhu.37, PHP.eB AAV, and others. An AAV vector may be deleted of one or all wild-type AAV genes, but still contain functional inverted terminal repeat (ITR) nucleic acid sequences. Functional ITR sequences are necessary for replication, structure, and packaging of AAV virions. The ITR sequences may be wild-type or substantially identical sequences, or may be modified, e.g., by insertion, mutation, deletion, or substitution of nucleotides, so long as functionality is maintained. Such AAV vectors may be replicated and packaged into infectious viral particles when present in a host cell that expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0153] The genome of AAV is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique coding nucleotide sequences for nonstructural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid. The terminal 145 nt are self-complementary and organized such that an energetically stable intramolecular duplex can form, forming a T-shaped hairpin. Such a hairpin structure serves as an origin of viral DNA replication that acts as a primer for the cellular DNA polymerase complex. After wild-type AAV infection in mammalian cells, the Rep genes (i.e., Rep78 and Rep52) are expressed from the P5 and P19 promoters, respectively, and both Rep proteins have functions in replicating the viral genome. A splicing event in the Rep ORF actually leads to the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52 and Rep40). However, it has been shown that unspliced ​​mRNAs encoding the Rep78 and Rep52 proteins are sufficient for AAV vector production in mammalian cells, and that the Rep78 and Rep52 proteins are sufficient for AAV vector production in insect cells.

[0154] AAV VP proteins are known to determine the cellular tropism of AAV virions. VP protein-encoding sequences are much less conserved among different AAV serotypes than the Rep proteins and genes. The ability of Rep and ITR sequences to cross-complement corresponding sequences in other serotypes allows for the production of homotypic AAV particles that contain the capsid proteins of one serotype (e.g., AAV5) and also the Rep and / or ITR sequences of another AAV serotype (e.g., AAV2). As used herein, homotypic AAV particles may be referred to as "x / y" typology, where "x" indicates the source of the ITRs and "y" indicates the serotype of the capsid, e.g., a 2 / 5 AAV particle has an AAV2 ITR and an AAV5 capsid.

[0155] An AAV vector may comprise one or more polynucleotide sequences of interest (one or more transgenes) flanked by at least one AAV ITR. Thus, in one aspect, the present disclosure relates to a nucleic acid vector construct comprising one or more nucleotide sequences encoding one or more peptide sequences of wild-type or mutant Aβ peptides disclosed herein, where the nucleic acid vector construct is a recombinant AAV vector, thereby comprising at least one AAV ITR flanked by one or more nucleotide sequences encoding one or more peptide sequences of wild-type or mutant Aβ peptides. In some embodiments of the nucleic acid vector construct, the one or more nucleotide sequences encoding one or more peptide sequences of wild-type or mutant Aβ peptides are flanked on either side by an AAV ITR.

[0156] Any suitable serotype of AAV may be used as a vector, and the vector may contain one or more polynucleotide sequences of interest. In some embodiments, the AAV vector containing one or more polynucleotide sequences of interest is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAvDJ, AAVrhlO.XX, AAVrh.8, AAVrh.10, AAVrh.43, AAVpi.2, AAVhu.11, AAVhu.32, AAVhu.37, or PHP.eB AAV vector. In some embodiments, the vector can cross the blood-brain barrier. In some embodiments, the vector can cross the blood-brain barrier includes AAV9, PHP.eB, AAVrh.8, AAVrh.10, or AAVrh.43. In some embodiments, vectors capable of crossing the blood-brain barrier can be delivered, for example, intravenously, intracerebrally and / or intraventricularly to efficiently and widely transduce neurons in the adult CNS. In some embodiments, adenoviral vectors are modified to reduce host responses. See, e.g., Russell J. Gen. Virol. 81:2573-2604 (2000); U.S. Patent Publication No. 2008 / 0008690; and Zaldumbide et al., Gene Therapy 15(4):239-46 (2008); which are specifically incorporated herein by reference in their entirety.

[0157] One of skill in the art can generate AAV vectors containing one or more polynucleotide sequences of interest flanked by at least one AAV ITR through standard recombinant techniques (e.g., Maniatis et al., 1988 and Ausubel et al., 1994, the entireties of which are specifically incorporated herein by reference).

[0158] Vectors may also contain other components or functions that further modulate gene transfer and / or gene expression or that provide beneficial properties to target cells. Such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue-specific binding); components that affect uptake of vector nucleic acid by cells; components that affect localization of polynucleotides within cells after uptake (e.g., agents that mediate nuclear localization); and components that affect expression of the polynucleotide.

[0159] Such components may also include markers, such as detectable and / or selectable markers, that may be used to detect or select cells that have taken up and expressed the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (e.g., the use of certain viral vectors that have components or functions that mediate binding and uptake) or the vector may be modified to provide such functions. A wide variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, it may be stably replicated by the cell during mitosis as an autonomous structure, integrated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0160] The eukaryotic expression cassette contained in the vector contains, inter alia, regulatory elements including (5' to 3') a eukaryotic transcriptional promoter operably linked to the protein-coding sequence, splice signals including intervening sequences, transcription termination / polyadenylation sequences, post-transcriptional regulatory elements, and an origin of replication.

[0161] A. Promoter / Enhancer A "promoter" is a control sequence that is a region of a nucleic acid sequence where initiation and rate of transcription are controlled. A promoter may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of a nucleic acid sequence. Terms: "operably located," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0162] Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, although in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a separate element above the start site itself serves to fix the start position. Additional promoter elements regulate the frequency of transcription initiation. Typically, additional promoter elements are located in the region 30-110 bp upstream of the start site, and many promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control of" a promoter, the 5' end of the transcription start site is positioned in the transcriptional reading frame "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0163] Spacing between promoter elements is often flexible so that promoter function is preserved when elements are inverted or moved relative to one another. For example, in the thymidine kinase promoter, spacing between promoter elements may be increased to 50 bp apart before activity begins to decrease. Promoters may allow individual elements to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0164] The promoter may be one that is naturally associated with the nucleic acid sequence, such as by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. These promoters may be referred to as "endogenous". Similarly, enhancers may be one that is naturally associated with a nucleic acid sequence located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer may also refer to an enhancer that is not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other virus, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to synthetically generating promoter and enhancer nucleic acid sequences, PCR may also be used in conjunction with the compositions disclosed herein. TMThe sequences can be generated using recombinant cloning and / or nucleic acid amplification techniques, including (see U.S. Pat. Nos. 4,683,202 and 5,928,906, respectively, which are specifically incorporated herein by reference in their entireties.) It is also contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, may also be used.

[0165] Of course, it is important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer and cell type combinations for protein expression (e.g., Sambrook et al., 1989, specifically incorporated herein by reference in its entirety). The promoter used may be constitutive, cell-specific, tissue-specific, inducible and / or useful under appropriate conditions to direct high levels of expression of the introduced DNA segment, e.g., advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0166] Additionally, any promoter / enhancer combination [e.g., Eukaryotic Promoter Data Base (EPDB) through the World Wide Web (epd.isb-sib.ch / )] may be used to drive expression. Non-limiting examples of other potential promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as beta-actin promoter (Ng, 1989; Quitsche et al., 1989), GADPH promoter (Alexander et al., 1988; Ercolani et al., 1988), metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and linked response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoter (tre) near the minimal TATA box. It is also possible to use the human growth hormone promoter sequence (for example the human growth hormone minimal promoter described in Genbank, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC 45007). A particular example may be the phosphoglycerate kinase (PGK) promoter.

[0167] In some embodiments, expression of the polynucleotide is regulated by a constitutive promoter, hi some embodiments, the constitutive promoter is CAG (also known as CAGGS or CBA), EF-1ALPHA, ubiquitin, or CMV.

[0168] In some embodiments, expression of the polynucleotide is regulated by a cell-specific promoter. In some embodiments, the cell-specific promoter is a neuron-specific promoter. In some embodiments, the neuron-specific promoter comprises human synapsin I (SYN) promoter, mouse calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, rat tubulin alpha I (Ta1), rat neuron-specific enolase (NSE) promoter, human platelet-derived growth factor-beta chain (PDGF) promoter, or THY1 (CD90) promoter. In some embodiments, the cell-specific promoter is human synapsin I.

[0169] In some embodiments, the expression of the polynucleotide is regulated by a tissue-specific promoter. In some embodiments, the tissue-specific promoter is a choroid plexus-specific promoter. In some embodiments, the choroid plexus-specific promoter comprises a Prlr promoter, a Spint2 promoter, or an F5 promoter. In some embodiments, the tissue-specific promoter is a liver-specific promoter. Liver-specific promoters are described, for example, in the literature [see LM Kattenhorn et al., Hum. Gene Ther. 27(12):947-961 (2016), specifically incorporated herein by reference in its entirety].

[0170] b. Protease cleavage sites / self-cleavage peptides and internal ribosome binding sites Suitable protease cleavage sites and self-cleaving peptides are known to those of skill in the art (see, for example, Ryan et al., 1997; Scymczak et al., 2004). Examples of protease cleavage sites are the cleavage sites of furin protease, potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus Nla protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Rice tungro spherical virus) 3C-like protease, PYFV (Parsnip yellow spot virus) 3C-like protease, thrombin, factor Xa and enterokinase. Due to the high cleavage stringency, the cleavage site of the tobacco etch virus (TEV) protease can be used. In some embodiments, the protease cleavage site is the cleavage site of the furin protease.

[0171] Exemplary self-cleaving peptides (also referred to as "cis-acting hydrolytic element", CHYSEL; see deFelipe (2002)) are derived from potyvirus and cardiovirus 2A peptides. Particular self-cleaving peptides may be selected from 2A peptides from FMDV (foot and mouth disease virus), equine rhinitis A virus, TaV (Thosea asigna virus) and porcine Teschovirus.

[0172] Specific initiation signals may also be used for efficient translation of coding sequences in polycistronic messages. These signals include the ATG initiation codon or adjacent sequences. For example, the initiation signal may include the Kozak consensus sequence having an amino acid sequence including GCCACCAUGGG (SEQ ID NO: 34). See Kozak, 1987; Harte et al., 2012. Exogenous translational control signals including the ATG initiation codon may need to be provided. One of skill in the art would be able to easily determine this and provide the necessary signals. It is well known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons may be natural or synthetic. Efficiency of expression may be improved by including appropriate transcriptional enhancer elements.

[0173] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to generate multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements in two members of the picornavirus family (poliovirus and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES elements in mammalian messages (Macejak and Sarnow, 1991). IRES elements may be linked to heterologous open reading frames. Multiple open reading frames may be transcribed together, each separated by an IRES, generating polycistronic messages. By virtue of the IRES element, each open reading frame may have access to ribosomes for efficient translation. Multiple genes may be efficiently expressed using a single promoter / enhancer to transcribe a single message (see US Pat. Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference).

[0174] c. multiple cloning site A vector may contain a multiple cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites that may be used with standard recombinant techniques to digest the vector (e.g., Carbonelli et al., 1999; Levenson et al., 1998; and Cocea, 1997, specifically incorporated herein by reference in their entireties). "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in the nucleic acid molecule. Many such restriction enzymes are commercially available. The use of such enzymes is well understood by those of skill in the art. Frequently, vectors are linearized or fragmented using a restriction enzyme that cuts within the MCS so that an exogenous sequence can be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments that may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0175] D splicing site Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns in the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure proper processing of the transcript for protein expression (see, e.g., Chandler et al., 1997, incorporated herein by reference).

[0176] e. Termination signal A vector or construct may contain at least one termination signal. A "termination signal" or "terminator" is comprised of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is considered. A terminator may be necessary in vivo to achieve a desired message level.

[0177] In eukaryotic systems, the termination region may also contain specific DNA sequences that allow site-specific cleavage of the nascent transcript to expose a polyadenylation site. This signals specialized endogenous polymerases to add a stretch of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are likely to be more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, the terminator contains a signal for RNA cleavage, and the termination signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements can serve to improve message levels and minimize read through to other sequences in the cassette.

[0178] Contemplated terminators include any known transcription terminator described herein or known to one of skill in the art, including, but not limited to, a genetic termination sequence, such as, for example, the bovine growth hormone terminator, or a viral termination sequence, such as, for example, SV40. In certain embodiments, the termination signal can be the absence of a transcribable or translatable sequence, for example, due to sequence truncation.

[0179] f polyadenylation signal Expression, particularly in eukaryotic expression, will generally include a polyadenylation signal to ensure proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to successful implementation, and any sequence may be used. Exemplary embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, which are convenient and known to function well in a variety of target cells. Polyadenylation can increase the stability of the transcript or facilitate cytoplasmic transport.

[0180] G. Post-transcriptional regulatory elements Vectors for use in the present disclosure may also include one or more post-transcriptional regulatory elements (PRE). Examples of PREs include the Woodchuck Hepatitis Virus PRE (WPRE), Hepatitis B Virus PRE, and intron A of the human cytomegalovirus immediate early gene. See Sun et al., 2009 and Mariati et al., 2010 for additional examples and details. In certain embodiments, the PRE is a WPRE. A WPRE is a DNA sequence that, when transcribed, creates a tertiary structure to enhance expression of a gene delivered by a viral vector.

[0181] h. origin of replication To propagate the vector in a host cell, the vector may contain one or more origins of replication (variously referred to as "ori"), such as the oriP of EBV as described above, or a nucleic acid sequence corresponding to a genetically engineered oriP (having a similar or improved function in differentiation programming, which is a specific nucleic acid sequence from which replication begins). Alternatively, origins of replication of other extrachromosomal replicating viruses, as described above, or autonomously replicating sequences (ARS) may be used.

[0182] 3. Vector Transmission Introduction of genetic modification or exogenous nucleic acid into cells may be performed using any suitable method for nucleic acid transfer for cell transformation, as described herein or as known to those skilled in the art. Methods for introducing and expressing genes into cells are known in the art. In relation to expression vectors, the vectors may be readily introduced into host cells, e.g., mammalian, bacterial, yeast or insect cells, by any method in the art. For example, expression vectors may be transferred to host cells by physical, chemical or biological means.

[0183] These methods include direct transfer of DNA, e.g., ex vivo transfection (Wilson et al., 1989; Nabel et al., 1989); transduction; viral transduction; injection (U.S. Pat. Nos. 5,994,624; 5,981,274; 5,945,100; U.S. Pat. No. 5,789,215, incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, and U.S. Pat. No. 5,789,215, incorporated herein by reference). Nos. 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each of which is incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); calcium phosphate precipitation (Graham and Van Der Eb, 1973;Chen and Okayama, 1987;Rippe et al., 1990); using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); direct sonic loading (Fechheimer et al., 1987); nucleofection; lipofection or liposome-mediated transfection (Nicolau and Sene, 1982;Fraley et al., 1979;Nicolau et al., 1987;Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991) and recipient-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); macroinjection or nanoparticle guns (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference); agitation with silicon carbide fibers (Kaeppler et al., Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); desiccation / inhibition-mediated DNA absorption (Potrykus et al., 1985); heat shock (Froger and Hall, 2007); and combinations of these methods. Such techniques may be applied to stably or transiently transform organelle(s), cell(s), tissue(s) or organism(s).

[0184] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle gun technology, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0185] Biological methods for introducing polynucleotide of interest into host cells may include the use of DNA and RNA vectors, into which polynucleotide of interest or transgene can be inserted.Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus, etc. (see, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, etc.).

[0186] Chemical means for introducing polynucleotides into host cells include macromolecule complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Nanoparticles are also contemplated. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0187] Methods for gene therapy and methods for transferring genes to a subject, for example using adeno-associated viruses, are described in the patent documents US6,967,018, WO2014 / 093622, US2008 / 0175845, US2014 / 0100265, EP2432490, EP2352823, EP2384200, WO2014 / 127198, WO2005 / 122723, WO2008 / 137490, WO2013 / 1421, 14, WO2006 / 128190, WO2009 / 134681, EP2341068, WO2008 / 027084, WO2009 / 054994, WO2014059031, US7,977,049 and WO2014 / 059029, each of which is incorporated herein by reference in its entirety.

[0188] A. Liposome-mediated transfection One exemplary delivery vehicle is a lipid and / or liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo or in vivo). In other aspects, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome through a linking molecule associated with the liposome and all of the oligonucleotides, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with the lipid, associated with the lipid, contained in a suspension in the lipid, contained or complexed in a micelle, or otherwise associated with the lipid. The lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any particular structure in solution. For example, they may exist in bilayer structures, micelles or "collapsed" structures. They may also simply be interspersed in the solution and form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols and aldehydes, as well as lipid droplets that occur naturally in the cytoplasm.

[0189] In certain embodiments, the nucleic acid may be entrapped in lipid complexes, such as liposomes. Liposomes are porous structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have various lipid layers separated by aqueous medium. Lipid layers form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before a closed structure is formed, trapping water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The amount of liposomes used may vary depending on the properties of the liposomes as well as the cells used, for example, about 5 to about 20 μg vector DNA per 1 to 10 million cells may be considered.

[0190] Liposome-mediated nucleic acid transfer and expression of foreign DNA in vitro has been very successful (see references: Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated transfer and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0191] In certain embodiments, the liposome may be complexed with hemagglutinating virus (HVJ), which has been shown to promote fusion with the cell membrane and facilitate cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed with or used with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet other embodiments, the liposome may be complexed with or used with both HVJ and HMG1. In other embodiments, the delivery vehicle may include a ligand and a liposome.

[0192] Lipids suitable for use in various embodiments may be obtained from commercial sources. For example, Lipofectamine may be obtained from Thermo Fisher Scientific, Waltham, Massachusetts, USA; dimyristyl phosphatidylcholine ("DMPC") may be obtained from Sigma, St. Louis, Missouri, USA; dicetyl phosphate ("DCP") may be obtained from K&K Laboratories, Plainview, New York, USA; cholesterol ("Choi") may be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc., Birmingham, Alabama, USA. Lipid stock solutions in chloroform or chloroform / methanol may be stored at about -20°C. Chloroform may be used as the only solvent since it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having a porous structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have many lipid layers separated by aqueous medium. The lipid layers form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before a closed structure is formed, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5:505-510). However, compositions having structures that differ from the normal porous structure in solution are also included. For example, lipids may assume a micellar structure or simply exist in heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0193] b. Electroporation method In a particular embodiment, the nucleic acid is introduced into the cells via electroporation, which involves exposing a cell suspension and DNA to a high-voltage electric discharge. The recipient cells can be made more susceptible to transformation by mechanical wounding. The amount of vector used can also vary depending on the characteristics of the cells used, for example, about 5 to about 20 μg of vector DNA per 1 to 10 million cells can be considered.

[0194] Transfection of eukaryotic cells using electroporation has been quite successful: mouse pre-B lymphocytes have been transfected with the human kappa immunoglobulin gene (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0195] C. Calcium phosphate method In another embodiment, the nucleic acid is introduced into the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA using this technique (Graham and Van Der Eb, 1973). This method has also been used to transfect mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells with the neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes with a variety of marker genes (Rippe et al., 1990).

[0196] d.DEAE-dextran method In another embodiment, the nucleic acid is delivered into the cells using DEAE-dextran followed by polyethylene glycol. Reporter plasmids have been introduced in this manner into mouse myeloma and erythroleukemia cells (Gopal, 1985).

[0197] 4. Selectable or screenable markers Regardless of the method of introducing exogenous nucleic acid into a host cell or exposing the cell to an inhibitor of the invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence within the host cell. Such assays include "molecular biological" assays familiar to those of skill in the art, such as, for example, Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides, e.g., immunological means (ELISA and Western blot) or assays described herein to confirm agents falling within the scope of the present disclosure.

[0198] In certain embodiments, cells containing exogenous nucleic acid can be identified in vitro or in vivo by including a marker in the expression vector or exogenous nucleic acid. Such a marker would confer a distinguishable change to the cells so that cells containing the expression vector can be easily identified. In general, a selectable marker may confer an attribute that allows for selection. A positive selectable marker is one whose presence allows for selection, whereas a negative selectable marker is one whose presence prevents selection. An example of a positive selectable marker is a drug resistance marker.

[0199] In addition to markers that confer a phenotype that allows for the differentiation of transformants based on the fulfillment of a condition, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes may be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Additional examples of selectable and screenable markers are well known to those skilled in the art.

[0200] Selectable markers may include reporter gene types used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of transfection or other manipulations to introduce foreign DNA into cells. Selectable markers are often antibiotic resistance genes; cells that have been manipulated to introduce foreign DNA will grow in medium containing the antibiotic, and cells that are able to grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include the Abicr gene of Tn5, which confers antibiotic resistance to geneticin, or the Neo gene.

[0201] Screenable markers may include reporter genes that allow researchers to distinguish between desired and undesired cells. Certain embodiments of the invention utilize reporter genes to indicate a particular cell lineage. For example, the reporter gene may be located within an expression element under the control of a ventricular- or atrial-selective regulatory element that is typically associated with the coding region of a ventricular- or atrial-selective gene for co-expression. The reporter allows cells of a particular lineage to be subjected to drug or other selection pressure or to be isolated without jeopardizing cell viability.

[0202] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). Vector-containing cells may be separated by FACS, for example, using fluorescent-tagged antibodies against cell surface proteins or substrates that can be converted to fluorescent products by vector-encoded enzymes.

[0203] In a particular embodiment, the reporter gene is a fluorescent protein. A wide range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning nearly the entire visible light spectrum (see Table 1 for non-limiting examples). Mutagenesis efforts on the original Aequorea victoria jellyfish green fluorescent protein have produced new fluorescent probes with a variety of hues from blue to yellow, which are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins emitting in the orange and red spectral regions have been developed from the marine sea anemone Discosoma striata and reef corals belonging to the Anthozoa genus. Other species have been mined to produce similar proteins with blue-green, green, yellow, orange and deep red fluorescence emissions. Research and development efforts are underway to improve the brightness and stability of fluorescent proteins to enhance their overall utility.

[0204] TIFF2024500768000016.tif231168TIFF2024500768000017.tif98168

[0205] III. Treatment of neurodegenerative diseases Aspects of the present disclosure relate to compositions and methods of using such compositions to treat or prevent subjects suffering from neurodegenerative diseases, disorders or conditions.In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Parkinson's disease dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down's syndrome, and / or pathological aging.In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0206] In certain embodiments, the disclosed methods further include treating a subject diagnosed with a neurodegenerative disease, disorder, or condition. In certain embodiments, the disclosed methods further include treating a subject diagnosed with symptoms of a neurodegenerative disease, disorder, or condition. In certain embodiments, the disclosed methods further include treating a subject identified as at risk for having a neurodegenerative disease, disorder, or condition. Using testing and diagnostic methods known in the art and described herein, a subject can be diagnosed with, or diagnosed as having, symptoms of, or identified as at risk for a neurodegenerative disease, disorder, or condition.

[0207] In some embodiments, the method further comprises determining that the subject is in need of treatment comprising a therapeutically effective amount of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof. In some embodiments, the vector encoding the Aβ peptide can encode a minigene that allows for protein expression of the Aβ peptide, and in some embodiments, the minigene can include a nucleotide sequence corresponding to an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:8, SEQ ID NOs:13-17, SEQ ID NOs:35-38, or SEQ ID NOs:66-85, or any value derivable therein. In some embodiments, the method further comprises providing the subject with a treatment comprising a therapeutically effective amount of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, if the subject is determined to be in need of such a treatment. In some embodiments, determining the subject in need of a treatment comprising a therapeutically effective amount of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, comprises diagnosing the subject with a neurodegenerative disease, disorder, or condition. In some embodiments, determining the subject in need of a treatment comprising a therapeutically effective amount of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, comprises diagnosing the subject as having symptoms of a neurodegenerative disease, disorder, or condition. In some embodiments, determining the subject in need of a treatment comprising a therapeutically effective amount of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, comprises identifying a subject at risk of having a neurodegenerative disease, disorder, or condition.

[0208] In some embodiments, the disclosed method comprises administering to a subject suffering from a neurodegenerative disease, disorder or condition a therapeutically effective amount of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof. As disclosed herein, a neurodegenerative disease may be associated with aggregation or oligomerization of Aβ peptide, and administration of a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof, has been unexpectedly shown to surprisingly prevent or reduce the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils or plaques. Additionally, administration of a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof, can surprisingly prevent or reduce the cytotoxicity of endogenous Aβ peptide aggregates. Thus, in some embodiments, compositions and corresponding methods for treating a subject suffering from a neurodegenerative disease, disorder or condition with a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof, are disclosed. In additional embodiments, a method is disclosed for inhibiting aggregation of endogenous Aβ peptides in vivo, comprising contacting at least one endogenous Aβ peptide in vivo with a therapeutically effective amount of an Aβ peptide variant expressed from a vector encoding the Aβ peptide variant, where the vector is optionally included in a composition. In additional embodiments, a method is disclosed for promoting aggregation of endogenous Aβ peptides in vivo, comprising contacting at least one endogenous Aβ peptide in vivo with a therapeutically effective amount of an expressed wild-type Aβ peptide or an Aβ peptide variant from a vector encoding the wild-type Aβ peptide or an Aβ peptide variant, where the vector is optionally included in a composition. In some embodiments, the neurodegenerative disease, disorder, or condition is characterized by abnormal aggregation of endogenous Aβ peptides. In some embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease.

[0209] The subject may have, for example, a condition having abnormal aggregation of Aβ peptides as a symptom and / or mechanism. Embodiments of the present disclosure include the treatment or prevention of any medical condition in which modulation of Aβ peptide aggregation is beneficial. In certain embodiments, a therapeutically effective amount of one or more compositions comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof is provided to an individual for attenuating Aβ peptide aggregation in the individual, or for delaying or reversing Aβ peptide aggregation in the individual. In certain embodiments, a therapeutically effective amount of one or more compositions comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof for promoting Aβ peptide aggregation is provided to an individual. In certain embodiments, medical conditions treated or prevented with a composition comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof include neurodegenerative diseases, disorders, or conditions characterized by aggregation of Aβ peptides. In certain embodiments, aggregation of Aβ peptides is not treated with a composition of the present disclosure. In some cases, a composition comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof treats or prevents a medical condition in an individual, for example, by ameliorating, inhibiting, slowing or reversing the aggregation of Aβ peptide. In some cases, a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof treats or prevents a medical condition in an individual, for example, by promoting the aggregation of Aβ peptide. In some embodiments, the amelioration, inhibition, slowing or reversal of Aβ peptide aggregation can attenuate tau protein seeding.

[0210] A subject may have a condition having as symptoms and / or mechanisms, for example, abnormal amyloid plaque formation, protein misfolding, increased levels of tau protein and / or increased levels of phosphorylated tau protein, increased tau protein and / or Aβ peptide seeding, neuroinflammation, cognitive decline, neurodegeneration, neuronal loss, and / or synaptic loss.Embodiments of the present disclosure include the treatment or prevention of any medical condition in which modulation of amyloid plaque formation, protein misfolding, levels of tau protein, phosphorylation of tau protein, seeding rate of tau protein and / or Aβ peptide seeding, neuroinflammation, cognitive decline, neurodegeneration, neuronal loss, and / or synaptic loss is beneficial. In certain embodiments, an individual is provided with a therapeutically effective amount of one or more compositions comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, for attenuating amyloid plaque formation, protein misfolding, tau protein levels, tau protein phosphorylation, tau protein and / or Aβ peptide seeding rate, neuroinflammation, cognitive decline, neurodegeneration, neuronal loss, and / or synaptic loss in the individual. In certain embodiments, medical conditions treated or prevented with a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof include neurodegenerative diseases, disorders, or conditions that may lead to protein misfolding, endogenous Aβ peptide aggregation, amyloid plaque formation, neuroinflammation, neurodegeneration, neuronal loss, or synaptic loss; abnormal tau levels, tau phosphorylation, or phosphorylated tau levels; tau seeding or endogenous Aβ peptide seeding; and / or cognitive decline. In certain embodiments, protein misfolding, aggregation of endogenous Aβ peptide, formation of amyloid plaques, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; abnormal tau levels, tau phosphorylation, or phosphorylated tau levels; tau seeding or endogenous Aβ peptide seeding; and / or cognitive decline are not treated with the compositions of the present invention.In some cases, the composition comprising a vector encoding an Aβ peptide variant, or a fragment or functional derivative thereof, treats or prevents a medical condition in an individual by ameliorating or inhibiting, for example, protein misfolding, aggregation of endogenous Aβ peptide, formation of amyloid plaques, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; abnormal tau levels, tau phosphorylation, or phosphorylated tau levels; tau seeding or endogenous Aβ peptide seeding; and / or cognitive decline.

[0211] Embodiments of the present disclosure include compositions and methods for treating or preventing cognitive decline as a result of protein misfolding, aggregation of endogenous Aβ peptides, formation of amyloid plaques, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; abnormal tau levels, tau phosphorylation, or phosphorylated tau levels; tau seeding or endogenous Aβ peptide seeding; and / or neurodegenerative disorders. In certain cases, delivery of a composition comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof to an individual prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils, or plaques or cytotoxicity of endogenous Aβ peptide aggregates. In certain cases, delivery of a composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof to an individual promotes the formation of plaques of endogenous Aβ peptide oligomers, protofibrils, fibrils, or endogenous Aβ peptide aggregates.

[0212] In some embodiments, a vector encoding a wild-type Aβ peptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:2), or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, a wild-type Aβ peptide of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:2, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:2 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVFFAE (SEQ ID NO:39), or any value derivable within these ranges.

[0213] In some embodiments, the vector encoding the Aβ peptide variant, or a fragment or functional derivative thereof, comprises a variant of SEQ ID NO:2 that corresponds to the wild-type Aβ peptide amino acid sequence.

[0214] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000018.tif13168, or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, the Aβ peptide variant of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:3, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:3 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVDFAE (SEQ ID NO:10), or any value derivable within these ranges.

[0215] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000019.tif11168, or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, the Aβ peptide variant of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:4, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:4 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVFPAE (SEQ ID NO:11), or any value derivable within these ranges.

[0216] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000020.tif11168, or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, the Aβ peptide variant of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:5, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:5 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVDFAE (SEQ ID NO:10), or any value derivable within these ranges.

[0217] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000021.tif11168, or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, the Aβ peptide variant of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:6, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:6 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLVPFAE (SEQ ID NO:12), or any value derivable within these ranges.

[0218] In some embodiments, the Aβ peptide variant comprises: TIFF2024500768000022.tif11168, or a fragment or functional derivative thereof, or any value derivable therein. In some embodiments, the Aβ peptide variant of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:7, or a fragment or functional derivative thereof. In some embodiments, a fragment or functional derivative of SEQ ID NO:7 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to KLPFFAE (SEQ ID NO:65), or any value derivable within these ranges. In some embodiments, the Aβ peptide variant comprises, consists of, or does not essentially consist of SEQ ID NO:7.

[0219] In some embodiments, a wild-type Aβ peptide or an Aβ peptide variant comprising, consisting of, or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a 1 to 22 amino acid truncation. In some embodiments, the N-terminal truncation comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid truncation.

[0220] In some embodiments, a wild-type Aβ peptide or an Aβ peptide variant comprising, consisting of, or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a 1 to 27 amino acid truncation. In some embodiments, the C-terminal truncation comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acid truncation.

[0221] In some embodiments, a wild-type Aβ peptide or an Aβ peptide variant comprising, consisting of, or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 comprises an N-terminal truncation and a C-terminal truncation, in some embodiments, the N-terminal truncation comprises a 1-22 amino acid truncation and the C-terminal truncation comprises a 1-27 amino acid truncation. In some embodiments, N-terminal truncations include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid truncations and C-terminal truncations include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acid truncations.

[0222] The wild-type or mutant Aβ peptide may be encoded by a vector encoding a minigene that allows for protein expression of the Aβ peptide. In some embodiments, the minigene may comprise a nucleotide sequence corresponding to an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:8, SEQ ID NOs:13-17, SEQ ID NOs:35-38, or SEQ ID NOs:66-85, or any derivable value within these ranges.

[0223] Also disclosed in some embodiments is a pharmaceutical composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof. In some cases, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof is provided as a monotherapy for an individual, while in other cases, the individual is provided with one or more additional therapies for treating or preventing protein misfolding, aggregation of endogenous Aβ peptide, formation of amyloid plaques, neuroinflammation, neurodegeneration, neuronal loss or synapse loss; abnormal tau levels, tau phosphorylation, or phosphorylated tau levels; tau seeding or endogenous Aβ peptide seeding; and / or cognitive decline; or in other cases, one or more additional therapies for promoting Aβ peptide aggregation, amyloid plaque formation, and / or fibril formation are provided to the individual.

[0224] The one or more additional therapies may be of any type, but in certain cases, the one or more additional therapies are one or more additional neurodegenerative disease therapeutic agents, such as Alzheimer's disease drugs. In some embodiments, the Alzheimer's disease drugs include aducanumab, donepezil, rivastigmine, galantamine, memantine, or tacrine. In some embodiments, the Alzheimer's disease drugs include additional and / or alternative drugs and biologics that reduce Aβ levels or prevent aggregation (i.e., secretase inhibitors / modulators, anti-Aβ manual immunization, anti-Aβ active immunization, or metal chelators); drugs and biologics that reduce tau levels or prevent tau aggregation or reduce pathological phosphorylation of tau (i.e., kinase inhibitors, anti-tau manual immunization, anti-tau active immunization, or antisense oligonucleotides); drugs and biologics that stabilize microtubules and and preparations that reduce neurodegeneration; drugs and preparations that block or modify inflammatory responses; drugs and preparations that reduce neuropsychiatric symptoms; drugs and preparations that enhance cognition (i.e., neurotransmitter inhibitors, modulators, activators); drugs and preparations that preserve or improve vascular function; or drugs and preparations that modify cellular metabolism (see, for example, references KG Yiannopoulou & S.G. Papageorgiou, J. Cent. Nerv. Sys. Dis. 12:1179573520907397 (2020); and J. Cummings et al., Alzheimer's Dement. (NY) 5:272-293 (2019)).

[0225] In some embodiments, the one or more additional therapies include one or more therapies for treating disorders or associated diseases of aging, such as cardiovascular disease, diabetes, atherosclerosis, obesity, cancer, infection, and neurological disorders. Any well-established indicator of the progression of aging can be used. In some embodiments, the one or more therapies for treating disorders or associated diseases of aging have the effect of reducing the incidence of cancer, slowing or alleviating cardiovascular diseases such as atherosclerosis; slowing and / or alleviating osteoporosis; improving glucose tolerance or reducing the incidence of related diseases such as diabetes and obesity; improving or reducing the decline in memory function and other cognitive functions; improving or reducing declining neuromuscular coordination; and improving or reducing the decline in immune function. The improvement of age-related disorders can also be the result of a reduction in symptoms in affected subjects or a reduction in the incidence of a disease or disorder in a population compared to an untreated population. The one or more therapies have the effect of treating and / or preventing various age-related conditions and diseases as assessed by specific markers and aging disorders. Thus, in an additional aspect, the present invention relates to the treatment or prevention in a subject of a disorder or marker of aging selected from the group of at least decreased cardiovascular function, osteoporosis, arthritis, glucose intolerance, insulin resistance, decreased memory, loss of neuromuscular coordination, increased cardiovascular disease, decreased cardiac, circulatory or pulmonary function and decreased lifespan, or a combination thereof.

[0226] IV. Administration of Therapeutic Compositions In certain embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is formulated as a pharmaceutical composition for therapeutic administration. In some embodiments, the disclosed methods include administering a neurodegenerative disease therapy to a subject or patient. The compositions of the present disclosure may be used for in vivo, in vitro, or ex vivo administration. In some embodiments, the neurodegenerative disease therapy includes a protein-based therapy, which may be a wild-type Aβ peptide or an Aβ peptide mutant therapy. In some embodiments, the neurodegenerative disease therapy includes a polynucleotide-based therapy, which may be a therapy comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide mutant or a fragment or functional derivative thereof. In some embodiments, the neurodegenerative disease therapy includes one or more neurodegenerative disease drugs. Any of these neurodegenerative disease therapies may be excluded. Combinations of these therapies may also be administered.

[0227] The therapies provided herein may include the administration of a combination of therapeutic compositions, such as a first neurodegenerative disease therapy (e.g., a wild-type Aβ peptide or an Aβ peptide variant, or a polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant) and one or more additional neurodegenerative disease therapies (e.g., a neurodegenerative disease drug). The therapies may be administered in any suitable manner known in the art. For example, the first and one or more additional neurodegenerative disease therapies may be administered sequentially (at different times) or simultaneously (at the same time or at about the same time; also "concurrently" or "substantially simultaneously"). In some embodiments, the first and one or more additional neurodegenerative disease treatment therapies may be administered in separate compositions. In some embodiments, the first and one or more additional neurodegenerative disease treatment therapies may be in the same composition. The different therapies may be administered in one composition or in more than one composition, such as two compositions, three compositions, or four compositions. Various combinations of formulations may be used.

[0228] In some embodiments, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof and the one or more additional neurodegenerative disease drugs are administered substantially simultaneously. In some embodiments, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof and the one or more additional neurodegenerative disease drugs are administered sequentially. In some embodiments, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof are administered prior to the administration of the one or more additional neurodegenerative disease drugs. In some embodiments, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof are administered after the administration of the one or more additional neurodegenerative disease drugs.

[0229] In some embodiments, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variant or a fragment or functional derivative thereof is delivered to the subject once. In some embodiments, the composition comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variant or a fragment or functional derivative thereof is delivered to the subject several times, for example, once a day, more than once a day, once a week, more than once a week, once a month, more than once a month, once a year, or more than once a year. In some embodiments, the wild-type Aβ peptide or Aβ peptide variant is administered to the subject several times. In some embodiments, the vector or polynucleotide encoding the wild-type Aβ peptide or Aβ peptide variant is administered to the subject once. The multiple treatments may or may not have the same formulation and / or route of administration.

[0230] In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered after the initiation of Aβ peptide oligomer, protofibril or fibril formation. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered after the initiation of amyloid plaque formation. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered before the initiation of Aβ peptide oligomer, protofibril or fibril formation. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered before the initiation of amyloid plaque formation.

[0231] A. Carriers In some embodiments, the pharmaceutical compositions of the present disclosure include an effective amount of one or more compositions comprising a polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant or variant dissolved or dispersed in a pharma- ceutically acceptable carrier. The terms "pharmaceutical" and "pharmacologically acceptable" are used interchangeably herein and refer to molecular entities and compositions that, when properly administered to a subject, such as a human, do not cause side effects, allergies or other untoward reactions and do not interfere with the therapeutic methods of the present disclosure. The preparation of pharmaceutical compositions comprising at least one vector encoding an Aβ peptide variant or a fragment or functional derivative thereof or an additional active ingredient will be known to those skilled in the art in light of the present disclosure, as exemplified in the literature [see Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, specifically incorporated herein by reference in its entirety]. It will also be understood that formulations for administration to a subject must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0232] The term "pharmaceutical acceptable carrier" as used herein includes any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, analogs, and combinations thereof, as known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, specifically incorporated herein by reference in its entirety). Except where any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Compositions containing wild-type Aβ peptide or Aβ peptide variants or polynucleotides encoding same may contain different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether sterilization is required for the route of administration, such as injection.

[0233] Further, according to the present disclosure, the compositions of the present disclosure suitable for administration may be provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., paste, or solid carriers. The use of a carrier is appropriate for carrying out the methods of the present disclosure, except where any conventional vehicle, agent, diluent, or carrier is deleterious to the recipient or to the therapeutic effect of the composition contained therein. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, alcohols, and the like, or combinations thereof. The compositions may also include various antioxidants to retard oxidation of one or more components. In addition, prevention of microbial action may be effected by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0234] According to the present disclosure, the composition may be combined with the carrier in any convenient and substantial manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine for those of skill in the art. The composition comprising the wild-type Aβ peptide or Aβ peptide variant or a polynucleotide encoding the same may be lyophilized.

[0235] In certain embodiments of the present disclosure, the composition is thoroughly combined or mixed with a semi-solid or solid carrier. Mixing may be performed in any convenient manner, such as grinding. Stabilizers may also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., gastrointestinal denaturation. Examples of stabilizers used in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0236] In further embodiments, the present disclosure may include the use of pharmaceutical lipid vehicle compositions comprising compositions comprising wild-type Aβ peptide or Aβ peptide variants or polynucleotides encoding one or more such lipids, and an aqueous solvent. The term "lipid" as used herein will be defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. Such a wide range of compounds is well known to those skilled in the art, and the term "lipid" is not limited to any particular structure by its use herein. Examples of lipids include compounds that contain long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., engineered or produced by humans). However, lipids are generally biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, and polymeric lipids, and combinations thereof. Of course, compounds other than the lipids specifically described herein that one of skill in the art would understand to be lipids are also included in the compositions and methods of this disclosure.

[0237] Those skilled in the art should be familiar with the range of techniques that may be used to disperse a composition in a lipid vehicle.For example, a composition or compositions comprising wild-type Aβ peptide or Aβ peptide variants or a polynucleotide encoding the same may be dispersed in a solution containing lipid, dissolved in lipid, oiled with lipid, mixed with lipid, covalently bound to lipid, contained in suspension in lipid, contained or complexed in micelles or liposomes, or associated with lipid or lipid structures by any means known to those skilled in the art.The dispersion may or may not form liposomes.

[0238] The composition(s) containing the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof may be formulated into a composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example salts formed with free amino groups of the proteinaceous composition, or salts formed with inorganic acids, for example hydrochloric acid or phosphoric acid, or organic acids, for example acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases, for example sodium, potassium, ammonium, calcium or ferric hydroxides; or organic bases, for example isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The dosage forms are easily administered in a variety of dosage forms, such as those formulated for parenteral administration, for example as infusions or aerosols for delivery to the lungs, or those formulated for ingestible administration, for example as drug-releasing capsules.

[0239] B. Route of Administration The therapeutic agent of the present invention may be administered by the same or different administration routes. The administration route of the composition may be, for example, intravenous, intracerebral, intracranial, intramuscular, subcutaneous, topical, oral, intramucosal, intradermal, transdermal, intraperitoneal, intraarterial, intraorbital, by implantation, intravaginal, intrarectal, intraspinal, intraarticular, intraventricular, intralaminar or intranasal; by inhalation, injection, injection, continuous injection, via catheter, by irrigation, directly to the target cells, by localized irrigation; by cream or lipid composition (e.g., liposome); by other methods or any combination of the above methods known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, specifically incorporated herein by reference in its entirety).

[0240] In some embodiments, the composition(s) comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered systemically or locally. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered to the central nervous system via retro-orbital injection. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered systemically to the central nervous system via peripheral injection. In some embodiments, the peripheral injection is an intravenous injection. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, is delivered to the cerebrospinal fluid (CSF). In some embodiments, the composition is delivered to the cerebrospinal fluid by non-surgical injection. In some embodiments, the non-surgical injection into the cerebrospinal fluid comprises a non-surgical intrathecal injection. In some embodiments, the composition is delivered to the cerebrospinal fluid by neurosurgical injection. In some embodiments, the neurosurgical injection into the cerebrospinal fluid comprises a neurosurgical injection into the cisterna magna. In some embodiments, the composition comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof is delivered to the ventricular system. In some embodiments, the composition is delivered to the ventricular system by neurosurgical injection. Delivery of a composition to the cerebrospinal fluid and / or the ventricular system is described, for example, in W. A. ​​Liguore et al., Molecular Therapy 27(11):2018-2037, which is incorporated herein by reference in its entirety. In some embodiments, the composition delivered to the central nervous system, CSF and / or the ventricular system crosses the blood-brain barrier.

[0241] In some embodiments, compositions comprising the wild-type Aβ peptide or Aβ peptide variants disclosed herein may be formulated to improve the in vivo stability of the wild-type Aβ peptide or Aβ peptide variants and / or the uptake or absorption of the wild-type Aβ peptide or Aβ peptide variants by cells, as described in A. L. Rewis and J. Richard, Therapeutic Delivery 6(2):149-163 (2015), the entire contents of which are incorporated herein by reference. For example, the wild-type Aβ peptide or Aβ peptide variants may be formulated with an absorption enhancer, such as acylcarnitine, sodium octanoate, sodium caprate, SNAC, SNAD, or 5-CNAC, to increase the absorption of the wild-type Aβ peptide or Aβ peptide variants by cells. In some embodiments, the in vivo stability of the wild-type Aβ peptide or Aβ peptide variant and / or the absorption or formulation for enhancing absorption of the wild-type Aβ peptide or Aβ peptide variant by cells will depend on the route of administration of the composition, e.g., oral or injection.

[0242] 1. Parenteral route Thus, in some embodiments, the composition(s) comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof may be administered via a parenteral route. The term "parenteral" as used herein includes routes that bypass the digestive tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example, but not limited to, retro-orbital, intracerebral, intracranial, intravenous, intradermal, intramuscular, intraarterial, intrathecal, subcutaneous, or intraperitoneal (see U.S. Patent Nos. 6,7537,514; 6,613,308; 5,466,468; 5,543,158; 5,641,515; and 5,399,363, each of which is specifically incorporated herein by reference in its entirety).

[0243] Solutions of the active compounds as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and oils. Under typical storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (e.g., U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of particle size as required in the case of dispersion, and by the use of surfactants. Prevention of microbial action may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Injectable compositions may be absorbed for extended periods of time by using in the composition preparations that delay absorption, for example, aluminum monostearate and gelatin.

[0244] For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, the sterile aqueous medium that may be used will be known to those skilled in the art in light of the present disclosure. For example, a dose can be dissolved in an isotonic NaCl solution and injected at the proposed site of administration (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any event determine the appropriate dose for the individual subject. For human administration, formulations must also meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0245] Sterile injectable solutions can be prepared by mixing the required amount of active compound in a suitable solvent with various other ingredients listed above, and then, if necessary, sterilizing by, for example, filtration. Generally, dispersions are prepared by mixing various sterilized active ingredients in a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying techniques, which produce a powder of active ingredient and any additional desired ingredients from a previously sterile-filtered solution. Powdered compositions are combined with liquid carriers, such as, for example, water or saline, with or without stabilizers.

[0246] 2. Digestive Pathway In certain embodiments of the present disclosure, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof are formulated to be administered via the digestive route. The digestive route includes all possible routes of administration where the composition comes into direct contact with the digestive tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, bucally, rectally or sublingually. Thus, these compositions may be formulated with an inert diluent or an assimilable edible carrier, enclosed in a hard- or soft-shell gelatin capsule, compressed into tablets, or directly integrated into the food of the meal.

[0247] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579; and 5,792,451, each of which is incorporated by reference in its entirety). The tablets, troches, pills, capsules, and the like may also include the following: Suitable additives include: binders such as gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants such as corn starch, potato starch, alginic acid, or combinations thereof; lubricants such as magnesium stearate; Sweeteners, such as sucrose, lactose, saccharin, or combinations thereof; flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of materials. A variety of other materials may be present as coatings or may otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain a carrier, such as a liquid carrier in addition to the above types of materials. Gelatin capsules, tablets, or pills may be enteric coated. Enteric coatings prevent denaturation of the composition in the stomach or upper intestine, where the pH is acidic. See, for example, U.S. Patent No. 5,629,001. Once reaching the small intestine, the internal basic pH dissolves the coating and the composition is released to be secreted into specialized cells, such as epithelial intestinal cells and Peyer's patch to be absorbed by M cells. An elixir syrup may contain the active compound, a sweetener such as sucrose, preservatives such as methyl and propylparabens, a dye and a flavoring, such as cherry or orange flavor.Of course, any material used in preparing any dosage unit form should be pharma- ceutically pure and substantially non-toxic in the amounts employed. The active compounds may also be included in sustained release preparations and dosage forms.

[0248] For oral administration, the composition(s) comprising the vector encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, toothpaste, buccal tablet, oral spray, or sublingual oral-administration form. For example, a mouthwash can be prepared by including a required amount of active ingredient in a suitable solvent, such as sodium borate solution (Dobell's Solution). Alternatively, the active ingredient can be incorporated into an oral solution, such as a solution containing sodium borate, glycerin, and potassium bicarbonate, dispersed in dentifrice, or added in a therapeutically effective amount to a composition that may include water, binders, abrasives, flavoring agents, elastomers, and humectants. Alternatively, the composition may be made in the form of a tablet or solution that can be placed under the tongue or dissolved in the mouth.

[0249] Additional dosage forms suitable for other modes of administration in the digestive tract include suppositories. Suppositories are solid dosage forms of various weights and shapes, generally used for medicinal purposes, for insertion into the rectum. After insertion, the suppository is softened, dissolved or dissolved with a co-fluid. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, about 0.5% to about 10% (by weight), preferably about 1% to about 2% (by weight) of the active ingredient.

[0250] 3. Other routes In other embodiments of the present disclosure, the composition(s) comprising a vector encoding a wild-type Aβ peptide or an Aβ peptide variant, or a fragment or functional derivative thereof, may be formulated for administration via a variety of other routes, e.g., topical (i.e., transdermal), mucosal (nasal, vaginal, etc.) and / or inhalation.

[0251] Pharmaceutical compositions for topical administration may include active compounds formulated for pharmaceutical application such as ointments, pastes, creams, or powders. Ointments include all tenacious, absorbent, emulsion, and water-soluble base compositions for topical application, whereas creams and lotions are compositions that include only emulsion bases. Drugs administered topically may contain a penetration enhancer to promote the absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidones, and laurocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorbent, emulsion, or water-soluble ointment base. Topical formulations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide a homogenous mixture. Transdermal administration of the present disclosure may also include the use of a "patch." For example, a patch may deliver one or more active agents in a continuous manner at a predetermined rate for a fixed period of time.

[0252] In certain embodiments, the pharmaceutical composition(s) comprising the vector encoding the Aβ peptide variant or a fragment or functional derivative thereof may be delivered by eye drops, intranasal spray, inhalation and / or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs through nasal aerosol sprays are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212, each of which is incorporated herein by reference in its entirety. Similarly, drug delivery using intranasal microparticle resins (see, for example, Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (see, for example, U.S. Patent No. 5,725,871, incorporated herein by reference in its entirety) is also well known in the pharmaceutical arts. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is ​​described, for example, in U.S. Patent No. 5,780,045, specifically incorporated herein by reference in its entirety.

[0253] The term aerosol refers to a colloidal system of finely divided solids in liquid particles dispersed in a liquefied or pressurized gas propellant. A typical aerosol of the present disclosure for inhalation will consist of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a compatible solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers vary according to the pressure requirements of the propellant. Administration of the aerosol varies according to the age, weight and severity of symptoms and response of the subject.

[0254] C. Medication The appropriate dosage of the composition(s) of the present disclosure administered to a subject can be determined by physical and physiological factors such as body weight, severity and course of the condition, type of disease being treated, clinical condition of the individual, previous or concurrent therapeutic interventions, clinical history of the individual and response to treatment, idiopathic nature of the subject, route of administration and the discretion of the attending physician. Depending on the dosage and route of administration, the preferred dosage and / or frequency of administration of an effective amount may vary depending on the subject's response. The practitioner responsible for administration will in any case determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0255] In certain embodiments, the pharmaceutical composition may contain, for example, up to or at least about 0.000001 to up to or at least about 10% (by weight) of the active compound. In other embodiments, the active compound may contain from about 0.001% to about 1% of the unit weight, or, for example, from about 0.01% to about 0.1%, and any range derivable within these ranges. Of course, the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations, should be considered by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be preferred.

[0256] The treatment may include multiple "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, the specific route and dosage form are within the skill of the clinical practitioner in determining the amount. A unit dose need not be administered in a single infusion, but may include continuous inputs over a period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0257] The amount administered according to the number of treatments and unit dose depends on the desired therapeutic effect. An effective dose is understood to mean the amount necessary to achieve a particular effect. Such doses may also be administered multiple times throughout the day and / or over the course of several days, weeks or months.

[0258] In some embodiments, a single dose of wild-type Aβ peptide or Aβ peptide variant is administered. In some embodiments, multiple doses of Aβ peptide variant are administered. In some embodiments, a single dose of wild-type Aβ peptide or Aβ peptide variant is administered. In some embodiments, the wild-type Aβ peptide or Aβ peptide variant is administered at a dose of at least, up to, or about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range or value derivable therein. In certain embodiments, an effective dose of the Aβ peptide variant is about, at least about, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range inducible therein. In certain embodiments, the wild-type Aβ peptide or Aβ peptide variant administered to the subject is metabolized in the body as a metabolic therapeutic agent, in which case the blood levels can be indicative of the amount of the formulation. Alternatively, to the extent that wild-type Aβ peptide or Aβ peptide mutants are not metabolized by the subject, the blood levels discussed herein can refer to unmetabolized wild-type or mutant Aβ peptide.

[0259] In some embodiments, a single dose of polynucleotide encoding wild-type Aβ peptide or Aβ peptide variant is administered. In some embodiments, multiple doses of polynucleotide encoding wild-type Aβ peptide or Aβ peptide variant are administered. In some embodiments, an effective dose of polynucleotide encoding wild-type Aβ peptide or Aβ peptide variant is administered. In some embodiments, polynucleotide encoding wild-type Aβ peptide or Aβ peptide variant is administered at least, at most, or about 1×10 per kg of subject body weight. 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or 1×10 18 In some embodiments, the polynucleotide encoding the wild-type Aβ peptide or an Aβ peptide variant is administered at a dose of 1×10 per kg of body weight of the subject. 8 〜1×10 18 In some embodiments, the polynucleotide encoding the wild-type Aβ peptide or the Aβ peptide variant is administered at a dose of 1×10 per kg of the subject's body weight. 11 〜1×10 14 In some embodiments, the polynucleotide encoding the wild-type Aβ peptide or an Aβ peptide variant is administered at a dose of 1×10 per kg of body weight of the subject. 12 〜1×10 15 The dose is the number of copies of the polynucleotide.

[0260] In some embodiments, the polynucleotide encoding the wild-type Aβ peptide or the Aβ peptide variant is comprised in a vector. In some embodiments, the polynucleotide encoding the wild-type Aβ peptide or the Aβ peptide variant is comprised in a vector encoding a minigene that allows for protein expression of the mutant Aβ peptide. In some embodiments, an effective dose of a vector comprising a polynucleotide encoding the wild-type Aβ peptide or the Aβ peptide variant is administered. In some embodiments, the vector is administered at least, at most, or at about 1×10 per kg of body weight of the subject. 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or 1×10 18 The vector genome is administered at a dose of 1×10 per kg of subject body weight or any inducible range or value within these ranges. In some embodiments, the vector is administered at a dose of 1×10 per kg of subject body weight. 8 〜1×10 18 In some embodiments, the vector is administered at a dose of 1×10 vector genome per kg of subject body weight. 11 〜1×10 14 In some embodiments, the vector is administered at a dose of 1×10 vector genome per kg of subject body weight. 12 〜1×10 15 The vector genome is administered at a dose of 100 mg / kg / day.

[0261] In some embodiments, a single dose of the one or more additional neurodegenerative disease medications is administered. In some embodiments, multiple doses of the one or more additional neurodegenerative disease medications are administered. In some embodiments, an effective dose of the one or more additional neurodegenerative disease medications is administered. In some embodiments, the one or more additional neurodegenerative disease drugs are administered at a dose of at least, up to, or about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range or value derivable therein. In certain embodiments, the effective dose of the one or more additional neurodegenerative disease medications is about, at least about, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 11 A dose that provides a blood level of 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range inducible therein. In certain embodiments, one or more additional neurodegenerative disease drugs administered to the subject are provided as metabolic therapeutic agents that are metabolized in the body, in which case the blood level can indicate the amount of the formulation. Alternatively, unless one or more additional neurodegenerative disease drugs are metabolized by the subject, the blood levels discussed herein can represent unmetabolized wild-type or mutant Aβ peptide.

[0262] Precise amounts of a therapeutic composition depend on the judgment of the practitioner and will be different for each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (palliative vs. therapeutic), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy to which the subject may be subjected.

[0263] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg of body weight may be converted and expressed in analogous concentration units of μg / ml or mM (blood concentration). It is also understood that uptake is species and organ / tissue dependent. Conversion factors and physiological assumptions applicable in connection with absorption and concentration measurements are well known and allow one of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy and results described herein.

[0264] V.Kit Certain aspects of the present disclosure also relate to kits that include the disclosed compositions or compositions for implementing the disclosed methods. In some embodiments, the kits may be used to assess one or more biomarkers (e.g., Aβ peptide oligomers, protofibrils, fibrils, plaques, misfolded proteins, tau proteins, phosphorylated tau proteins, seeded tau proteins and / or seeded Aβ peptides, neuroinflammatory markers, markers of cognitive decline, neurodegeneration markers, markers of neuronal loss, and / or markers of synaptic loss). In certain embodiments, the kits may be used to measure expression of Aβ peptides or other proteins in vitro or in vivo. In certain embodiments, the kit comprises at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any derivable value or range and combination therein. In some embodiments, there is a kit for assessing activity of a biomarker in a cell.

[0265] The kit may contain components that may be individually packaged or disposed in containers, such as tubes, bottles, vials, syringes, or other suitable container means.

[0266] Individual components may be provided in the kit in concentrated amounts; in some embodiments, a component is provided individually at the same concentration as it is in solution with the other components. Concentrations of components may be provided at 1×, 2×, 5×, 10×, or 20× or greater.

[0267] Kits for using the probes, synthetic nucleic acids, non-synthetic nucleic acids, synthetic peptides, non-synthetic peptides and / or inhibitors of the present disclosure for prognostic or diagnostic applications are included as part of the present disclosure.Specifically contemplated are any such molecules corresponding to any of the biomarkers identified herein, including nucleic acid primers / primer sets and probes identical to or complementary to all or a portion of the biomarker, which may include the coding sequence of the biomarker as well as non-coding sequences of the biomarker.

[0268] In certain aspects, negative and / or positive control nucleic acids, probes and inhibitors are included in some kit embodiments. The kits may also include samples that are negative or positive controls for the methylation of one or more biomarkers.

[0269] In other words, any embodiment of the disclosure that includes a particular biomarker is also contemplated to include embodiments that include a biomarker that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical in sequence to the mature sequence of the particular nucleic acid.

[0270] It is understood that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined. The originally submitted claims are considered to include multiple dependent claims on any submitted claim or combination of submitted claims. EXAMPLES

[0271] The following examples are included to illustrate embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that function well in the practice of the invention. However, those of skill in the art should recognize in light of this disclosure that many changes can be made in the specific examples disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0272] Example 1 - Gene therapy using Aβ mutants for amyloid reduction Peptide inhibitors fulfilling four therapeutic criteria have been identified in vitro, and the top two peptide candidates have been examined in vivo in animal models of Aβ amyloidosis. Minigene vectors were generated to express mutant Aβ peptides at the cell membrane where release into the extracellular space is regulated by endogenous proteases. This was followed by AAV delivery to neonatal mouse brain for widespread lifelong neuronal expression to test efficacy for Aβ reduction in the APP / PS1 mouse model of Alzheimer's disease (AD). This study yielded the counterintuitive result that peptides differing from pathogenic Aβ by merely one or two amino acids effectively reduced Aβ fibrillization, destabilizing existing fibrils and lowering oligomer toxicity for short periods in vitro, while reducing amyloid aggregation and neuroinflammation when used chronically in vivo.

[0273] This study also takes the first step in applying virus-mediated gene therapy to overcome past limitations in advancing peptide Aβ inhibitors from bench to brain. The larger impact of this study lies in the potential application of this strategy to other protein misfolding diseases, including toxic species of misfolded proteins that induce neurodegeneration, which have been avoided for technical reasons that peptide therapy can currently overcome through expression engineering and viruses.

[0274] A. The F20P and F19D / L34P mutants suppress the aggregation of wild-type Aβ42. The central hydrophobic region of Aβ (17Leu-Val-Phe-Phe-Ala21) controls the rate of monomer assembly and forms β-sheet hairpins in mature fibrils. Given its importance in fibril formation, in some embodiments, targeted amino acid substitutions in this region generate peptides that can prevent the aggregation of wild-type Aβ42 peptides. Based on previous studies on this domain, five other Aβ42 mutant peptides were examined: V18P, F19D, F19P, F19D / L34P, and F20P. It was first confirmed using Thioflavin-T analysis that none of the five mutants aggregated in fibrils by themselves (Figure 1A). We then tested whether any of the five peptides could competitively inhibit the fibrillization of wild-type Aβ42 when the two peptides were mixed in an equimolar ratio before initiating the fibrillization reaction (Figure 1B). Four of the five mutant peptides reduced fibrillization of wild-type Aβ, whereas one (V18P) exacerbated aggregation. Two peptide mutants, F20P and F19D / L34P, chosen for further studies, were shown to completely prevent aggregation. These two peptides were tested for their potential to degrade wild-type A42 when added after fibril formation. Both mutants degraded wild-type fibrils in a concentration-dependent manner, but F19D / L34P was much more effective at disassembling fibrils than F20P, achieving >80% ThT signal loss at the highest concentration tested (Figure 1C, 1D).

[0275] Finally, we investigated the possibility that mutant Aβ peptides could attenuate the cytotoxicity induced by wild-type Aβ oligomers. For this experiment, wild-type Aβ42 was used alone or mixed in an equimolar ratio with mutant peptides before oligomer assembly. After 24 hours, peptide solutions were added to cell culture medium of murine neuroblastoma 2a cell line (N2a). Oligomers assembled from wild-type Aβ42 caused nearly 50% cell loss compared to cultures treated with oligomerization buffer alone (Figure 1E). In contrast, F20P and F19D / L34P by themselves did not significantly alter cell viability. Notably, cell viability increased from 50% in the control group with wild-type Aβ42 alone to about 70% when wild-type peptides were co-cultured with the mutants during oligomer assembly. Thus, in some embodiments, the F20P and F19D / L34P mutants meet the minimal in vitro criteria for candidate aggregation inhibitors.

[0276] B. Design of vectors for secreting Aβ mutants in mammalian cells Next, the Aβ peptide mutants were tested in the brain at levels high enough to be therapeutically effective. An expression strategy was designed to stably express mutant Aβ at the cell membrane, where release into the extracellular space is regulated by endogenous γ-secretase. Previous studies achieved this goal by fusing the signal peptide of the amyloid precursor protein (APP) to the APP C-terminal fragment (β-CTF), but while this construct generated extracellular Aβ, overexpression of β-CTF inadvertently led to Aβ-independent lysosomal-autophagy pathology. Therefore, we sought to avoid potential lysosomal complications by using the smallest β-CTF fragment required for γ-secretase cleavage. We targeted the cell membrane using the Gaussia luciferase signal peptide, which was fused to a series of β-CTF fragments. The longest fragment was 54 amino acids in length and contained Aβ42 and the entire APP transmembrane domain plus two intracellular lysine residues (Figure 2A). Four additional constructs truncated the transmembrane domain by two amino acids each; the shortest construct contained only 45 residues (Aβ42+3 additions).

[0277] The different constructs were transfected in N2a cells and secreted Aβ was measured in the conditioned cell culture medium using immunoprecipitation followed by immunoblotting. The intact APP transmembrane domain plus two intracellular lysines was found to be necessary and sufficient to achieve Aβ secretion at levels comparable to those observed with the construct encoding the full-length CTF (Figure 2A). Addition of the γ-secretase inhibitor LY411575 blocked the release of Aβ mutants into the medium in a concentration-dependent manner, confirming that Aβ secretion is dependent on γ-secretase activity (Figure 2A).

[0278] This minimal expression construct was cloned into an AAV delivery vector for subsequent in vivo expression under the control of the ubiquitous CAG promoter (Figure 2B). A woodchuck poliovirus response element was included at the 3' end to stabilize the mRNA, followed by a bovine growth hormone polyA sequence. Finally, mass spectrometry was used to confirm whether this vector truly produces Aβ40 / 42 when expressed in cells. After transfecting N2a cells with the viral Aβ F20P vector, human Aβ was immunoprecipitated from the culture medium against the WT peptide. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was performed without enzymatic digestion to confirm whether the full-length peptide was made. Through this analysis, we confirmed the presence of peptides with the appropriate mass for Aβ40 F20P (Figure 2C) and Aβ42 F20P (data not shown) along with the correct sequence for each peptide (Figure 2D).

[0279] C. Expression of Aβ mutants in vivo using AAV We confirmed whether the peptides were expressed and secreted in vivo by injecting them into wild-type mice. All Aβ F19D / L34P and F20P expression constructs were packaged in AAV8 and injected into the lateral ventricles of neonatal (P0) mice. Mice were euthanized 3 to 4 weeks after injection to investigate the expression levels and distribution of the virus. Viral spread was assessed by immunostaining with a human-specific APP antibody (6E10) to detect mutant Aβ. Viral expression was biased to the frontal cortex, and subsequent analysis was focused on this region (Figure 3A). Co-immunostaining with 6E10 for mutant Aβ detection and Y188 for endogenous mouse APP detection confirmed that the virus-delivered precursor peptide was located at the cell membrane next to endogenous APP (Figure 3B).

[0280] To test for in vivo expression of the full-length peptide, we used IP followed by MS on homogenates from WT mice transduced with Aβ F20P. Both Aβ40 and Aβ-42 F20P from brain homogenates showed identical elution times as Aβ isolated from cell culture media; both peptides were again identified at the expected mass and sequence confirmed (Figure 3C, 3D; data for Aβ42 F20P are presented). Notably, partial cleavage products that may have been immunoprecipitated with 6E10 (e.g., intact TM domains without the signal peptide or γ-secretase-cleaved Aβ still attached to the signal peptide) were not shown in the spectrum.

[0281] The in vivo production of each mutant Aβ peptide was also measured by ELISA. The human-specific capture antibody and end-specific detection antibody in this assay ensured that the peptides measured were 1) human, and therefore virus-delivered, mutant Aβ, and 2) mature Aβ that had undergone γ-secretase cleavage. F20P was well expressed in vivo. Similar to native APP, this construct produced Aβx-40 peptide at levels many times greater than Aβx-42 (FIG. 3E). In contrast, F19D / L34P produced high levels of Aβx-42 but no detectable Aβx-40, indicating that in some embodiments, the L34P substitution abolishes end-specific Aβ40 antibody binding. These results suggest that in some embodiments, viral transduction with a minimal expression vector achieves cell membrane localization and γ-secretase cleavage to release mutant Aβ peptides into the brain.

[0282] D. Viral-delivered mutant peptides can reduce Aβ burden in APP / PS1 transgenic mice Viruses encoding Aβ F20P or F19D / L34P were injected into newborn (P0) APP / PS1 mice and non-transgenic littermates. Animals were harvested from this model at 7.5 months of age, shortly after amyloid deposition had begun. Cortical tissue from non-transgenic siblings was used to confirm that virally expressed Aβ was still produced at this age (Figure 3F). AAV-injected non-transgenic mice showed identical relative levels of Aβ40:42 at 3 weeks, with increased x-42 in both mutants and increased x-40 detected only in F20P (Figure 3G).

[0283] In APP / PS1 animals, plaque burden was assessed by Aβ immunostaining in the frontal cortex of one hemisphere and by Aβ ELISA in the other hemisphere. Compared to uninjected APP / PS1 control mice, mice treated with F20P showed nearly 75% reduction in Aβ deposition (Figure 4A, 4B). Consistent with the immunostaining, levels of insoluble Aβx-40 and Aβx-42 were also significantly reduced in mice treated with F20P (Figure 4C). The effect of F19D / L34P expression in APP / PS1 mice was more variable than F20P. Insoluble Aβx-40 levels were significantly reduced in APP / PS1 mice expressing F19D / L34P, but neither Aβx-42 nor plaque burden were significantly reduced (Figure 4B, 4C). Thus, although both F20P and F19D / L34P significantly reduced total Aβ accumulation, F20P was more effective at inhibiting aggregation in APP / PS1 mice. F20P- or F19D / L34P-treated non-transgenic animals did not accumulate detectable levels of human Aβ for 7.5 months after expression (data not shown; total Aβ40+42, soluble+insoluble, non-injected 100.7±45.5 (n=6); F19D / 34P 72.3±23.6 (n=11); F20P 158.2±35.2 pg / ml (n=10); ANOVA F(2,24)=2.02, p=0.15).

[0284] Since biophysical analysis suggested that at least one of the mutants, F19D / L34P, could not only prevent Aβ assembly but also promote the disassembly of existing Aβ fibrils in vitro (Figure 1C), we tested whether the mutants could assist in plaque clearance if administered after amyloid initiation. We achieved viral transduction and expression in the adult brain using newly evolved AAV serotypes such as PHP.eB, allowing for CNS transduction through peripheral injection. Injection of AAV PHP.eB was optimized using fluorescent proteins as a readout for transduction efficiency and spatial distribution throughout the brain (Figure 5A). The AAV PHP.eB serotype was found to be consistent with diffusion and approach the density achieved by neonatal AAV8 injection, suggesting that it delivers mutant Aβ at levels sufficient for use in vivo. Viral transduction also allowed us to test whether alternative routes of administration that may be used clinically could be matched with efficacy for neuronal transduction. Viral transduction of the choroid plexus has been used for gene therapy in mouse models of lysosomal storage diseases, AD and ALS. We found that ependymal cells of the choroid plexus can be easily transduced by AAV1, which is used in this application to test the transduction potential of mutant Aβ to the CSF (Figure 5B).

[0285] E. Expression of mutant Aβ reduces generalized neuroinflammation but can induce minor astrocytosis in APP / PS1 mice Amyloid plaques induce a pronounced neuroimmune response in which hypertrophic astrocytes and microglial cells migrate or divide to surround the core deposit. In most conditions, the degree of glial induction parallels the degree of amyloid load. Generally speaking, treatments that slow plaque accumulation also ameliorate these changes in glial morphology and localization. We therefore investigated whether the clustering of hypertrophic astrocytes and microglial cells, which typically defines the extent of core Aβ deposits, decreases with plaque load in mice treated with mutant Aβ. We focused our analysis on the F20P mutant because it has a more pronounced effect on plaque load than F19D / L34P. Both glial fibrillary protein (GFAP)-positive astrocytes and Iba1-positive microglial cells prominently outline amyloid plaques in the cortex of untreated APP / PS1 mice (Figure 5A, 5B). Indeed, the surface area of ​​both GFAP and Iba1 exceeds that of Aβ immunostaining in untreated transgenic mice (FIG. 5C). Lifelong treatment with virally delivered AβF20P significantly reduced staining for all of these markers, suggesting that in some embodiments, neuroinflammation decreases along with amyloid burden.

[0286] Increased cortical GFAP staining was also observed in virus-injected non-transgenic mice compared to uninjected controls (Figure 5A, 5C). GFAP-positive astrocytes were primarily restricted to a band adjacent to the corpus callosum, and the effect was specific for astrocytes: Iba1 levels in non-transgenic mice were not altered by virus exposure (Figure 5B, 5C). An identical pattern of pericallosal GFAP staining could be identified in F20P-treated APP / PS1 mice, but this region also contained plaque-associated astrocytosis that confounded quantification (Figure 5A).

[0287] Co-immunostaining for human Aβ (6E10) and GFAP was performed in 7.5 month old NTG mice. Some astrocytes expressing human Aβ were detected, but these co-labeled cells comprised only a small fraction of the GFAP+ population. These data suggest that in some embodiments, viral expression of mutant Aβ peptides may induce mild astrogliosis by itself, but the net effect of the amyloidosis model is to reduce the severity of chronic neuroinflammation commensurate with a reduction in plaque burden.

[0288] Example 2 - Deciphering the biophysical mechanism for the interaction between mutant and wild-type Aβ peptides The conformations adopted by the mutant peptides in solution, both in isolation and in the presence of wild-type Aβ, are determined to ensure that these properties are compatible with in vivo use.

[0289] A. Analysis of the structures adopted by Aβ peptides upon prolonged in vitro incubation. The structures formed by each mutant peptide will be assessed using four main types of analysis: circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), A11 / OC dot blot analysis, and transmission electron microscopy (TEM). Initial experiments will determine how the secondary and tertiary structures formed by the isolated F20P and F19D / L34P peptides change over time under various incubation conditions, and whether one of the two assembles in a quaternary structure indicative of aggregation. The secondary structure of each peptide will be monitored by far-infrared CD spectroscopy, and the tertiary / quaternary structure by near-ultraviolet spectroscopy, using spectral and thermal scans at various protein concentrations and solvent conditions. Kinetic CD spectroscopy measurements will complement equilibrium analysis to assess the time evolution of peptide folding and unfolding. Data collected by CD spectroscopy will be used to guide the timing of induction, algebraic growth, and plateau phases for each peptide, and samples will be removed for SEC to determine whether the monomeric peptides are converted to small oligomers and larger structures. Separate overdue samples are removed for A11 / OC dot blots to test for the formation of non-fibrillar and pre-fibrillar oligomeric states. A final set of samples is collected at steady-state reaction (>24 hours) for negative stain TEM imaging to confirm 1) the presence or absence and, where appropriate, periodicity and width of structured fibrils, 2) the appearance of smaller regular structures indicative of oligomers or protofibrils, or 3) irregular structures indicative of amorphous aggregates previously observed in proline-substituted mutants of amyloidogenic peptides.

[0290] B. Analysis of structures generated upon co-incubation of wild-type Aβ monomers with Aβ mutants. CD spectroscopy, SEC, A11 / OC dot blot and TEM are used to assess the structures formed by co-incubation of F20P or F19D / L34P with wild-type Aβ.

[0291] C. Percentage of mutant peptide required to prevent aggregation of wild-type Aβ42 and the minimum concentration required to disassemble pre-formed fibrils In some embodiments, equimolar mixtures of mutant Aβ peptides prevent wild-type Aβ42 aggregation, but lower mutant:wild-type ratios are sufficient. Most peptide inhibitors require stoichiometric or higher concentrations to prevent wild-type Aβ aggregation, but some candidates show low efficacy at inhibitor:wild-type ratios as low as 1:50. ThT assays are used to determine whether F20P or F19D / L34P can reduce or prevent wild-type Aβ42 fibrillization at substoichiometric concentrations up to 1:50 mutant:wild-type. Parallel experiments can be used to confirm the minimum concentration of mutant peptide required for disassembly of pre-formed Aβ42 fibrils. In some embodiments, as little as 5 μM F19D / L34P is sufficient to reduce ThT fluorescence of fibrils made from 10 μM WT Aβ starting material; in some embodiments, even lower concentrations of F19D / L34P achieve the same effect, and F20P may be equally efficient at disassembling fibrils.

[0292] D. Analysis of structures emerging upon degradation of wild-type Aβ fibrils by mutant Aβ peptides In some embodiments, wild-type Aβ fibrils lose ThT fluorescence over time upon addition of F19D / L34P peptide, suggesting that in some embodiments, this mutant enhances fibril disassembly. The structures generated by disassembly of wild-type Aβ fibrils are determined in the presence of mutant peptides. Wild-type Aβ42 fibrils are generated and exposed to 10 μM F20P or F19D / L34P for analysis by CD spectroscopy, SEC, dot blot and TEM as described herein. Parallel experiments are performed using the lowest concentrations of F20P or F19D / L34P that can significantly reduce ThT fluorescence in the above disassembly assay.

[0293] E. Effect of co-incubation of mutant peptides with wild-type Aβ on cytotoxicity The structures formed by the F20P and F19D / L34P mutants can reduce the cytotoxicity of wild-type Aβ42 aggregates. In some embodiments, co-incubation of wild-type Aβ42 with one of the mutants during oligomerization reduces subsequent cell death in N2a cultures. Studies are performed in cell lines and primary neuronal cultures, and the degradation products of Aβ fibrils by Aβ mutant peptides are tested in both the two cell systems. The reaction products for Aβ42 assembly and degradation are tested at equimolar and minimal effective peptide ratios of 0.1-10 μM for each mutant, and cell viability is measured by MTT assay.

[0294] Discussion These experiments provide a biophysical understanding of how mutant peptides prevent aggregation of wild-type Aβ and promote disassembly of pre-formed fibrils. CD spectra are evaluated for all of the equilibrium and kinetic reactions at different temperature, concentration, and solvent conditions to identify the physical structures that emerge upon interaction of wild-type and mutant peptides. In some embodiments, compared to wild-type Aβ, the isolated mutants remain in a predominantly random coil state, and co-incubation with wild-type reduces the appearance of structured oligomeric and fibril forms. In some embodiments, SEC analysis confirms the predominance of monomeric peptides versus oligomeric or fibril states for both the isolated Aβ mutants and for co-incubation with wild-type Aβ. In some embodiments, TEM analysis of end-stage incubations detects fibrils only from wild-type Aβ42, but not from the mutants alone or co-incubation with wild-type Aβ42.

[0295] Alternatively, in some embodiments, SEC analysis shows the appearance of oligomeric species with a decrease in monomers, but no substantial increase in larger species, and a shift in the CD spectra indicates a greater proportion of β-sheet structure over time, but not to the extent observed upon fibril formation. In some embodiments, ring-shaped or small fibrillar structures are observed by TEM, which may also react with A11 / OC. In some embodiments, such intermediates also occur after degradation of preformed fibrils. In some embodiments, these intermediates are less toxic than those formed by wild-type Aβ.

[0296] Example 3 - Determining the effect of dose, timing and route of mutant Aβ administration on efficacy in vivo

[0297] A. Analysis of lifelong mutant Aβ expression in APP mice In some embodiments, lifelong expression of mutant Aβ peptides has a substantial effect on plaque development in APP / PS1 mice. An additional group of APP / PS1 mice is treated with AAV-encoded scrambled Aβ peptide. This additional control ensures that the effects seen with treatment with mutant Aβ are due to the peptide and not a side effect of viral transduction. It is also determined whether expression of mutant Aβ affects APP expression or processing in vivo. In some embodiments, the amyloid-lowering mechanism of mutant Aβ occurs through binding of wild-type Aβ after release from cells, and full-length APP, sAPP and CTFs are measured by Western blot to ensure this is the case. Finally, in some embodiments, a second APP model, APP NL-F Knock-in mouse lines confirm the findings from APP / PS1 mice. Because KI mice develop plaques later than APP / PS1 mice, KI animals offer the opportunity to test whether long-term expression of mutant Aβ at levels higher than the endogenous peptide has unexpected effects on pathology or neuronal survival. NL-FBoth KI mice will be intracerebroventricularly (ICV) injected with mutant or scrambled AAV at P0 and harvested at 7.5 or 15 months for analysis. Outcome measures for this and subsequent experiments will be histological plaque burden (Aβ and thioflavin-S), human Aβ ELISA and human oligomeric Aβ ELISA (IBL, 82E11).

[0298] B. Ratio of mutant to wild-type Aβ required for efficacy (dosage) In some embodiments, an equimolar mixture of mutant and wild-type Aβ is sufficient to block aggregation of the wild-type peptide (FIG. 1B). Thus, in some embodiments, the effective in vivo dose for mutant Aβ is quite low. The in vivo dose-response relationship between mutant Aβ production and total Aβ accumulation was examined. The expression of the virus in vivo is mosaic in that virtually all cells are not transduced, but because the peptide is secreted, only a portion of the cells need to be transduced to expose the mutant peptide over a wide area. In some embodiments, the density of virus expression in the mouse brain is easily adjusted by controlling the titer of the injected virus. Here, we determine whether there is a threshold level of mutant Aβ required to slow plaque formation in vivo. ELISA is used to empirically determine the ratio of F20P mutant:wild-type Aβ using the levels of mutant Aβ at each titer in non-transgenic mice to compare with the levels of wild-type human Aβ measured in untreated young APP / PS1 mice. The dose-response relationships determined from this experiment are applicable to the APP / PS1 model, but in some embodiments may also provide valuable information regarding the relative exposure of mutant Aβ required for efficacy. APP / PS1 and non-transgenic mice are injected intracerebroventricularly with F20P AAV at P0 and harvested for analysis 7.5 months later. Virus dilutions are known from previous in vitro studies and range from approximately 0.05 to 1× the titers previously used.

[0299] C. Ventricular administration exerts a parenchymal delivery effect In some embodiments, mutant Aβ does not need to be expressed by neurons to inhibit the aggregation of wild-type Aβ in the extracellular space. In some embodiments, expression in any cell type with access to interstitial fluid is sufficient to deliver mutant Aβ widely throughout the brain. In some embodiments, the ventricular membrane lining of the lateral ventricles is ideal for delivering secreted proteins to the brain through the CSF. The sufficiency of delivery of mutant Aβ to the CSF to reduce Aβ aggregation through the cortex, as observed with neuronal secretion, is tested. APP / PS1 and non-transgenic animals are injected with AAV1-encoded F20P or scrambled Aβ to transduce the choroid plexus. Injections are performed between P3 and P7, after the ventricular membrane-brain barrier has matured. Non-transgenic animals are used to measure the concentration of mutant Aβ reaching the cortex after ependymal transduction, to compare with that achieved by neuronal transduction. APP / PS1 mice are harvested at 7.5 months for analysis.

[0300] D. Mutant Aβ maintains advantage when introduced after plaque initiation In vitro studies show that, in some embodiments, mutant Aβ promotes degradation when introduced after fibril formation. The potential of mutant Aβ to clear pre-existing aggregates in the brain is examined. This experiment utilizes the AAV serotype PHP.eB to broadly transduce cells in the adult brain. APP / PS1 and non-transgenic siblings are injected retro-orbitally at 7.5 months of age with PHP.eB virus carrying F20P mutant Aβ or a scrambled peptide. The human synapsin promoter is used for neuron-specific expression; viral titers for injection are 1x10 11 〜1×10 12Empirically matched for each preparation within the posted range of gc / mouse. Non-transgenic animals are used to measure concentrations of mutant Aβ in the cortex after adult injection to compare with those achieved by treatment at P0. Animals are treated and then harvested at 3 and 6 months for analysis and compared to mice harvested without treatment at 7.5 months of age.

[0301] Discussion This experiment examines how dose, timing and route of administration affect the in vivo efficacy of mutant Aβ. In some embodiments, expression of F20P, and to a lesser extent, F19D / L34P, reduces plaque burden and Aβ burden in an expanded cohort of APP / PS1 mice compared to non-injected APP / PS1 mice and all newly added controls expressing scrambled Aβ peptide. In some embodiments, the same effects are seen in APP NL-F These later onsets, observed in APP / PS1 mice, meanwhile, overexpress mutant Aβ for a longer period before recovery, producing more mutant Aβ than non-transgenic mice.

[0302] In some embodiments, there is a minimum ratio of mutant to wild-type Aβ required to slow plaque formation in vivo, which is roughly similar to the minimum ratio required for in vitro efficacy. Above this ratio, in some embodiments, a graded degree of plaque reduction is observed, albeit with a less well-defined dose-response relationship than in vitro. The concentration of mutant Aβ reaching the cortex through the CSF using non-transgenic animals is compared to the concentration achieved by neuronal expression to determine whether CSF delivery achieves cortical levels of mutant Aβ that are equal to or exceed the minimum effective dose for neuronal expression. If not, delivery is improved using alternative serotypes for ventricular expression (i.e., AAV4) or alternative promoters active in the choroid plexus (i.e., Prlr, Spint2, F5).

[0303] These experiments will be expanded into cognitive testing, synaptic analysis and electrophysiology in subsequent studies to establish a fundamental understanding of Aβ species from exposure to mutant peptides, identify optimal therapeutic regimens to mitigate or reverse Aβ accumulation in vivo and determine whether unintended neuroinflammation can interfere with physiological measurements.

[0304] Example 4 - Interrogation of the neuroimmune reaction to variant Aβ as a possible accomplice to plaque reduction In some embodiments, neonatal viral transduction induces a subtle, cell-specific neuroimmune response in the brain, but analysis is limited to morphological markers and does not show any underlying molecular changes. RNA profiling is used to evaluate a broad range of neuroimmune markers in the context of amyloid pathology or remodeling due to viral expression alone. The evolution of the neuroimmune profile with age is examined to determine whether transduction of adult brains induces a more pronounced response than was initially observed in mice injected as neonates. Histological analysis of astrocytes and microglial cells is completed to determine how plaque burden, viral exposure, and cellular response interact after treatment with mutant Aβ post-intervention at various stages of disease. Effects in some embodiments are due to astrocyte reactivity, whereas effects in other embodiments are due to biophysical effects of mutant Aβ versus wild-type peptide. In some embodiments, the biophysical effects overcome the astrocyte response. Empirical testing is performed to establish the utility of gene therapy as a platform for autoinhibition in Alzheimer's disease and other protein aggregation diseases.

[0305] A. Upregulation of transcriptional markers of neuroinflammation upon viral delivery of mutant Aβ and the effect of age at viral delivery on the response. In this experiment, we address whether introduction of AAV expressing a non-native protein induces a neuroinflammatory response in the brain. We initially focus on non-transgenic animals to avoid the confounding effect of plaque-associated gliomas in APP / PS1 animals. Animals for this and subsequent experiments are generated as in Example 3: a small portion of frontal cortex (approximately 20-40 mg) is collected from one hemisphere of each animal for total RNA extraction, and the remaining cortex is reserved for ELISA and Western studies as in Example 3. The contralateral hemisphere is fixed for histological studies. Here, the transcriptional profile of non-transgenic control mice is compared to siblings treated with AAV-F20P or scrambled peptide at P0 or 7.5 months. Non-transgenic animals injected at P0 and harvested at 15 months are also included to evaluate the impact of extended viral expression on the neuroinflammatory response. Transcription is profiled using the Nanostring nCounter Neuroinflammation Panel for Mouse, which covers 756 genes spanning a broad range of potential neuroimmune responses. Unlike RNA sequencing, this platform directly digitally measures mRNA molecules for linear quantification without template amplification. Expression data is analyzed using nSolver software to compare gene expression between animals treated with F20P or scrambled peptide for each treatment / recovery age combination and uninjected controls. Second-level analyses are acquired from the Baylor College of Medicine Multi-Omics Data Analysis Core for pathway and gene set enrichment. RNA sequencing is also available, providing a broader readout for potential alterations.

[0306] B. Effects of virally transmitted Aβ mutants on neuroinflammatory responses We addressed whether delivery of AAV and its non-natural peptide cargo alters the neuroinflammatory response to amyloid deposits. NL-FCortical transcriptional profiles of mice were analyzed using uninjected APP / PS1 and APP NL-F Compare with Sibling. Nanostring N-Counter Neuroinflammation Panel is used for expression profiling of neuroimmune responses in APP models.

[0307] C. Diverse morphologies and distributions of neuroinflammatory cells upon viral expression of mutant Aβ. Examining the cellular profile of the neuroimmune response to viral delivery of mutant Aβ. Immunofluorescence was performed on non-transgenic, APP / PS1 and APP NL-F It was used to detect microglial cells (Iba1 as a pan-microglial marker, P2RY12 for homeostatic microglial cells, CD68 for phagocytic microglial cells, and MHCII for inflammatory microglial cells) and astrocytes (Aldh1l1 as a pan-astrocytic marker, GFAP preferential for reactive astrocytes) in mice. Mice were harvested after injection of F20P or scrambled peptide at P0 or 7.5 months for comparison with uninjected mice. Outcome measurements included qualitative morphology and quantitative cell number and spatial distribution of cortical neuroimmune cells per unit area in association with amyloid plaques in non-transgenic animals or in the APP model.

[0308] Discussion This work investigates the neuroinflammatory response to viral delivery of non-native proteins by transcriptional and cellular markers. These experiments also examine whether the reduction in amyloid observed in pilot studies is due to a neuroinflammatory response to treatment. In some embodiments, mild astrocytosis may accompany viral delivery in non-transgenic mice, but both astrocytosis and microgliosis are reduced in proportion to plaque burden in F20P-treated APP / PS1 animals. In some embodiments, these molecular and histological experiments allow resolution of such discrepancies and support a mechanism based on Aβ steric inhibition rather than neuroinflammatory phagocytosis.

[0309] In some embodiments, if mRNA or histological profiles suggest elevated inflammation in animals treated with mutant Aβ, the profiles of mice treated via neuronal transduction are compared to those of mice treated via CSF. Since CSF delivery limits viral exposure but not the spread of mutant Aβ, comparison with neuronal expression must pinpoint whether the virus or the peptide is responsible. An alternative in vitro study directly tests microglial / astrocyte responses to mutant Aβ peptides using purified primary cell cultures and recombinant proteins. To exclude the role of neuroinflammation in plaque reduction, treatment efficacy in the presence and absence of astrocytes / microglia is also compared.

[0310] Finally, in some embodiments, the mRNA panel indicates that additional cell types, such as circulating macrophages, T- or B-cells, are involved in any neuroinflammatory response, and FACS sorting of brain tissue can be used to distinguish the cellular components and extent of neuroinvasion. The pathway of delivery (i.e., ventricular rather than neuronal) or capsid (clinically used AAV9 vs. experimentally used AAV8 / PHP.eB) may be altered for future studies to mitigate any treatment-associated neuroinflammation.

[0311] In some embodiments, if the mRNA marker panel or histological staining confirms an increased response in virus-treated animals, additional experiments are optionally performed to map the time course of neuroinflammatory changes following P0 or adult viral transduction.

[0312] Example 5 - Mutant Aβ blunts seeding of AD aggregation. We tested whether gene therapy with mutant Aβ could reduce the potential for pathogenic protein seeds to accelerate disease progression in vivo. These experiments provide an opportunity to test whether mutant Aβ can be restricted to prevent the formation of seeds from monomers or even inhibit the growth of existing seeds. Furthermore, the experiments test pathogenic seeds in human Alzheimer's disease tissue, which represents a more diverse and realistic challenge than genetic models alone. The first experiment examines whether preventative expression of mutant Aβ can blunt the formation of plaques that develop upon inoculation with human Alzheimer's disease extracts enriched for Aβ seeds. The final experiment tests the potential of Aβ reduction to attenuate secondary seeding of tau in amyloid-bearing mice.

[0313] A. Mechanism of effect of mutant Aβ on the rate and extent of amyloid seeding in pre-deposited mice We test whether lifelong expression of mutant Aβ can be induced by inoculation of exogenous Alzheimer's disease brain extracts to alleviate accelerated amyloidosis. We produce Aβ extracts for in vivo seeding from two pathologically confirmed Alzheimer's disease subjects and one age-matched healthy control. We acquire frozen frontal cortex samples. Protein extracts for injection are homogenized in PBS and sonicated for clarification. Extracts are characterized by A11 / OC dot blot for oligomeric Aβ, ELISA for total Aβ concentration, and assay of protein misfolding circulating amplification (PMCA) for seeding amount. At birth, APP / PS1 mice were administered AAVs encoding F20P mutant or scrambled Aβ, and 1 month later, brain extracts from Alzheimer's disease or healthy controls were bilaterally injected into the hippocampus and upper cortex. Animals are harvested 3-5 months later, prior to the normal initiation of plaques in this model, for Aβ immunohistochemistry and ELISA to assess the severity and spread of Aβ aggregation in animals expressing F20P vs. scramble.

[0314] B. Mutant Aβ affects seeded tau pathology in amyloid-bearing mice. The final experiment will test whether the presence of mutant Aβ can prevent or blunt the formation of tau neuropathology in amyloid-containing mice. Tau extracts for injection are produced from cortical grey matter by differential centrifugation of sarkosyl-insoluble brain homogenates as originally described by the Lee group. Extracts are characterized by PHF1 Western blot and ELISA for total tau concentration and tested for seeding potential using tau RD FRET biosensor cells (available from ATCC). Once the Alzheimer's-tau extracts perform as expected in vitro, in vivo experiments are performed by administering AAV encoding F20P or scrambled Aβ to neonatal APP / PS1 mice. After 12 months, Alzheimer's (AD)-tau extracts are injected bilaterally into the hippocampus and upper cortex to ensure sufficient amyloid burden. Animals are harvested after 3 or 6 months for tau immunohistochemistry (AT8, AT180, MC1) and amyloid immunostaining and histology (thioflavin-S) to measure the spread and total area of ​​phospho-tau immunostaining relative to amyloid burden in animals expressing F20P versus scramble.

[0315] Discussion In some embodiments, lifelong expression of mutant Aβ may reduce amyloid formation in APP / PS1 mice and reduce the seeding of plaques and phospho-tau neurites by Alzheimer's disease extracts. In some embodiments, mutant Aβ limits the expansion of injected AD-Aβ seeds as well as newly generated seeds. Similarly, in some embodiments, mutant Aβ reduces the appearance of phospho-tau neurites by attenuating the formation of amyloid plaques necessary to promote secondary seeding of tau. Alternatively, in some embodiments, mutant Aβ is unable to attenuate seeding by exogenous aggregates, suggesting that in some embodiments, mutant Aβ is sufficient to slow seed formation in APP mice, but once present, it may not be able to prevent seed growth or interact with seeds present in AD extracts; this is unlikely due to the predicted mechanism of steric hindrance during fibril expansion, which must occur regardless of the starting material.

[0316] These experiments provide an important foundation for future studies to test how the timing of treatment relative to inoculation can control efficacy. Tests will be conducted to determine whether mutant Aβ can interfere with seeding if it is delivered after plaque initiation. Over time, the effects of dose and route of administration on the progression of amyloid to tau will be examined.

[0317] Example 6 - Exemplary Methods Preparation of WT Aβ42 or mutant peptide stocks. Synthetic Aβ42 WT or mutant peptides were purchased from Biomatik (Wilmington, DE). To prepare aggregate-free Aβ42 WT or mutant peptide stock solutions, powdered peptides were dissolved in 50% acetonitrile, frozen, and lyophilized overnight to remove residual trifluoroacetic acid. Lyophilized Aβ42 peptides were dissolved in HFIP (#105228, Sigma-Aldrich, St. Louis, MO). The Aβ-HFIP solution was incubated at room temperature (RT) for 30 min before being divided into aliquots. HFIP was allowed to evaporate overnight in a fume hood and then transferred to a SpeedVac for 1 h to remove any remaining traces of HFIP. Tubes containing peptide films were stored over desiccant at -20 °C until use. Immediately prior to use in an experiment, lyophilized peptides were dissolved in DMSO to a final concentration of 5 mM and sonicated in a water bath sonicator for 10 min.

[0318] Preparation of oligomeric and fibrillar WT Aβ. Oligomeric Aβ was generated by dissolving peptides in Ham's F-12 medium (#30611040-1, Fisher Scientific, Pittsburgh, PA) at a final concentration of 100 μM WT Aβ42 peptide or 100 μM WT + 100 μM mutants and incubating for 24 h at 4°C without shaking. Fibrillar Aβ was generated by dissolving WT peptide in PBS at a final concentration of 100 μM and incubating for 24 h at 37°C without shaking.

[0319] ThT assay to test the kinetics of Aβ self-aggregation, competition with WT peptide and fibril disassembly. Self-aggregation of Aβ42 WT or mutant monomers was tested in PBS containing 5 μM thioflavin T (ThT) at a starting concentration of 10 μM. ThT fluorescence was measured using an Infinite M1000 Pro Plate Reader (Tecan, Mannedorf, Switzerland) at an excitation wavelength of 440 nm and an emission wavelength of 485 nm. Reactions were incubated at 37 °C without shaking and then shaken for 5 s before reading fluorescence. Competition between mutant and WT Aβ was performed similarly and tested at a final concentration of 10 μM using a starting concentration of 10 μM for each peptide in the mixture (1:1, WT:mutant) except for WT alone. Fibril disassembly was assessed by mixing 10 μl of WT Aβ42 fibrils (described above) 1:1 with 5, 10, or 20 μM monomeric F19D / L34P or F20P peptides in PBS containing 5 μM ThT. Fluorescence was measured every 24 h without shaking for 48 h at 37 °C.

[0320] Oligomeric Aβ toxicity assay. N2a cells were incubated at 1 × 10 4 The cells were cultured in EMEM (Eagle's minimum essential medium, #112-018-101, VWR, Radnor, PA) medium supplemented with U / ml penicillin / streptomycin (#15140-122, Life Technologies, Carlsbad, CA) and 10% fetal bovine serum (#MT35010CV, Fisher) at 37°C in 5% CO 2 Confluent cells were trypsinized and diluted with 1% N to minimize cell growth. 2After dilution in EMEM containing supplements, 5,000 cells were plated per well in a clear flat-bottom 96-well plate (#07-200-89, Fisher). 10 μl of each oligomer formulation (WT Aβ alone or WT+ mutants, as described above) or 10 μl of a 1:10 dilution was added to 90 μl of culture medium. Cell viability was measured 24 hours after treatment using the MTS assay (#G3582, Promega, Madison, WI) according to the manufacturer's instructions. Briefly, 20 μl of AQueous One Solution Reagent was added directly to the culture wells and incubated at 37°C, 5% CO 2 After incubation at ambient for 2 hours, assays were performed by recording absorbance at 490 nm using an Epoch 2 spectrophotometer (Biotek, Winooski, VT).

[0321] Plasmid constructs. PCR was used to add the Gaussia luciferase signal peptide (GLSP, amino acids MGVKVLFALICIAVAEA, corresponding to SEQ ID NO: 20) to the N-terminus of Aβ-CTF. First, synthetic DNA for GLSP with partial Aβ sequence was prepared using oligo GLSP-1 5'ATGGGCGTGAAGGTCCTGTTCGCCCTGATTTGCATCGCCGTCGCAGAGGCAGATGCAGA 3' (SEQ ID NO: 40) and oligo GLSP-2 5'TCTGCATCTGCCTCTGCGACGGCGATGCAAATCAGGGCGAACAGGACCTTCACGCCCAT 3' (SEQ ID NO: 41). The oligonucleotides were annealed by incubation in a thermal cycler programmed to start at 95°C for 2 minutes, followed by gradual cooling to 25°C. Second, a plasmid containing the human APP wild-type sequence (pBS-hAPPwt-IRES-GFP) was amplified with forward primer 5′GTCGCAGAGGCAGATGCAGAATTCCGACATGAC 3′ (SEQ ID NO: 42) and reverse primer 5′GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG 3′ (SEQ ID NO: 43) to generate an Aβ-CTF with a partial GLSP sequence. Then, the GLSP-Aβ-CTF was generated by Gibson assembly combining the Aβ-CTF + partial GLSP to the GLSP + partial Aβ using the forward primer to add the Kozak sequence 5′GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 44) and reverse primer 5′GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG 3′ (SEQ ID NO: 45) from the two templates. The generated GLSP-Aβ-CTF fragment was cut with HindIII and EcoRV and subcloned into pAAV containing a CAG promoter and a WPRE to generate pAAV-GLSP-Aβ-CTF.

[0322] The GLSP-Aβ-CTF deletion series was generated by cloning various GLSP-Aβ-CTF deletions into pAAV, which were amplified by PCR from full-length pAAV-GLSP-Aβ-CTF using a set of reverse primers and a common forward primer, then cut with HindIII and EcoRV and ligated into pAAV.

[0323] Forward primer: Aβ-GLSP-F (KOZAK): 5′ GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 46).

[0324] Reverse primer: Aβ-R(KK): 5′ GCGCGgatatcTTACTACTTCTTCAGCATCACCAAGGTG 3′ (SEQ ID NO: 47); Aβ-R(ML): 5′ GCGCGgatatcTTACTACAGCATCACCAAGGTGATGA 3′ (SEQ ID NO: 48); Aβ-R(LV): 5′ GCGCGgatatcTTACTACACCAAGGTGATGACGATCA 3′ (SEQ ID NO: 49); Aβ-R(IT): 5′ GCGCGgatatcTTACTAGGTGATGACGATCACTGTCG 3′ (SEQ ID NO: 50); Aβ-R(IV): 5′ GCGCGgatatcTTACTAGACGATCACTGTCGCTATGA 3′ (SEQ ID NO: 51); Aβ-R(IA): 5′ GCGCGgatatcTTACTACGCTATGACAACACCGCCCA 3′ (SEQ ID NO:52);

[0325] To construct pAAV-GLSP-Aβ(F19D / L34P)-KK containing the F19D / L34P substitution, two PCR reactions were performed. The GLSP-Aβ(F19D / L34P)-KK fragment was amplified from pAAV-GLSP-Aβ-KK using the forward primer 5′ GCGCGaagcttGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 53) and the reverse primer 5′ ACCATGGGTCCAATGATTGCACCTTTGTTTGAACCCACATCTTCTGCAAAGTCCACCAA 3′ (SEQ ID NO: 54). The second fragment of GLSP-Aβ(F19D / L34P)-KK was amplified using forward primer 5′ TTGGTGGACTTTGCAGAAGATGTGGGTTCAAACAAAGGTGCAATCATTGGACCCATGGT 3′ (SEQ ID NO: 55) and reverse primer 5′ GCGCGGATATCTTACTACTTCTTCAGCATCACCAAGGTG 3′ (SEQ ID NO: 56). The resulting fragments were joined by Gibson assembly using forward primer 5′ GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 57) and reverse primer 5′ GCGCGGATATCTTACTACTTCTTCAGCATCACCAAGGTG 3′ (SEQ ID NO: 58). The resulting insert was cut with HindIII and EcoRV and subcloned into pAAV to generate pAAV-GLSP-Aβ(F19D / L34P)-KK.

[0326] Two PCR reactions were performed on pAAV-GLSP-Aβ(F20P)-KK containing the F20P substitution. A fragment of GLSP-Aβ(F20P)-KK was amplified from pAAV-GLSP-Aβ-KK using forward primer 5′ GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 59) and reverse primer 5′ TCTTCTGCAGGGAACACCAATTTTTG 3′ (SEQ ID NO: 60). A second fragment of GLSP-Aβ(F20P)-KK was amplified using forward primer 5′ CAAAAATTGGTGTTCCCTGCAGAAGA 3′ (SEQ ID NO: 61) and reverse primer 5′ GCGCGGATATCTTACTACTTCTTCAGCATCACCAAGGTG 3′ (SEQ ID NO: 62). The resulting fragments were joined by Gibson assembly using the forward primer 5′ GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 63) and the reverse primer 5′ GCGCGGATATCTTACTACTTCTTCAGCATCACCAAGGTG 3′ (SEQ ID NO: 64). The resulting insert was digested with HindIII and EcoRV and subcloned into pAAV to generate pAAV-GLSP-Aβ(F20P)-KK. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).

[0327] Harvesting of Aβ peptides in N2a conditioned medium. N2a cells were cultured at 1 × 10 4Cells were grown in 6-well plates until approximately 90% confluent using DMEM (Dulbecco's modified Eagle's medium, #12-604F, VWR) supplemented with U / ml penicillin / streptomycin and 10% fetal bovine serum. Cells were subsequently transfected with 2.5 μg / well of sequentially deleted APP-CTF sequences (e.g., pAAV-GLSP-Aβwt-KK) using Lipofectamine LTX (#15338030, Fisher) or 14 μg / dish (10 cm) of pAAV-GLSP-Aβ(F20P)-KK. After 24 h, cells were washed with 1× Dulbecco's PBS and serum-reduced medium composed of DMEM and 0.2% fetal bovine serum was added, after which the cells were incubated at 37°C in 5% CO. 2 Cells were then allowed to recover at 4°C for 24 h. Conditioned medium was collected after 24 h and centrifuged at 10,000 rpm for 5 min at 4°C (2 ml / well x 3 wells per IP reaction). To test the dependence of Aβ release on γ-secretase activity, cells were transfected as above and subsequently medium was replaced with 0.1 or 1 nM LY411575 (#SML0506, Sigma). Supernatants were collected and eluted with protease inhibitor (#5892970001, Sigma) and 0.01% NaN 3 (#S2002, Sigma) to prevent proteolysis.

[0328] Aβ IP of conditioned medium for immunoblotting. Protein G Dynabeads (50 μl, #10003D, Fisher) were loaded with mouse anti-Aβ antibody 6E10 (1 μg, #SIG-39320, BioLegend, San Diego, CA) for 1 h at room temperature. Conditioned medium of transfected N2a cells (6 ml) was incubated with 50 μl of Dynabead-6E10 complexes on a rotator overnight at 4°C. The next day, beads were collected using magnetic separation and the supernatant was removed. Beads were washed three times with PBS containing 0.02% Tween-20. Immunoprecipitated complexes were eluted with 50 mM glycine pH 2.8. After this, samples were denatured with an equal volume of 2× Laemmli sample buffer at 70°C for 10 min and electrophoresed on a 16.5% Criterion Tris-Tricine gel (#3450063, Bio-Rad, Hercules, CA). Proteins were transferred to nitrocellulose using a Trans-Blot Turbo Transfer System (#170-4159, Bio-Rad). Membranes were blocked with PBS containing 0.1% Tween-20 and 5% nonfat dry milk for 1 h at room temperature and probed overnight at 4°C with 6E10 diluted 1:5000 in blocking solution. Antibody binding was detected with a mouse anti-IgG secondary antibody conjugated with IRDye (#26-32210, LI-COR, Lincoln, NE) diluted 1:20,000 in block. Blots were imaged with an Odyssey Fc Imager and analyzed with Image Studio software (LI-COR).

[0329] Aβ IP of conditioned medium for MS. Approximately 60 mL of N2a conditioned medium containing Aβ F20P was incubated with 10 μg mouse anti-Aβ antibody 6E10 (#SIG-39320; BioLegend, San Diego, CA, USA), split into two batches of 30 mL each, and incubated overnight at 4 °C on a rotator. The next day, Protein G Dynabeads (100 μL; #10003D; Fisher Scientific) were added to the medium and incubated for 2 h at 4 °C. To remove the supernatant, the beads were collected by magnetic separation and then washed three times with PBS containing 0.02% Tween-20, followed by two times with PBS without detergent. Finally, the immunoprecipitated complexes were eluted with 80 μL of 10% formic acid solution for MS analysis.

[0330] MS. Brain homogenates and immunoprecipitates eluted with culture medium expressing Aβ F20P were dried in a SpeedVac and resuspended in 50 μL of 20% formic acid. 1 μL of the IP reaction was loaded onto a 10 cm, 100 μm inner diameter C3 column (ZORBAX 300SB-C3; 300 Å 5 μm), self-packed in fused silica, and pulled to form a nanoelectrospray ejector. Online high performance LC (HPLC) was performed on a Thermo Scientific U3000 RSLCnano ProFlow system. A 50 min linear gradient from 0% buffer B to 35% buffer B was run with Buffer A: 2% acetonitrile, 0.1% formic acid and Buffer B: 98% acetonitrile and 0.1% formic acid. The column eluent was introduced into a Thermo Scientific Orbitrap Fusion Lumos by nanoelectrospray ionization. A static spray voltage of 2,200 V and an ion delivery tube temperature of 320° C. were set for the source.

[0331] MS1 was performed with an Orbitrap at a resolution setting of 60k in positive mode with quadrupole isolation. An automatic gain control (AGC) target of 5.0e6 with a maximum injection time of 200ms, two microscans and a scan range of 350-2,000 mass-to-charge ratio (m / z) were used. Target precursor m / z selected for MS2 fragmentation included monoisotopic and most abundant masses for +4, +5 and +6 Aβ F20P 38, 40 and 42 amino acid unmodified ions. Higher energy collisional dissociation (HCD) fragmentation with a normalized collision energy of 45% was used. MS2 acquisition was performed with an Orbitrap using a resolution setting of 15k, an AGC target of 1e6, a maximum injection time of 100ms, a scan range of 150-2,000 m / z and three microscans. Next, α-secretase cleaved Aβ 1-16 masses were specifically targeted for MS analysis along with the uncleaved and partially cleaved precursors (with or without the signal peptide and remaining TM domain) to improve sensitivity for these targets. The parameters were replicated with a target mass list containing such alternatively cleaved peptides.

[0332] Data processing was performed by a custom analysis suite as described above. Aβ F20P 38, 40 and 42 peptides, α-secretase-cleaved Aβ 1-16 along with uncleaved and partially cleaved precursor peptides (with or without signal peptide and residual TM domain) were used as t...

Claims

1. 1. A pharmaceutical composition for the prevention or treatment of a neurodegenerative disease, disorder or condition comprising a vector encoding an Aβ peptide variant, wherein the Aβ peptide variant comprises an amino acid sequence having at least 80% identity to: or a fragment or functional derivative thereof.

2. 2. The pharmaceutical composition of claim 1, wherein the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:

3.

3. 2. The pharmaceutical composition of claim 1, wherein the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:

4.

4. 2. The pharmaceutical composition of claim 1, wherein the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:

5.

5. 2. The pharmaceutical composition of claim 1, wherein the Aβ peptide variant comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:

6.

6. 2. The pharmaceutical composition of claim 1, wherein the vector encoding the Aβ peptide mutant encodes a nucleotide sequence corresponding to an amino acid sequence comprising a truncated beta-carboxyl-terminal fragment (β-CTF) of amyloid precursor protein.

7. 2. The pharmaceutical composition of claim 1, wherein the truncated β-CTF is fused to a signal peptide sequence, the signal peptide sequence comprising a nucleotide sequence corresponding to an amino acid sequence comprising a Gaussia luciferase signal peptide or a nucleotide sequence corresponding to an amino acid sequence comprising a mouse immunoglobulin heavy chain signal peptide.

8. 7. The pharmaceutical composition of claim 6, wherein the truncated β-CTF comprises an Aβ peptide variant sequence, a transmembrane domain sequence and a cytoplasmic sequence.

9. 9. The pharmaceutical composition of claim 8, wherein the cytoplasmic sequence comprises a nucleotide sequence corresponding to an amino acid sequence for membrane anchoring, facilitating gamma-secretase cleavage, and / or extracellular release of the Aβ peptide variant.

10. The pharmaceutical composition of claim 1, wherein the vector comprises an amino acid sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to any one selected from the group consisting of SEQ ID NO:8, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:

80.

11. The pharmaceutical composition of claim 1, wherein expression of the vector is regulated by a constitutive promoter, a cell-specific promoter or a tissue-specific promoter.

12. 2. The pharmaceutical composition of claim 1, wherein the vector is an adenovirus, lentivirus, retrovirus or adeno-associated virus vector.

13. The pharmaceutical composition of claim 1 , wherein the vector is an AAV vector.

14. 2. The pharmaceutical composition of claim 1, wherein the vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAvDJ, AAVrhlO.XX, AAVrh.8, AAVrh.10, AAVrh.43, AAVpi.2, AAVhu.11, AAVhu.32, AAVhu.37, or PHP.eB AAV.

15. The pharmaceutical composition of claim 1, wherein the composition prevents or reduces protein misfolding, endogenous Aβ peptide aggregation, amyloid plaque formation, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; reduces tau levels, tau phosphorylation or phosphorylated tau levels; slows tau seeding or endogenous Aβ peptide seeding; or promotes cognitive improvement.

16. The pharmaceutical composition of claim 1, which prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils or plaques.

17. The pharmaceutical composition of claim 1, which prevents or reduces the cytotoxicity of endogenous Aβ peptide aggregates.

18. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease, disorder or condition is one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Parkinson's disease dementia, vascular dementia, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, Down's syndrome, and pathological aging.

19. 10. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharma- ceutically acceptable carrier.

20. 20. The pharmaceutical composition of claim 19, wherein the pharma- ceutically acceptable carrier comprises a liposome, a polymeric micelle, a microsphere or a nanoparticle.

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