Transfer of Aβ mutants to suppress 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 No. 63 / 127,815, filed on 18 December 2020, the disclosures of which are included in their entirety by reference.

[0002] This invention was made possible with government support granted by NS092615, AG054160, AG056028, and AG058188 by the National Institutes of Health. The government has specific rights to this invention.

[0003] The aspects of this disclosure relate at least to 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. Pathological features of Alzheimer's disease include the presence of neurofibrillary tangles and amyloid deposits in the patient's brain. Beta-amyloid peptides (Aβ) and their aggregated forms are the main components of amyloid plaques. Aβ1-42 are particularly prone to aggregation, forming oligomers, protofibrils, insoluble fibrils, and plaques. Engineered antibody therapy is effective in reducing Aβ aggregation; however, the side effect profile and the requirement for repeated intravenous transmission hinder the widespread use of engineered antibodies for the treatment of neurodegenerative diseases such as Alzheimer's disease. Therefore, there is a need in the industry for novel therapeutic approaches with high specificity, low toxicity, and extended in vivo half-lives that can prevent the oligomerization and aggregation of Aβ peptides. [Overview of the project] [Problems that the invention aims to solve]

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

[0006] The embodiments of this disclosure include methods and compositions for treating subjects having neurodegenerative diseases, disorders or conditions, and / or for inhibiting the aggregation of Aβ peptides in vivo. The methods of this disclosure may include one, two, three, four, or five or more of the following steps: providing a subject with an Aβ peptide variant; providing the subject with one or more additional therapies for neurodegenerative diseases, disorders or conditions; diagnosing a subject having a neurodegenerative disease, disorder or condition; diagnosing a subject having symptoms of a neurodegenerative disease, disorder or condition; and diagnosing a subject at risk of having a neurodegenerative disease, disorder or condition. Specific embodiments of this disclosure may exclude one or more of the prior art elements and / or steps.

[0007] In some embodiments, the present invention discloses a method for treating or preventing a neurodegenerative disease, disorder, or condition from a subject, comprising the step of 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, the step of 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 down tau seeding or endogenous Aβ peptide seeding or promotes cognitive improvement. In some embodiments, the step of administering a vector encoding an Aβ peptide variant prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils or plaques. In some implementations, the step of administering a vector encoding an Aβ peptide variant prevents or reduces the cytotoxicity of the endogenous Aβ peptide aggregate.

[0008] In some aspects, the present invention describes a method for inhibiting the aggregation of endogenous Aβ peptides in vivo, comprising the step of contacting at least one endogenous Aβ peptide in vivo with an Aβ peptide variant expressed in a therapeutically effective amount from a vector encoding the Aβ peptide variant (the vector being present in the composition). In some embodiments, the method for inhibiting the aggregation of endogenous Aβ peptides treats or prevents neurodegenerative diseases, disorders, or pathological conditions in a subject organism. In some embodiments, the method for inhibiting the aggregation of endogenous Aβ peptides prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils, or plaques. In some embodiments, the method for inhibiting the aggregation of endogenous Aβ peptides prevents or reduces the cytotoxicity of endogenous Aβ peptide aggregates.

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

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

[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 contains an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 3, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant contains an N-terminal cleavage that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 3, or contains SEQ ID NO: 3. In some embodiments, the N-terminal cleavage contains 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 cleavage. In some embodiments, an Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 3, or containing SEQ ID NO: 3, includes a C-terminal cleavage. In some embodiments, the C-terminal cleavage includes 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 cleavages. In some embodiments, a fragment or functional derivative of SEQ ID NO: 3 contains 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 with KLVDFAE (SEQ ID NO: 10).

[0013] In some embodiments, the Aβ peptide variant contains an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 4, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant contains an N-terminal cleavage that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 4, or that contains SEQ ID NO: 4. In some embodiments, the N-terminal cleavage contains 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 cleavage. In some embodiments, an Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 4, or containing SEQ ID NO: 4, includes a C-terminal cleavage. In some embodiments, the C-terminal cleavage includes 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 cleavages. In some embodiments, a fragment or functional derivative of SEQ ID NO: 4 contains 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 with KLVFPAE (SEQ ID NO: 11).

[0014] In some embodiments, the Aβ peptide variant contains an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 5, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant contains an N-terminal cleavage that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 5, or that contains SEQ ID NO: 5. In some embodiments, the N-terminal cleavage contains 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 cleavage. In some embodiments, an Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 5, or containing SEQ ID NO: 5, includes a C-terminal cleavage. In some embodiments, the C-terminal cleavage includes 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 cleavages. In some embodiments, a fragment or functional derivative of SEQ ID NO: 5 contains 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 with KLVDFAE (SEQ ID NO: 10).

[0015] In some embodiments, the Aβ peptide variant contains an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 6, or a fragment or functional derivative thereof. In some embodiments, the Aβ peptide variant contains an N-terminal cleavage that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 6, or that contains SEQ ID NO: 6. In some embodiments, the N-terminal cleavage contains 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 cleavage. In some embodiments, an Aβ peptide variant having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 6, or containing SEQ ID NO: 6, includes a C-terminal cleavage. In some embodiments, the C-terminal cleavage includes 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 cleavages. In some embodiments, a fragment or functional derivative of SEQ ID NO: 6 contains 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 with 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. In some embodiments, the vector encodes a polynucleotide encoding the Aβ peptide variant.

[0017] In some embodiments, the vector or the polynucleotide encoded by the vector encodes an Aβ peptide variant or a fragment or functional derivative thereof. In some embodiments, the vector or the 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 containing a cleaved beta-carboxyl-terminal fragment (β-CTF) of amyloid precursor protein. In some embodiments, the cleaved β-CTF is fused to a signal peptide sequence. In some embodiments, the signal peptide sequence contains a nucleotide sequence corresponding to an amino acid sequence containing a Gaussial luciferase signal peptide or a nucleotide sequence corresponding to an amino acid sequence containing a mouse immunoglobulin heavy chain signal peptide. In some embodiments, the cleaved β-CTF contains an Aβ peptide variant sequence, a transmembrane domain sequence, and a cytoplasmic sequence. In some embodiments, the transmembrane domain sequence contains a nucleotide sequence corresponding to an amino acid sequence containing SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 86, or SEQ ID NO: 87. In some embodiments, the cytoplasmic sequence contains 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 containing two lysine residues. In some embodiments, the cytoplasmic sequence corresponds to an amino acid sequence containing three lysine residues. In some embodiments, the cytoplasmic sequence corresponds to an amino acid sequence containing two lysine residues following an arginine residue in the 5'-3' direction.

[0018] In some embodiments, the minigene contains an amino acid sequence that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with 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 contains an amino acid sequence that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with 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 contains an amino acid sequence that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with 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 contains an amino acid sequence that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with 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 contains an amino acid sequence that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with 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 contains an amino acid sequence that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80.

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

[0020] In some implementations, the vector contains a polynucleotide encoding an Aβ peptide or a fragment or functional derivative thereof. In some implementations, the vector is a viral vector or a non-viral vector. In some implementations, the vector is an adenovirus, lentivirus, retrovirus, or adeno-associated virus vector. In certain implementations, the vector is an AAV vector. In some implementations, the vector is 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 implementations, the vector is AAV9, PHP.eB, AAVrh.8, AAVrh.10, or AAVrh.43.

[0021] In some implementation methods, 1 × 10 per kg of the subject's body weight 8 〜1×10 18 The vector genome dose is administered to the subject. In some implementations, approximately 1 × 10⁶ doses are administered per kg of the subject's body weight. 11 to about 1×10 14 The vector genome dose is administered to the subject. In some implementations, approximately 1 × 10⁶ doses are administered per kg of the subject's body weight.12 to about 1×10 15 A dose of the vector genome is administered to the target organism. In some implementations, the vector transduces the target organism's cells, causing them to express minigenes.

[0022] In some embodiments of the methods and compositions disclosed herein, the compositions further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises liposomes, polymer micelles, microspheres, or nanoparticles.

[0023] In some implementations, the composition is delivered systemically or locally. In some implementations, the composition is delivered systemically to the central nervous system via peripheral infusion. In some implementations, peripheral infusion is intravenous infusion. In some implementations, the composition is delivered to the cerebrospinal fluid (CSF). In some implementations, the composition is delivered to the CSF by non-surgical infusion. In some implementations, non-surgical infusion to the CSF includes non-surgical intrathecal infusion. In some implementations, the composition is delivered to the CSF by neurosurgery. In some implementations, neurosurgery infusion to the CSF includes neurosurgery infusion into the cisterna magna. In some implementations, the composition is delivered to the ventricular system. In some implementations, the composition is delivered to the ventricular system by neurosurgery. In some implementations, the composition crosses the blood-brain barrier.

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

[0025] In some implementations, the subject is provided with an effective dose of one or more additional therapies for neurodegenerative diseases, disorders, or conditions. In some implementations, one or more additional therapies include Alzheimer's disease drugs. In some implementations, the Alzheimer's disease drugs include aducanumab, donepezil, rivastigmine, galantamine, memantine, or tacrine.

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

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

[0028] When used in combination with the term "includes," the word "one" or "one" may mean "one," but it also coincides with the meanings 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, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C. In other words, "and / or" functions as an inclusion or exclusion.

[0030] The words “comprising” (and all including forms such as “comprising” and “including”), “having” (and all having forms such as “having” and “having”), “including” (and all including forms such as “comprising” and “including”), or “containing” (and all containing forms such as “containing” and “containing”) are comprehensive or unrestricted and do not exclude any further elements or stages of method that are not mentioned.

[0031] Compositions and methods for use may “include,” “essentially constitute,” or “consist of” any component or step disclosed throughout the specification. Compositions and methods “essentially constitute” any component or step disclosed shall limit the scope of the claim to specific materials or steps that do not substantially affect the basic and novel features of the claimed invention.

[0032] In connection with therapeutic, diagnostic, or physiological purposes or effects, any method may be described as a “use” claim term, such as “use” of any compound, composition, or formulation discussed herein to achieve or embody the described therapeutic, diagnostic, or physiological purposes or effects.

[0033] It should be considered that any embodiment discussed herein may be embodied in connection with any method or composition of the present invention, and vice versa. Furthermore, the compositions of the present invention may be used to achieve the methods of the present invention.

[0034] Further objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while illustrating specific embodiments of the present invention, are given merely as illustrations, since a variety of variations and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from the detailed description.

[0035] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention may be better understood by referring to one or more of these drawings together with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0036] [Figure 1]Figures 1A–1E show that mutant Aβ peptides reduce fibrillation and cytotoxicity of wild-type Aβ. Figure 1A shows the thioflavin-T (ThT) test for auto-aggregation of mutant peptides compared to wild-type (WT) Aβ42. None of the five mutants showed auto-aggregation during the reaction; for comparison, WT Aβ42 (green) reached the ThT-binding stall phase within 1 hour. Figure 1B shows a competitive test to examine the inhibition of WT Aβ42 aggregation. Each mutant was mixed with WT Aβ42 in a 1:1 ratio and incubated with ThT. V18P worsened the aggregation of the WT peptide; the other four mutants reduced fibrillation. F20P (red) and F19D / L34P (blue) prevented aggregation and were selected for further study. Figure 1C shows the fibril degradation test for F19D / L34P. WT Aβ42 fibrils were exposed to varying concentrations of the monomeric F19D / L34P peptide. Incubation with F19D / L34P produced a concentration-dependent decrease in ThT fluorescence consistent with the degradation of Aβ fibrils, compared to incubation with fibrils alone. Figure 1D shows the degradation of fibrils against F20P, as described for F19D / L34P. F20P also reduced ThT fluorescence in a concentration-dependent manner, but was considerably less effective than F19D / L34P. Figure 1E shows MTS analysis, demonstrating that, unlike the wild-type Aβ42 oligomer (green), both F20P (red) and F19D / L34P (blue) were similar to the untreated control group (black) without inducing toxicity in the N2a cells themselves. 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 the oligomeric Aβ reaction added to N2a medium for testing. This corresponds to 0.1 or 10 μM monomeric WT Aβ42 starting material and the same amount of mutant peptide (if shown). ANOVA, *p<0.05, **p<0.01 Bonferroni post-hoc test for comparison with WT Aβ42 alone. All data are shown mean ± SEM. [Figure 2]Figures 2A–2D show expression constructs optimized for efficient secretion of mutant Aβ. Figure 2A shows a sequential deletion strategy used to identify the shortest CTF fragment sufficient for γ-secretase cleavage. The constructs were transfected with N2a cells, and Aβ was recovered from the culture medium and secretion was measured. The optimal construct contained the entire transmembrane domain and two intracellular lysine (KK) molecules. This minigene achieved Aβ secretion equivalent to that of the full-length CTF (left blot, arrows indicate uncleaved Aβ+ residual transmembrane (TM) domain compared to secreted Aβ with terminal residues for each construct). Secretion of wild-type Aβ42 using the 'KK' minimal construct was dose-dependently blocked by the β-secretase inhibitor LY411575 (right blot, arrows again indicate uncleaved Aβ+TM domain compared to secreted Aβ with nM GSI concentration). Figure 2B shows the design of an AAV vector for in vivo delivery of the mutant Aβ peptide. The expression cassette contains a Gaussial ciferase signaling peptide at the N-terminus of Aβ, followed by the Aβ42 variant and the minimal APP C-terminal transmembrane sequence, both regulated by the CAG promoter. Figure 2C shows the extracellular release of full-length variant Aβ using mass spectrometry of immunoprecipitated Aβ. Peptides secreted from the culture medium were isolated by 6E10 immunoprecipitation using N2a cells expressing Aβ F20P. The MS1 spectra of the eluted peptides show the expected masses for Aβ40 F20P and Aβ42 F20P (data not shown). The peak for complete Aβ40 F20P at m / z = 856.4441 in the +5 charge state shows a single isotope mass of 4,277.1841 Da, while the most abundant isotope shows a mass of 4,279.1851 Da, within a 10 ppm error of the expected mass. Figure 2D shows MS2 fragmentation of the peptide, which matches the mass of Aβ40 F20P, and sequence identity is confirmed by a fragment ion tolerance of less than 20 ppm. [Figure 3]Figures 3A–3G demonstrate that neonatal AAV injection generates neuronal expression of mutant Aβ. AAV encoding F19D / L34P or F20P Aβ was injected into the lateral ventricle of wild-type neonatal mice. Mice were harvested 3 weeks or 7 months later for immunostaining and / or ELISA analysis. Figure 3A shows anti-human Aβ immunostaining (6E10, green) collected 3 weeks after P0 injection of AAV-F20P demonstrates widespread viral expression in the cortex of sagittal sections. Figure 3B shows excellent co-staining between the virus-transmission mutant Aβ((F20P; 6E10, green) and endogenous mouse APP (Y188, red), suggesting membrane transduction of the mutant peptide in cortical neurons. Figure 3C shows mass spectrometry of immunoprecipitated Aβ to confirm the generation of the in vivo full-length mutant Aβ. Peptides were isolated from brain homogenized fluid by 6E10 immunoprecipitation using mice expressing Aβ F20P. The MS1 spectra of the eluted peptides show the expected masses for Aβ42 F20P (shown) and Aβ40 F20P. The peak of complete Aβ42 F20P at m / z=893.2675 with a +5 charge state (d=0.2) shows a single isotope mass of 4,461.3011 Da, compared to the most abundant isotope composition of 4,463.3136 The value is Da, and it is within a 15 ppm error of the expected mass. Figure 3D shows MS2 fragmentation of the peptide to confirm 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 homogenate from wild-type mice euthanized at 3 weeks of age. Both mutants produced human Aβ42, and Aβ40 was detected only in mice transduced with F20P. F20P produced higher levels of Aβ40 compared to uninjected mice, while F19D / L34P showed negligible levels of Aβ40 production, which is presumed 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 the construct encoding the wild-type γ-secretase site.Figure 3F shows that mutant human Aβ expression can still be detected by ELISA 7.5 months after P0 virus injection. The values ​​in the figure relate to the soluble fraction of frontal lobe homogenate from non-transgenic animals. The absolute values ​​in panels C and D cannot be directly compared because they were tested at different times using kits from different manufacturing lots. In Figure 3G, the Aβ40:42 ratio 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 shown mean ± SEM. n=2-10 / treatment. [Figure 4] Figures 4A–4C demonstrate that lifetime expression of mutant Aβ reduces plaque load and Aβ accumulation in APP / PS1 mice. APP / PS1 mice were injected with AAV encoding Aβ F19D / L34P or F20P into P0 cells and retrieved after 7.5 months. Figure 4A shows that Aβ immunostaining indicates reduced plaque accumulation in mice treated with the mutant Aβ peptide. Figure 4B shows that cortical plaque load, measured by %Aβ area, confirms that F20P mice have even less amyloid than untreated mice. n=5 uninjected, n=4 F19D / L34P, n=8 F20P. Figure 4C shows that MSD ELISA against human Aβ peptide in guanidine extracts of cortical tissue reflects plaque histology. Aβ40 levels were reduced in all two mutants, while F20P also showed reduced Aβ42 levels. The decrease in Aβ reached a meaningful level at F20P and showed a trend at 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 shown mean ± SEM. [Figure 5]Figures 5A-5B demonstrate the use of viral serotypes to control the spatial distribution and timing of transposable gene expression. Figure 5A shows the PHP.eB virus encoding a red fluorescent protein used to demonstrate CNS expression after peripheral injection. 1.6 × 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 ventricle of neonatal mice. Note the diffusion of the virus into the fourth ventricle. Inserts in the figures show the lateral ventricle (box-shaped) at a higher magnification. Alternative serotypes such as AAV4 or AAV5 may be used to provide specificity for ependymal cells when injected into the lateral ventricle. [Figure 6]Figures 6A–6D show that the mutant Aβ peptide reduces reactive gliosis corresponding to plaque loading. All APP / PS1 and wild-type (non-transgenic, NTG) mice were harvested at 7.5 months after injection of icv P0 virus to transmit the F20P mutant Aβ. Uninjected siblings were used for comparison. Figures 6A and 6B show GFAP immunostaining used to detect astrocytes; Iba1 to detect microglia cells. Fluorescent immunostaining shown in the bottom row was controlled with thioflavin-S to detect amyloid plaques. In APP / PS1 mice, F20P treatment reduced the size of neuroglial lesions and the number of peripheral cells compared to uninjected animals (top and bottom columns). In contrast, viral injection did not affect the morphology or density of microglia cells in NTG mice, but increased the number of GFAP+ cells along the corpus callosum (middle column). Figure 6C shows the quantification of colorimetric immunostaining for GFAP and Iba1, confirming the qualitative finding that F20P treatment reduced the corresponding glial cell staining area in APP / PS1 mice, but increased GFAP levels in NTG mice compared to the uninjected control group. Figure 6D shows that human Aβ viral expression was detected in a subgroup of forebrain astrocytes. Co-immunofluorescence for human Aβ (6E10, green) and GFAP (red) was dilute in NTG mice injected with F20P, detecting co-labeled cells (arrows). APP / PS1 n=5 uninjected, n=7-8 F20P; NTG n=5-6 uninjected, n=5 F20P. The small portion of GFAP+ astrocytes (red) co-labeled with anti-human Aβ antibody 6E10 (green) suggests that they were transduced at P0 by AAV injection. Bidirectional ANOVA, *p<0.05, **p<0.01, ****p<0.0001. Data are presented as mean ± SEM. [Modes for carrying out the invention]

[0037] This invention is based on the remarkable discovery that at least partially amyloid-beta (Aβ) peptide variants, vectors encoding the variants, or vectors encoding minigenes that enable the expression of the variant Aβ peptide protein can suppress the aggregation of endogenous Aβ peptides. Furthermore, it has been demonstrated that administration of the vectors encoding the variants or vectors encoding minigenes that enable the expression of the variant Aβ peptide protein remarkably prevents or reduces the cytotoxicity of endogenous Aβ peptide oligomer, protofibril, fibril or plaque formation and 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 variant or a vector encoding a minigene enabling the expression of a mutant Aβ peptide protein may prevent or reduce protein misfolding, aggregation of endogenous Aβ peptides, amyloid plaque formation, neuroinflammation, neuronal degeneration, neuronal loss or synaptic loss; may reduce tau levels, phosphorylated or phosphorylated tau levels; may slow tau seeding or endogenous Aβ peptide seeding, or may promote cognitive improvement.

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

[0039] I. Neurodegenerative diseases, disorders, and pathologies In certain embodiments, methods for treating neurodegenerative diseases are disclosed. As used herein, the terms “neurodegenerative disease, disorder, or condition” or “neurodegenerative disease” refer primarily to conditions affecting neurons in the human brain that result in progressive loss of neuronal structure or function (including neuronal death). Neurodegenerative diseases may, but are not limited to, the following types: Alzheimer’s disease, Parkinson’s disease, Parkinson’s dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down syndrome, and / or pathological aging. In certain 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 (known as dementia). Dementia is a loss of cognitive function, such as thinking, memory, reasoning, and behavioral abilities, to the extent that it impairs daily living and activities. Dementia ranges widely in severity from the mildest stage, where it is just beginning to affect a person's functioning, to the most severe stage, where a person must become completely dependent on others for basic daily living activities.

[0041] The causes of dementia can be diverse, depending on the type of brain changes that may occur. Dementia accounts for the largest share of neurodegenerative diseases, with Alzheimer's disease representing approximately 60–70% of dementia cases. In 2016, approximately 5.4 million Americans had Alzheimer's disease. Other types of dementia include Lewy body dementia, anterior temporal lobe disease, and vascular dementia. In some cases, patients may have mixed dementia, a combination of two or more types of dementia. 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, with overall atrophy and degeneration of parts of the temporal lobe, parietal lobe, frontal cortex, and cingulate gyrus. Degeneration may also be present in brainstem nuclei such as the locus coeruleus. Studies using MRI and PET have documented that Alzheimer's patients show a reduction in the size of certain brain regions compared to similar images of healthy elderly individuals as they progress from mild cognitive impairment to Alzheimer's disease. The pathology of Alzheimer's disease is primarily characterized by the presence of senile plaques and nerve fiber entanglements, which interfere with normal brain function and chemistry, leading to a significant deficiency of neurotransmitters and a progressive loss of brain function. Abnormal amounts of beta-amyloid and tau proteins form in the brain, beginning to engulf brain cells and form plaques and nerve fiber entanglements. Both amyloid plaques and nerve fiber entanglements are clearly visible under a microscope in the brains of Alzheimer's patients, particularly in the hippocampus. While some plaques and entanglements develop as a result of aging in many elderly people, the brains of Alzheimer's patients have a much larger amount of plaques and entanglements in specific brain regions, such as the temporal lobe.

[0043] The most common early symptom is difficulty remembering recent events. As Alzheimer's disease progresses, symptoms may include language problems, disorientation, mood swings, loss of motivation, mismanagement of self-care, and behavioral problems. More severely affected Alzheimer's patients often withdraw from family and society. Gradually, they lose physical function and ultimately die. The rate of progression can vary, but the general expected lifespan after an Alzheimer's diagnosis is 3 to 9 years.

[0044] A. The process of Alzheimer's disease The disease progression is divided into distinct stages, along with a progressive 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 testing. Mild cognitive impairment (MCI) is characterized by the onset of Alzheimer's disease symptoms. This has often been identified as a transitional stage between normal aging and dementia. MCI can present with a variety of symptoms, and when memory loss is the primary symptom, it is called "amnesic MCI" and is frequently considered a prodromal stage of Alzheimer's disease. The initial symptoms are often mistakenly attributed to aging or stress, but through detailed neuropsychological examination, mild cognitive impairment may appear up to eight years before the clinical criteria for diagnosing Alzheimer's disease are met. The most significant impairment is short-term memory loss, manifesting 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 impairments in semantic memory, semantic memory, and conceptual relationships, can also be symptoms of early Alzheimer's disease. Indifference may also be observed and remains the most persistent neuropsychiatric symptom as the disease progresses. Depressive symptoms, irritability, and reduced awareness of subtle memory impairments are also common.

[0045] Increased learning and memory impairment in Alzheimer's disease patients ultimately leads to a final diagnosis. In a small percentage of patients, difficulties with language, executive function, perception (cognitive impairment), or motor execution (motor impairment) are more pronounced than memory problems. Alzheimer's disease does not affect all memory capacity equally. Older memories of an individual's life (episodic memory), learned facts (semantic memory), and implicit memories (bodily memory of how to do things) are less affected than new facts and memories. Language problems are primarily characterized by vocabulary reduction and decreased word fluency, leading to a general deficit in oral and written language. At this stage, basic thoughts can generally be communicated adequately. Difficulty coordinating and planning specific movements, or motor impairment, may be present, but may be subtle, while performing fine motor tasks. As Alzheimer's disease progresses, patients may continue to perform many tasks independently, but may require assistance or supervision for cognitively demanding activities.

[0046] The gradual deterioration ultimately impairs the patient's independence, making it difficult for them to perform the most common activities of daily living. Language impairment may manifest as an inability to remember vocabulary, leading to frequent missubstitution or paraphrasing. Reading and writing abilities may be progressively lost. As Alzheimer's disease progresses, complex motor sequences may become poorly coordinated, increasing the risk of falls. Memory problems worsen, and long-term memory may be impaired. Behavioral and neuropsychiatric changes may become more widespread, including unstable influences leading to wandering, irritability, crying, unplanned outbursts of aggression, or resistance to care. Sundowning may also be present. Approximately 30% of Alzheimer's patients exhibit optical illusions, misidentification, and other delusional symptoms. Patients may also experience anosognosia, a loss of insight into the disease process and limitations.

[0047] In the final stages of Alzheimer's disease, patients may become completely dependent on caregivers. Language may diminish to mere phrases or single words, eventually leading to 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 the patient becomes 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 Based on studies of twins and families, the genetic inheritance range for Alzheimer's disease is between 49% and 79%. Approximately 0.1% of cases are known as early-onset familial Alzheimer's disease, a familial form of autosomal dominant inheritance that develops before age 65. The majority of autosomal dominant familial Alzheimer's disease cases may be due to a mutation 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 the production of beta-amyloid protein (A-beta protein or Aβ protein), the main component of senile plaques, or increase the ratio of Aβ42 protein (composed of 42 amino acids) to Aβ40 protein (composed of 40 amino acids). Some mutations alter the ratio between Aβ42 and other main forms of amyloid-beta protein, such as Aβ40, without increasing total Aβ levels.

[0049] Most cases of Alzheimer's disease do not exhibit autosomal dominant inheritance and are classified as sporadic Alzheimer's disease, where environmental and genetic differences may act as risk factors. The most well-known genetic risk factor is the inheritance of the apolipoprotein E (APOE) ε4 allele. 40 to 80% of Alzheimer's patients have at least one APOEε4 allele. The APOEε4 allele increases the risk of the disease up to 3 times in heterozygotes and up to 15 times in isozygotes. Genome-wide association studies (GWAS) have identified more than 30 additional gene regions that appear to influence 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 associated with the risk of Alzheimer's disease can be found in the literature, the full text of which is included as a reference in this application [c. BW Kunkle et al., Nature Genetics 51, 414-430 (2019)]. Alleles of the TREM2 gene are also associated with a 3 to 5 times higher risk of developing Alzheimer's disease. Microglia in the brain in some TREM2 mutants are unable to further regulate the amount of beta-amyloid present. According to a 2018 study by Mukherjee et al. (genetic data and cognitively defined subgroups of late-onset Alzheimer's disease; Mol Psychiatry (2018), specifically, which is included herein by full reference, divides Alzheimer's disease into six categories including memory, language, spatiotemporal and executive function and adds 30 SNPs), many single nucleotide polymorphisms (SNPs) are associated with Alzheimer's disease.

[0050] Familial Alzheimer's disease in Japanese bloodlines has been identified as being associated with a deletion mutation at codon 693 of APP, known as the Osaka mutation. Isozygotes carrying this mutation develop Alzheimer's disease. The mutation accelerates Aβ oligomerization, but the protein does not form amyloid fibrils. Mice expressing this mutation exhibit all the common pathologies of Alzheimer's disease caused by the Osaka mutation.

[0051] A substitution mutation of alanine to valine at codon 673 of the APP gene increases the risk of Alzheimer's disease in isozygote carriers, likely by promoting Aβ formation and thus increasing amyloid fibril development, whereas heterozygote carriers with A673V may be protected from Alzheimer's disease. This substitution is adjacent to the beta-secretase cleavage site and may lead to increased APP cleavage by beta-secretase through the amyloidogenesis pathway. In contrast, substituting the same alanine at position 673 with threonine (A673T) can prevent Alzheimer's disease. This substitution is also adjacent to the beta-secretase cleavage site and can reduce amyloid beta formation by 40% in vitro.

[0052] Furthermore, a theory has been proposed that extracellular Aβ precipitates are the root cause of Alzheimer's disease. This theory is supported not only by the location of the APP gene on chromosome 21, but also by the fact that many patients with chromosome 21 trisomy (Down syndrome) who have an additional gene copy show the earliest symptoms of AD by age 40. In addition, APOE4 is a major genetic risk factor for Alzheimer's disease (see references: e.g., M. Safieh et al., BMC Medicine 17:64 (2019), and CGFernandez et al., Front. Aging Neuroscience 11:14 (2019), specifically these references are included herein by reference in their entirety). Transformed mice expressing mutant forms of the human APP gene develop Alzheimer's disease-like brain pathology with fibrillary amyloid plaques and spatial learning defects.

[0053] Aβ precipitates are formed from Aβ (referred to as A-beta peptide or beta-amyloid) and are typically 39 to 43 amino acids long. Aβ peptides are fragments of larger proteins called transmembrane proteins (APPs) that span the cell membrane. APPs are thought to play a role in normal neuronal growth, survival, and post-injury recovery. In Alzheimer's disease, gamma-secretase and beta-secretase work together in the protein degradation process to break down APP into even smaller fragments. These fragments can self-assemble to form beta-amyloid fibrils, which are dense extracellular precipitates known as senile plaques or amyloid plaques. Plaques are dense, mostly insoluble precipitates consisting of beta-amyloid peptides and cellular material from outside and around the neuron. Experimental vaccines have shown to remove amyloid plaques in early human studies but have not had a significant effect on dementia, suggesting that non-plaque Aβ oligomers are the primary pathogenic form of Aβ. These toxic oligomers, also known as amyloid-derived diffusive ligands, disrupt neural communication by binding to surface receptors on neurons and altering synaptic structure.

[0054] Other studies have suggested that tau protein abnormalities initiate the Alzheimer's disease cascade (AD cascade). All neurons have a cytoskeleton that is partially composed of microtubules. When tau protein is phosphorylated, it stabilizes the microtubules. In Alzheimer's disease models, tau undergoes chemical changes and becomes hyperphosphorylated. Hyperphosphorylated tau begins to pair with other tau fibers. Ultimately, this forms tangles of nerve fibers inside the nerve cell body. Tangles (neurofibrils) are aggregates of microtubule-associated protein tau that are hyperphosphorylated and accumulate inside the cell. Once tangles occur, microtubules are broken down, the cell's cytoskeletal structure is destroyed, and the neuronal transport system collapses. Initially, this can cause malfunctions in biochemical communication between neurons, and later, it can lead to cell death. Pathogenic tau can also induce neuronal death through transposition factor dysregulation.

[0055] Age is a significant 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 specific parts of the brain, inflammation, vascular damage, the generation of unstable molecules that become free radicals, and mitochondrial dysfunction (disruption of intracellular energy production). Therefore, older age (60 years and older) is a risk factor for Alzheimer's disease.

[0056] Several additional theories have been proposed to explain the cause of Alzheimer's disease. For example, the inflammatory hypothesis suggests that Alzheimer's disease is triggered by spontaneously persistent progressive inflammation in the brain, which is neurodegenerative and reaching a peak. In some models, infectious viruses and microbial agents are thought to act in conjunction with Aβ to generate a positive feedback mechanism against neuroinflammation. (See reference [ALKomaroff, JAMA 324(3):239~240(2020)]). A neurovascular hypothesis has also been proposed, suggesting that impaired blood-brain barrier (BBB) ​​function may be involved. Cellular homeostasis of biometals such as ionic copper, iron, and zinc is also disrupted in Alzheimer's disease; these ions affect tau, APP, and APOE, and these dysregulations can induce oxidative stress that can contribute to the pathology. Another hypothesis suggests that during aging, dysfunction of oligodendrocytes and associated myelin contributes to axonal damage, inducing amyloid formation and tau hyperphosphorylation as side effects.

[0057] C. Diagnosis of Alzheimer's disease Alzheimer's disease is generally diagnosed based on the patient's medical history, relative history, and behavioral observations. The presence of characteristic neurological and neuropsychological features and the absence of alternative conditions support this diagnosis. Advanced medical imaging, including computed tomography or magnetic resonance imaging and single-photon emission computed tomography or positron emission tomography, can rule out other brain pathologies or subtypes of dementia. Furthermore, the progression to Alzheimer's disease can be predicted in prodromal stages characterized by mild cognitive impairment.

[0058] The research framework of the National Institute on Aging and Alzheimer's Association working group for diagnosing Alzheimer's disease currently uses a biomarker classification system that divides the main Alzheimer's disease biomarkers into three categories based on the type of pathological change: β-amyloid (A), pathological tau (T), and neurodegenerative (N). The ATN nomenclature presents a conceptual framework based on empirical observations of the relationships between amyloid, tau, and neurodegenerative markers over the past decade. "A" refers to amyloid-beta (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 tangled nerve fibers in parenchyma. "N" refers to neurodegenerative or neuronal damage and dysfunction measured, for example, by hippocampal volume or cortical volume or thickness. "A" + "T" is considered to have diagnostic specificity for Alzheimer's disease, whereas "N" is not specific to the diagnosis of Alzheimer's disease as it can reflect any number of etiologies other than Alzheimer's disease. The ATN system is described in detail in the literature (DS Knopman et al., Alzheimer's & Dement. 14(4):563~575 (2018), specifically the full text which is included as a reference in this application).

[0059] Assessment of intellectual function, including memory tests, can further characterize the disease state. Healthcare institutions have developed diagnostic criteria to standardize the diagnostic process. In 1984, the National Institute of Neurological and Communication 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, requiring that cognitive impairment and suspected dementia syndromes be confirmed by neuropsychological testing for possible or probable clinical diagnosis of Alzheimer's disease. Eight intellectual domains, such as memory, language, perceptual ability, attention, motor ability, orientation, problem-solving, and executive function, are most commonly impaired in Alzheimer's disease. These domains are identical to those listed in the NINCDS-ADRDA Alzheimer's criteria 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 clear histopathological confirmation.

[0060] In 2011, the National Institute on Aging / Alzheimer's Society released an updated approach for clinicians and scientists to provide advanced guidelines for advancing research on diagnosis and treatment. The 2011 guidelines differ from the 1984 diagnostic criteria in several key respects. These organizations recognize that Alzheimer's disease progresses through a spectrum of three stages: an asymptomatic early preclinical stage; an intermediate stage of mild cognitive impairment; and a final stage in which dementia symptoms appear. The 1984 criteria dealt with only one stage of the disease, which is the final stage of dementia; and extend the criteria for Alzheimer's dementia beyond memory loss, which is the first or sole primary symptom. These organizations recognize that other aspects of cognition, such as word-finding ability or judgment, may be impaired first. The 1984 criteria focused on memory loss as a core feature of Alzheimer's dementia; and reflect a greater understanding of the differences and relationships between Alzheimer's dementia and non-Alzheimer's dementia, as well as between Alzheimer's and other disorders that may influence disease development, such as vascular disease. In 1984, this relationship was not well recognized or understood, and the potential use of biomarkers—indicators of underlying brain disease—to diagnose Alzheimer's disease was acknowledged. However, guidelines explicitly state that biomarkers should be used almost exclusively in research rather than in clinical settings. These biomarkers did not exist when the original criteria were developed in 1984, and diagnosis was generally confirmed through post-mortem autopsy.

[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 yield normal results, except for obvious cognitive impairments that may not be indistinguishable from the outcome of other disease processes, including other causes of dementia. Additional neurological examinations are important in the differential diagnosis of Alzheimer's disease and other conditions. Interviews with family members are also used to assess the disease. Caregivers can provide important information not only about the patient's ability to perform daily living activities but also about the decline in the patient's mental function over time. The caregiver's perspective can be particularly important because Alzheimer's patients generally do not recognize their own shortcomings.

[0062] Supplementary tests can provide additional information about the characteristics of the disease or can be used to rule out other diagnoses. Blood tests can identify alternative causes of dementia other than Alzheimer's disease. Thyroid function tests, B12 assessment, syphilis exclusion, metabolic problem exclusion (including renal function, electrolyte levels, and diabetes tests), heavy metal (e.g., lead, mercury) levels, and anemia assessment are also common. Since depression can be a cause of, or even an early sign of, Alzheimer's disease or cognitive impairment, a depressive examination may also be used.

[0063] II. Aβ peptide variants In some embodiments, the disclosed composition comprises 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 enables the expression of the Aβ peptide protein. Thus, in some embodiments, the wild-type or mutant 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 enables the expression of the Aβ peptide protein.

[0064] Aβ peptides are fragments of larger proteins called transmembrane proteins (APPs) that can pass through the neuronal membrane. A representative mRNA APP sequence can be found in GenBank® accession number NM_000484 and contains the following amino acid sequence.

[0065] (Sequence ID: 1)

[0066] Gamma-secretase and beta-secretase work together in the protein degradation process to break down 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, cleaves within the transmembrane region of APP, producing numerous C-terminal fragment isomorphs of 30 to 51 amino acid residues in length. Separately from 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 form beta-amyloid fibrils that can self-assemble in dense extracellular deposits known as plaques. Aβ42 is also hydrophobic and therefore the most amyloidogenic peptide. However, the central amino acid sequence KLVFFAE (SEQ ID NO: 39) is known to form amyloid on its own and can form the core of amyloid fibril.

[0067] Accordingly, in certain embodiments of this disclosure, the disclosed compositions include wild-type Aβ peptide. In certain embodiments of this disclosure, the disclosed compositions include variants of wild-type Aβ peptide. In certain embodiments, wild-type Aβ peptide or Aβ peptide variants promote aggregation of endogenous Aβ peptide. In certain embodiments, wild-type Aβ peptide or Aβ peptide variants self-assemble as oligomers, protofibrils, fibrils, or plaques. In certain embodiments, Aβ peptide variants prevent aggregation of endogenous Aβ peptide. In certain embodiments, Aβ peptide variants do not self-assemble as oligomers, protofibrils, fibrils, or plaques. In certain embodiments, Aβ peptide variants reduce the cytotoxicity of remaining Aβ peptide aggregates. In certain embodiments, the full-length Aβ variants disclosed herein simultaneously allow targeting of multiple aggregation domains to increase affinity and specificity for Aβ.

[0068] In some embodiments, the 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, or any derivable value within these ranges. In some embodiments, the wild-type Aβ peptide of this disclosure comprises, constitutes, or is essentially composed of, DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 2), or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 2 contains 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 with KLVFFAE (SEQ ID NO: 39), or any derivable value within these ranges.

[0069] In some implementations, Aβ peptide variants are TIFF0007867713000001.tif12168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges, where bold residues indicate substitutions from the wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 3, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 3 contains 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 with KLVDFAE (SEQ ID NO: 10), or any derivable value within these ranges.

[0070] In some implementations, Aβ peptide variants are TIFF0007867713000002.tif12168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges, where bold residues indicate substitutions from the wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 4, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 4 contains 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 with KLVFPAE (SEQ ID NO: 11), or any derivable value within these ranges.

[0071] In some implementations, Aβ peptide variants are TIFF0007867713000003.tif10168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges, where bold residues indicate substitutions from the wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 5, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 5 contains 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 with KLVDFAE (SEQ ID NO: 10), or any derivable value within these ranges.

[0072] In some implementations, Aβ peptide variants are TIFF0007867713000004.tif10168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges, where bold residues indicate substitutions from the wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 6, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 6 contains 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 with KLVPFAE (SEQ ID NO: 12), or any derivable value within these ranges.

[0073] In some implementations, Aβ peptide variants are TIFF0007867713000005.tif11168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges, where bold residues indicate substitutions from the wild-type Aβ peptide. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 7, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 7 contains 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 with KLPFFAE (SEQ ID NO: 65), or any derivable value within these ranges. In some embodiments, the Aβ peptide variant contains, is composed of, or is not essentially composed of SEQ ID NO: 7.

[0074] In some embodiments, wild-type Aβ peptides or Aβ peptide variants that include, or are composed of, or essentially constitute, 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 include an N-terminal cleavage. In some embodiments, the N-terminal cleavage includes 1 to 22 amino acid cleavages. In some embodiments, the N-terminal cleavage includes 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 cleavages.

[0075] In some embodiments, wild-type Aβ peptides or Aβ peptide variants that include, or are composed of, or essentially constitute, 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 include a C-terminal cleavage. In some embodiments, the C-terminal cleavage includes 1 to 27 amino acid cleavages. In some embodiments, the C-terminal cleavage includes 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 cleavages.

[0076] In some embodiments, wild-type Aβ peptides or Aβ peptide variants that include, or are composed of, or are essentially composed 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 include N-terminal and C-terminal cleavage. In some embodiments, the N-terminal cleavage includes 1 to 22 amino acid cleavage, and the C-terminal cleavage includes 1 to 27 amino acid cleavage. In some embodiments, the N-terminal cleavage includes 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 cleavages, and the C-terminal cleavage includes 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 cleavages.

[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 the 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 the polynucleotide encoded by the vector encodes the whole APP beta-carboxyl-terminal fragment (β-CTF) and comprises 99 C-terminal amino acids of an amyloid precursor protein containing the wild-type Aβ peptide or Aβ peptide variant amino acid sequence fused to the signal peptide at the N-terminus of the β-CTF.

[0078] In some embodiments, the vector or the 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 containing a cleaved β-CTF fused to a signal peptide at the N-terminus of the β-CTF. In some embodiments, the cleaved β-CTF contains, all of its extracellular and transmembrane amino acids, a wild-type Aβ peptide or Aβ peptide variant amino acid sequence (or a fragment or functional derivative thereof), including the transmembrane domain amino acid sequence and the cytoplasmic amino acid sequence.

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

[0080] In some implementations, minigenes are TIFF0007867713000006.tif55168, or a fragment or functional derivative thereof, comprises 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, or any derivable value within these ranges, where bold text corresponds to an Aβ peptide variant amino acid sequence, underlined text corresponds to a transmembrane domain amino acid sequence, and unaltered text corresponds to a cytoplasmic amino acid sequence. In some implementations, the minigene contains, is composed of, or is essentially composed 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 implementations, minigenes are TIFF0007867713000007.tif55168, or a fragment or functional derivative thereof, comprises 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, or any derivable value within these ranges, where bold text corresponds to an Aβ peptide variant amino acid sequence, underlined text corresponds to a transmembrane domain amino acid sequence, and unaltered text corresponds to a cytoplasmic amino acid sequence. In some implementations, the minigene contains, is composed of, or is essentially composed 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 implementations, minigenes are TIFF0007867713000008.tif54168, or a fragment or functional derivative thereof, comprises 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, or any derivable value within these ranges, where bold text corresponds to an Aβ peptide variant amino acid sequence, underlined text corresponds to a transmembrane domain amino acid sequence, and unaltered text corresponds to a cytoplasmic amino acid sequence. In some implementations, the minigene contains, is composed of, or is essentially composed 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 implementations, minigenes are TIFF0007867713000009.tif55168, or a fragment or functional derivative thereof, comprises 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, or any derivable value within these ranges, where bold text corresponds to an Aβ peptide variant amino acid sequence, underlined text corresponds to a transmembrane domain amino acid sequence, and unaltered text corresponds to a cytoplasmic amino acid sequence. In some implementations, the minigene contains, is composed of, or is essentially composed 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 implementations, minigenes are TIFF0007867713000010.tif56168, or a fragment or functional derivative thereof, comprises 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, or any derivable value within these ranges, where bold text corresponds to an Aβ peptide variant amino acid sequence, underlined text corresponds to a transmembrane domain amino acid sequence, and unaltered text corresponds to a cytoplasmic amino acid sequence. In some implementations, the minigene contains, is composed of, or is essentially composed 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 implementations, minigenes are TIFF0007867713000011.tif55168, or a fragment or functional derivative thereof, comprises 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, or any derivable value within these ranges, where bold text corresponds to the wild-type Aβ42 peptide amino acid sequence, underlined text corresponds to the transmembrane domain amino acid sequence, and unaltered text corresponds to the cytoplasmic amino acid sequence. In some implementations, the minigene contains, is composed of, or is essentially composed 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 contains nucleotide sequences corresponding to the 13 C-terminal amino acids and an additional 10 amino acids of the Aβ peptide variant. Therefore, those skilled in the art will recognize that the amino acid sequence of the Aβ peptide variant contains 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: The file contains TIFF0007867713000012.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 contains 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, or any derivable value within these ranges.

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

[0088] As a non-restrictive example, in some embodiments, the signal peptide sequence includes a nucleotide sequence corresponding to an amino acid sequence containing 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: BATannous et al., Molecular Therapy 11(3):435~443 (2005), and S. Knappskog et al., J. of Biotechnology 128:705~715 (2007), specifically the full text of which is included as a reference herein]. In some embodiments, the amino acid sequence of the Gaussia luciferase signal peptide includes MGVKVLFALICIAVAEA (SEQ ID NO: 19). The corresponding nucleotide sequence encoding the Gaussial cyperage signal peptide of SEQ ID NO: 19 may include ATGGGCGTGAAGGTCCTGTTCGCCCTGATTTGCATCGCCGTCGCAGAGGCA (SEQ ID NO: 20).

[0089] As a second non-restrictive example, in some embodiments, the signal peptide sequence includes a nucleotide sequence corresponding to the amino acid sequence containing the 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 [references: BATannous et al., Molecular Therapy 11(3):435~443 (2005), and S. Knappskog et al., J. of Biotechnology 128:705~715 (2007), specifically, the full text of which is included as a reference herein]. In some embodiments, the amino acid sequence of the MoIgH signal peptide includes MGWSCIILFLVATATGVHS (SEQ ID NO: 21). The corresponding nucleotide sequence encoding the MoIgH signal peptide of SEQ ID NO: 21 is: It may also include TIFF0007867713000013.tif22168, where the bolded portion of sequence number 22 corresponds to the intronic non-coding sequence of the MoIgH signal peptide.

[0090] In some implementations, the minigene contains 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 with one of SEQ ID NO: 19 or SEQ ID NO: 21, or any derivable value within these ranges.

[0091] In some implementations, polynucleotides are included in the vector. In some implementations, the vector stably expresses wild-type or mutant Aβ peptides, or fragments thereof, or functional derivatives, at the cell membrane where the release of Aβ peptides into the extracellular space is regulated by endogenous γ-secretase.

[0092] A. Protein composition As used in this application, the terms "proteinic molecule," "proteinic composition," "proteinic compound," "proteinic chain," or "proteinic substance" generally refer to, but are not limited to, proteins consisting of approximately 200 or more amino acids or full-length endogenous sequences translated from genes; polypeptides of approximately 100 or more amino acids; and / or peptides of approximately 3 to approximately 100 amino acids. All of the terms "proteinic" as described above may be used interchangeably in this application.

[0093] As used herein, “protein,” “peptide,” or “polypeptide” means a molecule containing at least three amino acid residues. As used herein, the term “endogenous” means a version of a molecule that occurs naturally in a living organism. In some embodiments, endogenous peptides include wild-type peptides. In some embodiments, endogenous peptides include amino acid modifications from wild-type peptides. In some embodiments, wild-type versions of proteins or peptides are used, but in many embodiments of this disclosure, modified proteins or peptides are used. The terms can be used interchangeably. “Modified protein,” “modified polypeptide,” or “mutant” refers to a protein or peptide whose chemical structure, in particular its amino acid sequence, has been modified from that of a 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 may have multiple activities or functions). It is specifically considered that a modified / mutant protein or peptide may be modified with respect to one activity or function, but may retain wild-type activity or function in another aspect.

[0094] Where protein is specifically referred to herein, it generally refers to a native (endogenous) or recombinant (mutant or variant) protein or peptide. Proteins may be isolated directly from intrinsic 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 publicly known protocols. See, for example, the literature whose full text is specifically referenced herein [see: Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979)]. Alternatively, recombinant DNA technology 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 suitable for expression. In certain embodiments, there are isolated nucleic acid segments containing the nucleic acid sequence encoding the peptide and recombinant vectors. The term “recombinant” may also be used with the name of a peptide or a specific peptide, which generally refers to a peptide produced from a nucleic acid molecule manipulated in vitro, or a replica product of such a molecule.

[0095] In a particular mode of implementation, the size of at least one protein molecule is approximately 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, Approximately 43, approximately 44, approximately 45, approximately 46, approximately 47, approximately 48, approximately 49, approximately 50, approximately 51, approximately 52, approximately 53, approximately 54, approximately 55, approximately 56, approximately 57, approximately 58, approximately 59, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, approximately 500, approximately 525, approximately 550, approximately 575, approximately 600, approximately 625, approximately 65 The amino acid sequence may include, but is not limited to, 0, approximately 675, approximately 700, approximately 725, approximately 750, approximately 775, approximately 800, approximately 825, approximately 850, approximately 875, approximately 900, approximately 925, approximately 950, approximately 975, approximately 1000, approximately 1100, approximately 1200, approximately 1300, approximately 1400, approximately 1500, approximately 1750, approximately 2000, approximately 2250, approximately 2500 or more amino molecular residues, and any derivable range within these ranges, or derivatives of corresponding amino acid sequences described or referenced in this application.

[0096] The peptides described in this application 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 ,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113 ,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,14 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, or 1000 or more amino acids (or any derivable range within these ranges) may be fixed lengths.

[0097] In a particular embodiment, the proteinaceous composition comprises at least one protein, polypeptide, or peptide. It is considered that virtually any protein, polypeptide, or peptide-containing component described herein may be used in the compositions and methods disclosed herein. In an additional embodiment, the proteinaceous composition comprises a biocompatible protein, polypeptide, or peptide. As used herein, the term “biocompatible” means a substance that, when applied to or administered to a given organism in accordance with the methods and amounts described herein, does not cause meaningful side effects. Such inappropriate and undesirable effects are similar to serious toxicity or adverse immunological reactions. In a preferred embodiment, the biocompatible protein-, polypeptide-, or peptide-containing composition would 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] Protein compositions may be prepared by any technique known to those skilled in the art, including the expression of proteins, polypeptides, or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous substances. Nucleotide and protein, polypeptide, and peptide sequences for a variety of genes have been previously disclosed and can be found in computerized databases known to those skilled 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 / ). Coding regions for these known genes may be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art. Alternatively, a variety of commercially available formulations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0099] In certain embodiments, proteinaceous compounds may be purified. Generally, “purified” refers to a specific or protein, polypeptide, or peptide composition that has been fractionated to remove various other proteins, polypeptides, or peptides, the composition may be evaluated to substantially maintain its activity (e.g., by protein assay as is known to those skilled in the art with respect to the specific or target protein, polypeptide, or peptide).

[0100] The proteins and peptides suitable for use in the present invention may be self-proteins or peptides, but the present invention does not expressly limit the use of such self-proteins. As used herein, the terms “self-protein, polypeptide, or peptide” mean a protein, polypeptide, or peptide derived from or obtained from a living organism. The living organisms that may be used include, but are not limited to, cattle, reptiles, amphibians, fish, rodents, birds, dogs, cats, fungi, plants, or prokaryotes, and selected animal or human subjects are preferred. The “self-protein, polypeptide, or peptide” may be used as a component of a composition for application to a selected animal or human subject.

[0101] Preferred proteins, including wild-type or mutant Aβ peptides, are considered in that the compositions described herein are particularly suitable for use in treating or preventing neurodegenerative diseases.

[0102] To select other proteins, polypeptides, peptides, etc. for use in the methods and compositions of this disclosure, those skilled in the art would preferably select proteinaceous substances having one or more of the following properties: forming a solution with a high percentage of solubilized proteinaceous substance; having high viscosity (i.e., about 40 to about 100 poise); having the correct molecular charge to bind to the dye if it is a non-covalent mixture (i.e., anionic protein and cationic dye, or cationic protein and anionic dye); having the correct amino acids present to form covalent crosslinks (i.e., one or more tyrosine, histidine, tryptophan and / or methionine); and / or being biocompatible (i.e., in the case of mammals, of mammalian origin, preferably in the case of humans, of human origin, in the case of dogs, of canine origin, etc.; being autologous, non-allergenic and / or non-immunogenic).

[0103] The compositions described herein are considered to contain approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides, and / or proteins per ml. The protein concentration in the composition may be approximately, at least approximately, or at most approximately 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 derivable range within these ranges).

[0104] B. Biologically functional equivalents The structure of the polynucleotide and / or protein according to the present invention may be modified and / or altered, and such biological functional equivalents are also included in the present invention, while obtaining molecules having similar or improved properties.

[0105] 1. Modified polynucleotides and peptides A biologically functional equivalent may include a polynucleotide that has been manipulated to contain distinct sequences while maintaining the ability to encode a “wild-type” or standard protein or peptide, or a “mutant” protein or peptide. This can be achieved by degeneration of the gene code, i.e., the presence of multiple codons encoding the same amino acid. The term “functionally equivalent codon” is used herein to refer to codons encoding the same amino acid, such as six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” that represent biologically equivalent amino acid codons or changes in codons. In one example, a person skilled in the art may want to incorporate restriction enzyme recognition sequences into a polynucleotide without interfering with the polynucleotide’s ability to encode a protein.

[0106] In relation to functional equivalents, skilled technicians understand that the concept of a “biological functional equivalent” is inherent in the definition of a protein and / or polynucleotide, meaning that there is a limit to the number of changes that can be made within a defined portion of the molecule while maintaining a molecule with an acceptable level of equivalent biological activity. Accordingly, a biological functional equivalent is defined in this application as a protein (and polynucleotide) and / or other endogenous Aβ peptide polymer structure and / or protein (and polynucleotide) having substitutions or mutations in selected amino acids (or codons) that interfere with / interfere endogenous Aβ peptide aggregation or promote the degradation of endogenous Aβ peptide fibrils or other endogenous Aβ peptide polymeric structures, and / or other endogenous Aβ peptide polymeric structures and / or proteins (and polynucleotides) that do not self-aggregate, for example, by reducing the toxicity and / or aggregation of endogenous Aβ peptides. The biological functional equivalents may also include proteins (and polynucleotides) having substitutions or mutations in selected amino acids (or codons) that maintain the ability to promote the aggregation of endogenous Aβ peptides and / or endogenous Aβ peptide fibrils or other endogenous Aβ peptide polymeric structures and / or the aggregation of proteins (and polynucleotides), and / or proteins (and polynucleotides) having substitutions or mutations in selected amino acids (or codons) that self-aggregate, for example.

[0107] Generally, the shorter a molecule is, the fewer changes it can undergo within the molecule while maintaining its function. Longer domains may undergo intermediate levels of change. Full-length proteins will have the greatest tolerance to even greater levels of change. However, it must be understood that certain molecules or domains that are highly structure-dependent may be deformable or unacceptable.

[0108] In one example, polynucleotides may be biologically functional equivalents (and codes) with more significant changes. Certain amino acids can substitute for other amino acids in protein structures without a substantial loss of interaction and binding ability with structures such as antigen-binding domains of antibodies, binding sites on substrate molecules, and receptors.

[0109] Substitutional mutants typically involve replacing one amino acid with another at one or more sites within a protein and may be designed to modulate one or more properties of a polypeptide with or without loss of other functions or properties. The substitution may be conservative, i.e., one amino acid is replaced with one having a similar shape and charge. Conservative substitutions are well known in the industry and include, for example, changes from 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; and valine to isoleucine or leucine. So-called "conservative" changes do not interfere with the biological activity of the protein, because the structural changes do not affect the protein's ability to perform the function it was designed for. Accordingly, the inventors consider that a variety of changes can be made to the gene and protein sequences disclosed herein while still satisfying the objectives of the present invention. Alternatively, substitutions may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve chemically substituting one residue with another, such as replacing a nonpolar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitutions may also involve exchanging one member of an amino acid class with a member of another class.

[0110] In other embodiments, modification of polypeptide function is intended by introducing one or more substitutions. For example, certain amino acids can substitute for other amino acids in a protein structure without significant loss of interaction-binding ability. For instance, structures such as enzyme-catalyst domains or interacting components may have substituted amino acids to maintain such function. Since the biological functional activity of a protein is defined by its interaction ability and properties, specific amino acid substitutions may occur in the protein sequence and the basic DNA coding sequence, and nevertheless, proteins with similar properties can be produced. Accordingly, the inventors believe that a variety of changes may be made to the DNA sequence of a gene without significant loss of biological utility or activity.

[0111] Deletion mutants typically lack one or more residues in the native or wild-type protein. This may involve the deletion of individual residues or multiple adjacent amino acids. Stop codons may be introduced (by substitution or insertion) into the encoding nucleic acid sequence to produce a cleaved protein. For example, a peptide can be mutated by the cleavage or deletion of multiple adjacent amino acids, making it even shorter than its corresponding endogenous form.

[0112] Insertion mutations generally involve adding amino acid residues to the non-terminal site of a polypeptide. This may involve the insertion of one or more amino acid residues. Terminal additions may also be generated, which may include fusion proteins that are polymers or concatenators of one or more peptides or polypeptides described or referenced herein. For example, it can be considered that peptides may be modified by fusing or joining heterologous protein or polypeptide sequences that have a specific function (e.g., for targeting or localization, for enhanced activity, for purification purposes, etc.).

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

[0114] Furthermore, amino acid and nucleic acid sequences may each include additional N- or C-terminal amino acids, or additional residues such as 5' or 3' sequences, and are still essentially the same as one of the sequences disclosed herein, as long as the sequence meets the criteria presented above, including the maintenance of biological protein activity for protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences that may include, for example, a variety of non-coding sequences located on the sides of the 5' or 3' portion of the coding region.

[0115] The amino acid sequence variants in this disclosure may be, for example, substitution, insertion, or deletion variants. The regions or fragments of the polypeptides in this disclosure are 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, 5 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 It is considered that the amino acid sequence may have, at least, or at most 200 or more amino acid substitutions, adjacent amino acid additions, or adjacent amino acid deletions.Alternatively, any region or fragment of a polypeptide of the present disclosure may contain, or have an amino acid sequence that is identical, at least identical, or at most identical to any SEQ ID NO: 1 to 19, SEQ ID NO: 21, 23 to 39, and 65 to 87, or at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical to, any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at most identical, to any amino acid sequence that is at least identical, or at least or at least identical, to any amino acid sequence that is at least identical, or at least identical, or at least identical, or

[0116] Furthermore, in some implementations, the region or fragment is any sequence number: 1 to 19, sequence numbers: 21, 23 to 39 and 65 to 87 (where position 1 is at the N-terminus of the sequence number) 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, 5 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, 19 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, 4, 5, 6, 7, 8, 9, 10, 11, starting with 500,12、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, It contains an amino acid region consisting of 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, and 500 or more adjacent amino acids.

[0117] The polypeptides described herein may contain 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 mutant amino acids of any SEQ ID NOs: 1 to 19, SEQ ID NOs: 21, 23 to 39 and 65 to 87, or at least or up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,5 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, 15 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) adjacent amino acids and at least 60%, 6 They 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 described herein are 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, This may include 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 permutations (or any derivable range within these ranges).

[0119] The substitution is 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 ,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135, 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, 19 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 This may be done at 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 similarities of amino acid side-chain substitutions, such as their hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and / or type of amino acid side-chain substitutions shows that arginine, lysine, and / or histidine are all positively charged residues; alanine, glycine, and / or serine are all of similar size; and / or phenylalanine, tryptophan, and / or tyrosine all have generally similar shapes. Accordingly, in accordance with these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined in this application as biologically functional equivalents.

[0121] When making such changes to produce biologically functional equivalents, the hydropathy index of amino acids may be taken into consideration. The hydrophobic profile of a protein is calculated by assigning a numerical value ("hydrophobic index") to each amino acid and then averaging these values ​​along a repeating peptide chain. Each amino acid is assigned a value based on its hydrophobic 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 functions to proteins is generally understood in this field (Kyte and Doolittle, 1982). The relative hydrophobic properties of amino acids are accepted to contribute to the secondary structure of the resulting protein, which in turn defines the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Furthermore, it is known that certain amino acids may be substituted with other amino acids having similar hydrophobic indices or scores, while still maintaining similar biological activity. When making changes based on hydrophobic indices, in certain embodiments, this includes substitutions of amino acids with hydrophobic indices within ±2. In some aspects of this disclosure, this includes hydrophobic indices within ±1, and in other aspects of this disclosure, this includes hydrophobic indices within ±0.5.

[0122] It is understood in the art that substitutions of similar amino acids can be effectively carried out based on hydrophilicity. U.S. Patent No. 4,554,101, which is included in its entirety as reference to this application, states that the maximum local mean hydrophilicity of a protein, determined by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. In certain embodiments, the maximum local mean hydrophilicity of a protein, determined by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. The following hydrophilic 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 hydrophilic values, certain implementations include amino acid substitutions with hydrophilic values ​​within ±2, other implementations include hydrophilic values ​​within ±1, and other implementations include ±0.5. In some cases, epitopes can be identified from the primary amino acid sequence based on hydrophilicity. It is understood that amino acids may be substituted with other amino acids that have similar hydrophilicity values, and that biologically and immunologically equivalent proteins can still be produced.

[0123] Furthermore, those skilled in the art can consider structure-function studies to identify residues of similar polypeptides or proteins that are important for their activity or structure. From such a comparative perspective, the importance of amino acid residues in a protein corresponding to amino acid residues that are important for the activity or structure of similar proteins can be predicted. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0124] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence of this structure from similar proteins or polypeptides. In terms of such information, those skilled in the art can predict the alignment of amino acid residues for the three-dimensional structure. They can choose not to alter the amino acid residues expected to be on the protein surface, because such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can generate test mutants containing single amino acid substitutions at each of the target amino acid residues. These mutants can then be screened using standard tests for binding and / or activity, thus providing information gathered from such routine experiments, which allows those skilled in the art to determine amino acid positions that 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 this disclosure, amino acid substitutions are made to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) modify binding affinity for protein complex formation, (4) modify ligand binding affinity, and / or (5) confer or alter other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (conservative amino acid substitutions in certain embodiments) may be made in naturally occurring sequences. Substitutions may be made in protein portions outside of domains (one or more) that form intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not substantially alter the structural features of the protein or polypeptide (e.g., one or more substitute amino acids that do not disrupt the primary, secondary, or tertiary structures that characterize the native protein) may be used.

[0126] As mentioned above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substitutions, such as hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take into account the diverse properties mentioned 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 molecule” refers to any amino acid, amino acid derivative, or amino acid mimetic known to those skilled in the art. In certain embodiments, the residues of the protein molecule are sequential without any non-amino molecules interfering with the sequence of amino molecule residues. In other embodiments, the sequence may include one or more non-amino molecule mosquitoes. In certain embodiments, the sequence of residues of the protein molecule may be interrupted by one or more non-amino molecule mosquitoes. Peptides and polypeptides contain 20 “native” amino acids and their post-translational variations. However, in vitro peptide synthesis allows for the use of modified and / or specific amino acids.

[0128] Therefore, the term "proteinic composition" includes, but is not limited to, amino molecule sequences containing at least one of the 20 common amino acids, or at least one modified or unique amino acid, derived from naturally synthesized proteins, as shown in the table below.

[0129] TIFF0007867713000014.tif116168

[0130] 3. Mimics In addition to the biological functional equivalents discussed above, the inventors also consider that structurally similar compounds can be formulated to mimic the core portion of the peptide or polypeptide of the present invention. These compounds, which can be called peptidomimetics (mimicking peptides), are also functional equivalents, as they may be used in the same manner as the peptide of the present invention.

[0131] Specific mimetic molecules that mimic elements of the secondary and tertiary structures of proteins are described in the literature [see Johnson et al. (1993)]. The fundamental rationale for using peptide mimetic molecules is that the peptide backbone of a protein exists primarily to orient amino acid side chains in a way that facilitates molecular interactions such as those between antibodies and / or antigens. Therefore, peptide mimetic molecules are designed to allow molecular interactions similar to those of native molecules.

[0132] Some successful applications of the peptide mimicry concept focus on mimicking β-turns in proteins known to be highly antigenic. The structure of a similar β-turn within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetic bodies can be constructed to achieve similar spatial orientations of the essential elements of the amino acid side chains.

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

[0134] The beta-II turn was successfully mimicked using a cyclic L-pentapeptide and a D-amino acid. Weisshoff et al. (1999). Furthermore, the literature [Johannesson et al. (1999)] reports on a bicyclic tripeptide with reverse turn-inducing properties.

[0135] Methods for generating specific structures are disclosed in the industry. For example, alpha-helical mimes are described in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures further stabilize peptides or proteins thermally and increase their resistance to proteolysis. Six-membered, seven-membered, eleven-membered, twelve-membered, thirteen-membered, and fourteen-membered ring structures are disclosed.

[0136] Methods for generating structurally restricted beta-turns and beta-bulges are described, for example, in U.S. Patents 5,440,013; 5,618,914; and 5,670,155. Beta-turns allow for lateral substitutions with altered structures without corresponding changes in skeletal morphology and have suitable ends for integration into peptides by standard synthetic procedures. Other types of mimic turns include reverse turns and gamma turns. Reverse turn mimics are disclosed in U.S. Patents 5,475,085 and 5,929,237, and gamma turn mimics are described in U.S. Patents 5,672,681 and 5,674,976.

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

[0138] In certain embodiments, the vector may be configured to include an exogenous nucleic acid sequence that causes cells to express the proteinaceous compositions and peptide variants disclosed herein. Details of the components of these vectors and the methods of delivery are disclosed below.

[0139] In some embodiments, the compositions disclosed herein can be administered to cells to include one or more genetic modifications by cellular genetic engineering. Cells are “genetically modified,” “genetically altered,” or “transgenic” if exogenous nucleic acids or polynucleotides are delivered into the cell by any suitable artificial manipulation means, or if the cells are descendants of the original altered cell that inherited the polynucleotides.

[0140] In various embodiments, DNA constructs or vectors encoding peptide sequences of the wild-type or mutant Aβ peptide or its fragment or functional derivative disclosed herein are provided. Genetic modifications may also be introduced into cells. Such modifications include, for example, transducing cells with a vector encoding the wild-type Aβ peptide or Aβ peptide mutant or its fragment, or a functional derivative, to produce cells expressing the wild-type Aβ peptide or Aβ peptide mutant or its fragment, or a functional derivative. Viral vectors encoding peptide sequences of the wild-type Aβ peptide or Aβ peptide mutant or its fragment or a functional derivative disclosed herein enable continuous gene expression and Aβ peptide release.

[0141] The cells described herein include any cells into which the protein compositions and peptide variants disclosed herein and / or DNA constructs or vectors prepared to contain exogenous nucleic acid sequences so as to express the protein compositions and peptide variants disclosed herein can be introduced and expressed as described herein. It should be understood that the basic concepts of the invention as described herein are not limited by cell type. The cells described herein include eukaryotic cells, mammalian cells, animal cells, human cells, and the like. Furthermore, cells include all cells that are beneficial or preferred for regulating the production of functional proteins.

[0142] 1. Modification of peptides to improve exogenous signaling In some embodiments, the wild-type or mutant Aβ peptides disclosed herein may be modified to improve cellular uptake or absorption of the peptide. For example, in some embodiments, a cell-permeable peptide (CPP) attaches to the wild-type or mutant Aβ peptide to produce a cell-permeable and / or brain-permeable inhibitor 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 or large DNA fragments. The cargo is linked to the peptide through chemical linkage via covalent bonds or through non-covalent interactions. CPPs generally have an amino acid composition that contains a relatively large number of positively charged amino acids such as lysine or arginine, or has a sequence that includes an alternating pattern of polar, charged amino acids and nonpolar, hydrophobic amino acids. These two types of structures are called polyvalent cationic or amphiphilic, respectively. A third type of CPP is a hydrophobic peptide that contains only purely low-charge nonpolar residues or groups of hydrophobic amino acids important for cellular uptake.

[0143] In some implementations, the cell-permeable peptide is a retro-inverted version of the HIV protein transmission domain "TAT"; polyarginine (e.g., R8-R12); polyamine; MAP; MTS; MPG; penetratin; Pep-1; transportan; or VP22. In some implementations, the cell-permeable peptide is a retro-inverted version of the HIV protein transmission domain "TAT". In some implementations, the cell-permeable peptide is polyarginine. In some implementations, the cell-permeable peptide is polyamine. In some implementations, attaching the cell-permeable peptide to a wild-type or mutant Aβ peptide makes the wild-type or mutant Aβ peptide cell-permeable without vectorization.

[0144] The transactivating transcriptional activator (TAT) of human immunodeficiency virus 1 (HIV-1) penetrates the impermeable phospholipid bilayer of the cell membrane, allowing it to pass through biological barriers. (See reference: M. Green et al., Biochim Biophys Acta 1414:127-139 (1998), specifically the full text of which is included as a reference in this application). After being secreted in HIV-infected cells, TAT migrates to adjacent cells, altering gene transcription and spreading the disease. The first report demonstrating that Tat-derived peptides can deliver large proteins to other cell types and mammalian organs was published in 1994, demonstrating the chemical crosslinking and identification of 36 amino acid regions of HIV-1 that can facilitate the uptake of β-galactosidase into living cells as a chimera. See reference [S. Fawell et al., Proc Natl Acad Sci USA 91:664~668 (1994)]. This HIV TAT protein transduction domain (PTD) contains a sequence presumed to adopt a cluster of basic amino acid residues and an α-helical configuration. Numerous studies have aimed to describe whether shorter domains of the TAT peptide are sufficient for intracellular incorporation. The main determinant required for translocation has been identified as a cluster of basic amino acids, and it has been shown that peptides with an α-helical region can enter cells more efficiently, but the presumed α-helical domain is unnecessary. The cleaved multicationic peptide GRKKRRQRRR, containing RNA-binding and nuclear localization signal (NLS) motifs, has been identified as suitable for effective translocation into cells and tissues and is included in TAT. See reference [E. Vives E et al., J Biol Chem 272:16010~16017 (1997)]. Therefore, in some implementations, wild-type or mutant Aβ peptides are attached to the TAT sequence.The TAT sequence may include a peptide having an amino acid sequence comprising GRKKRRQRRR (SEQ ID NO: 23), YGRKKRRQRRR (SEQ ID NO: 24), GRKKRRQRRRPQ (SEQ ID NO: 25), and the 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 with any SEQ ID NO: 23 to 25, or any fragment or derivative thereof.

[0145] Oligo- and poly-arginines are structurally the simplest CPPs, with Arg being the only building block, and can be easily manufactured. When poly-arginines bind to phospholipid membranes in aqueous solution or due to strong side-chain charge repulsion and the lack of hydrophobic or amphiphilic structures, they adopt a random coil morphology, and membrane permeability depends primarily on electrostatic interactions with the lipid membrane 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 contain polymers of L- or D-arginines containing six or more arginine amino acids. In some implementations, polyarginine contains R8-R12, which corresponds to the amino acid sequence containing RRRRRRRR-RRRRRRRRRRRR (Sequence ID: 26) (Reference: G. Tunnemann, et al., J. Pept. Sci. 14:469-476 (2008), specifically the full text of which is included as a reference in this application).

[0146] Polyamines, such as putrescine, spermidine, and spermine, have been shown to increase protein permeability across the blood-nerve and blood-brain barriers. Polyamine transporters may be responsible for the transport of polyamine-mutant proteins. See, for example, the literature [references: JFPoduslo & G.L.Curran, J. Neurochem 66:5705~5709 (1996); JFPoduslo & G.L.Curran, J. Neurochem 67:734~741 (1996); and JFPoduslo, in addition to J. Neurobio 39(3):371~82 (1999), specifically the full text of which is included as a reference in this application]. Thus, systemic administration of polyamine-mutant wild-type or mutant Aβ peptides may be an efficient approach for delivering these therapeutic agents to the CNS for the treatment of a variety of neurological diseases, including Alzheimer's disease, and in some implementations, the wild-type or mutant Aβ peptide is covalently bound to the polyamine. In some applications, the polyamine is putrescine. In some applications, the polyamine is spermidine. In some applications, the polyamine is spermine.

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

[0148] In some implementations, cell-permeable and / or brain-permeable Aβ peptide variants include Aβ peptide variants in which the spontaneously occurring L-amino acid of the Aβ peptide variant is replaced by the D-enantiomer of the amino acid. See reference [JF. Poduslo et al., J. Neurobio 39(3):371~82 (1999)]. Peptides containing the D-residue are even more resistant to proteolysis. See reference [Robson, Nat Biotechnol 14:893~895 (1996), and Schumacher et al., Science 271:1854~1857 (1996)]. Furthermore, such modifications can make Aβ peptide variants less immunogenic, because the D-residue peptide is far less antigenic, while the L-amino acid peptide is efficiently processed for restricted presentation to the main histocompatibility complex class II- on T helper cells, thus generating an active humoral immune response that impairs the bioactivity of the drug. See reference [Herve, and J.Immunol.156:157~163(1997)]. Accordingly, in some embodiments, the spontaneously occurring L-amino acids of the Aβ peptide variants disclosed herein are replaced with D-enantiomers of the amino acids to improve the cell permeability 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 ligating a nucleic acid encoding the wild-type or mutant Aβ peptide or a portion thereof to a promoter, and contaminating the construct with an expression vector that is absorbed and expressed by cells. The vector may be suitable for replication and, optionally, integration within eukaryotes. Typical cloning vectors include transcription and translation terminators, start sequences, and promoters useful for regulating the expression of a target nucleic acid sequence. For example, nucleic acids 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-generating vectors, and sequencing vectors. Generally, suitable vectors include a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193). In some implementations, suitable vectors can cross the blood-brain barrier.

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

[0151] Numerous viral-based systems have been developed for gene transfer into mammalian cells. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses (including self-inactivating lentiviral vectors). For example, adenoviruses provide a convenient platform for gene transfer systems. Selected genes may be inserted into vectors using commercially known techniques and packaged into retroviral particles. The recombinant viruses may then be isolated and transferred to cells of an in vivo or in vitro target organism. Thus, in some implementations, nucleic acids encoding Aβ peptide variants are introduced into cells using recombinant vectors such as viral vectors containing, for example, lentiviruses, retroviruses, gamma-retroviruses, adeno-associated viruses (AAVs), herpesviruses, or adenoviruses.

[0152] In certain implementations, the vector is an AAV vector. “AAV vector” means a recombinant vector derived from adeno-associated virus serotypes 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 have one or all wild-type AAV genes deleted but still contain a functional reverse-terminal repeat (ITR) nucleic acid sequence. Functional ITR sequences are necessary for the replication, structure, and packaging of AAV virions. The ITR sequence may be a wild-type sequence or substantially identical to it, or it may be modified, for example, by nucleotide insertions, mutations, deletions, or substitutions, as long as the function is maintained. Such an AAV vector may be replicated and packaged with infectious viral particles when present in a host cell expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0153] The AAV genome is a linear single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Reverse-terminal repeats (ITRs) are located on the sides of the unique coding nucleotide sequences for unstructured replication (Rep) and structural replication (VP) proteins. VP proteins (VP1, -2, and -3) form a capsid. The terminal 145nt are self-complementary and configured to form an energetically stable intramolecular duplex that forms a T-shaped hairpin. Such a hairpin structure acts as the origin of viral DNA replication, acting 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 promoter and P19 promoter, respectively, and both Rep proteins have replication function for the viral genome. A splicing event in the Rep ORF actually triggers the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that unconjugated mRNA encoding the Rep78 and Rep52 proteins is sufficient for AAV vector production in mammalian cells. Furthermore, the Rep78 and Rep52 proteins are also sufficient for AAV vector production in insect cells.

[0154] The AAV VP protein is known to determine the cellular affinity of AAV virions. The VP protein-coding sequence is far less conserved than the Rep protein and gene within different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of anamorphic AAV particles containing the capsid protein of one serotype (e.g., AAV5) and the Rep and / or ITR sequences of another AAV serotype (e.g., AAV2). In this specification, anamorphic AAV particles may also be referred to as "x / y" types, where "x" indicates the source of the ITR and "y" indicates the serotype of the capsid, for example, a 2 / 5 AAV particle having the ITR of AAV2 and the capsid of AAV5.

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

[0156] Any compatible serotype of AAV may be used as a vector, and the vector may contain one or more polynucleotide sequences of interest. In some implementations, AAV vectors containing one or more polynucleotide sequences of interest are 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 vectors. In some implementations, the vectors can cross the blood-brain barrier. In some implementations, vectors that can cross the blood-brain barrier include AAV9, PHP.eB, AAVrh.8, AAVrh.10, or AAVrh.43. In some implementations, vectors capable of crossing the blood-brain barrier can be transmitted, for example, intravenously, intracerebrally, and / or intravenously, enabling efficient and widespread transduction of neurons in the adult CNS. In some implementations, adenovirus vectors are modified to reduce host response. See references [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); these references are specifically included in full in this application].

[0157] Those skilled in the art will be able to construct an AAV vector containing one or more polynucleotide sequences of interest with at least one AAV ITR located laterally, through standard recombinant techniques (e.g., Maniatis et al., 1988 and Ausubel et al., 1994, the literature of which is specifically included in its entirety as a reference).

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

[0159] Such components may also include detectable and / or sorting markers that can be used to detect or sort cells that absorb and express nucleic acids transmitted by the vector. Such components may be provided as a natural feature of the vector (e.g., the use of a specific viral vector having a component or function that mediates binding and uptake) or the vector may be modified to provide such a function. A wide variety of such vectors are publicly known in the art and are generally available. If the vector is maintained in a host cell, the vector 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 includes, in particular, a eukaryotic transcription promoter operably linked to a protein-coding sequence, a splice signal including an intervening sequence, a transcription termination / polyadenylation sequence, a post-transcriptional regulatory element, and a replication origin (5' to 3' direction).

[0161] a. Promoter / Enhancer A "promoter" is a regulatory sequence, which is a region of a nucleic acid sequence whose initiation and transcription rate are controlled. A promoter may contain a genetic element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind in order to initiate specific transcription of the nucleic acid sequence. "Operatable positioning," "operable linking," "under control," and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence and controls the transcription initiation and / or expression of the sequence in question.

[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, and in some promoters without a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the SV40 late gene, the individual elements above the start site help to fix the start site. Additional promoter elements regulate the transcription initiation frequency. Generally, additional promoter elements are located in the 30-110 bp upstream region of the start site, and many promoters have been shown to also contain functional elements downstream of the start site. To bring the coding sequence "under the control" of the promoter, the 5' end of the transcription start site is positioned in the "downstream" (i.e., 3') transcription read frame of the selected promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0163] The spacing between promoter elements is often flexible, so that the promoter's function is preserved when elements are inverted or moved relative to each other. For example, in a thymidine kinase promoter, the spacing between promoter elements may be increased to a 50 bp interval before activity begins to decrease. It appears that the promoter allows individual elements to function cooperatively or independently to activate transcription. Promoters may or may not be used with "enhancers," which are cis-action regulatory elements involved in the transcriptional activation of nucleic acid sequences.

[0164] The promoters may be naturally associated with the nucleic acid sequence, as can be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. These promoters may be noted as “endogenous.” Similarly, the enhancer may be naturally associated with the nucleic acid sequence located downstream or upstream of the sequence in question. Alternatively, certain advantages can be obtained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which means a promoter that does not normally associate with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer may also mean an enhancer that does not normally associate with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, any other virus, or promoters or enhancers isolated from prokaryotic or eukaryotic cells, and promoters or enhancers that are not “spontaneously generated,” i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions, and / or mutations that modify expression. In addition to synthetically generating the nucleic acid sequences of promoters and enhancers, PCR may be used in relation to the compositions disclosed herein. TMSequences can be generated using recombinant cloning and / or nucleic acid amplification techniques, including (see U.S. Patents 4,683,202 and 5,928,906, respectively, the full text of which is specifically included herein). Regulatory sequences that direct the transcription and / or expression of non-nuclear organelle sequences, such as mitochondria, may also be used.

[0165] Naturally, it is important to use promoters and / or enhancers that effectively direct the expression of a DNA segment in a selected organelle, cell type, tissue, organ, or organism for expression. Experts in the field of molecular biology are generally familiar with the use of promoter, enhancer, and cell type combinations for protein expression (e.g., Sambrook et al. 1989, specifically, the full text of which is included as a reference herein). 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, which is advantageous, for example, for the large-scale production of recombinant proteins and / or peptides. Promoters may be heterogeneous or endogenous.

[0166] Furthermore, any promoter / enhancer combination [e.g., the Eukaryotic Promoter Database (EPDB) via the World Wide Web (epd.isb-sib.ch / )] may also be used to drive expression. Other non-restrictive examples of potential promoters include early or late viral promoters, e.g., SV40 early or late promoter, cytomegalovirus (CMV) immediate early promoter, Roussarcoma virus (RSV) early promoter; eukaryotic cell promoters, e.g., 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 promoters (cre), serum response element promoters (sre), phorbol ester promoters (TPA), and response element promoters (tre) near the minimum TATA box. It is also possible to use a human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter listed in Genbank, accession number X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from ATCC, Cat. No. ATCC 45007). A specific example may be a phosphoglycerate kinase (PGK) promoter.

[0167] In some implementations, polynucleotide expression is regulated by constitutive promoters. In some implementations, these constitutive promoters are CAG (also known as CAGGS or CBA), EF-1ALPHA, ubiquitin, or CMV.

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

[0169] In some implementations, polynucleotide expression is regulated by tissue-specific promoters. In some implementations, the tissue-specific promoter is a choroid plexus-specific promoter. In some implementations, the choroid plexus-specific promoter includes the Prlr promoter, Spint2 promoter, or F5 promoter. In some implementations, the tissue-specific promoter is a liver-specific promoter. Liver-specific promoters are described, for example, in the literature [see L.M. Kattenhorn, as well as Hum. Gene Ther. 27(12):947-961 (2016), specifically the full text of which is included as a reference in this application].

[0170] b. Protease cleavage sites / Binding sites for self-cleaving peptides and internal ribosomes Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, e.g., Ryan et al., 1997; Scymczak et al., 2004). Examples of protease cleavage sites include furin protease, potivirus NIa protease (e.g., tobacco ecchi virus protease), potivirus HC protease, potivirus P1 (P35) protease, biovirus Nla protease, biovirus RNA-2-encoding protease, aftovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tunglobulin virus) 3C-like protease, PYFV (parsnip yellow spot virus) 3C-like protease, thrombin, factor Xa, and enterokinase cleavage sites. Due to its high cleavage stringency, the cleavage site of tobacco etch virus (TEV) protease can be used. In some implementations, the cleavage site of the protease is the cleavage site of furinprotease.

[0171] Exemplary self-cleaving peptides (also known as “cis-action hydrolysis elements,” see CHYSEL; de Felipe (2002)) are derived from potivirus and cardiovirus 2A peptides. Specific self-cleaving peptides may be selected from 2A peptides of FMDV (foot-and-mouth disease virus), equine rhinitis A virus, TaV (Thosea asigna virus), and porcine tescovirus.

[0172] Specific start signals may also be used in polycistron messages for efficient translation of coding sequences. These signals may include an ATG start codon or adjacent sequence. For example, the start signal may include a Kozak consensus sequence having an amino acid sequence containing GCCACCAUGGG (SEQ ID NO: 34). See [Kozak, 1987; Harte et al., 2012]. In some cases, it may be necessary to provide an exogenous translation control signal containing an ATG start codon. Those skilled in the art will be able to easily determine this and provide the required signal. It is well known that, in order to ensure translation of the whole insert, the start codon must be "in frame" with the reading frame of the coding sequence of interest. The exogenous translation control signal and start codon may be natural or synthetic. Expression efficiency may be improved by including appropriate transcriptional enhancer elements.

[0173] In certain implementations, the use of internal ribosome entry site (IRES) elements is employed to generate multiple gene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylation cap-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements for two members of the picornavirus lineage (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), and IRES elements for mammalian messages have also been described (Macejak and Sarnow, 1991). IRES elements may be ligated to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES to generate a polycistronic message. Thanks to the IRES element, each open reading frame may have access to a ribosome for efficient translation. Multiple genes may be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, respectively, as incorporated herein by reference).

[0174] c. Multicloning site The vector may include a multicloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites that may be used in conjunction with standard recombination techniques to degrade the vector (e.g., Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, in particular, the full text of which is included herein by reference). “Restriction enzyme digestion” means catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations on the nucleic acid molecule. Many of such restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Frequently, vectors are linearized or fragmented using restriction enzymes that cleave within the MCS so that an exogenous sequence can be ligated into the vector. “Ligation” means the process of forming a phosphodiester bond 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 field of recombinant art.

[0175] d. Splicing site Most transcribed eukaryotic RNA molecules undergo RNA splicing, removing introns in the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or apceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler 1997, as referenced herein).

[0176] e. Termination signal A vector or construct may contain at least one termination signal. The “termination signal” or “terminator” consists of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Therefore, in certain implementations, a termination signal that terminates the production of an RNA transcript is considered. A terminator may be necessary in vivo to achieve the desired message level.

[0177] In eukaryotic systems, the termination region may also contain a specific DNA sequence that allows for site-specific cleavage of a new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of approximately 200 A residues (polyA) to the 3' end of the transcript. The RNA molecule deformed with this polyA tail appears to be more stable and more efficiently translated. Thus, in other modes of implementation, including eukaryotes, the terminator contains a signal for RNA cleavage, and the termination signal facilitates polyadenylation of the message. Elements of the terminator and / or polyadenylation site can play a role in enhancing the message level and minimizing read-through to other sequences in the cassette.

[0178] The terminators considered include, but are not limited to, gene termination sequences such as the terminator of bovine growth hormone, or viral termination sequences such as SV40, as described herein or known to those skilled in the art. In certain embodiments, the termination signal may be, for example, the absence of a transcribable or translatable sequence due to sequence cleavage.

[0179] f. Polyadenylation signal Expression, particularly in eukaryotic expression, will generally involve polyadenylation signals to ensure proper polyadenylation of the transcript. The characteristics of the polyadenylation signal are not considered decisive for successful implementation, and any sequence may be used. Exemplary implementations include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, both 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-transfer regulatory elements The vectors used in this disclosure may also contain one or more post-transcriptional regulatory elements (PREs). Examples of PREs include the woodchuck hepatitis virus PRE (WPRE), the hepatitis B virus PRE, and intron A of the human cytomegalovirus pre-early gene. For additional examples and details, see the references [c. Sun et al., 2009 and Mariati et al., 2010]. In a particular mode of implementation, the PRE is a WPRE. A WPRE is a DNA sequence that, when transcribed, generates a tertiary structure to enhance the expression of the gene transmitted by the viral vector.

[0181] h. Origin of replication To propagate the vector in host cells, the vector may contain one or more replication site origins (variously referred to as "ori"), for example, nucleic acid sequences corresponding to the oriP of EBV or genetically engineered oriP as described above (which have similar or enhanced function in differentiation programming, specifically the nucleic acid sequence in which replication begins). Alternatively, replication origins of other extrachromosomal replicating viruses, or autonomous replication sequences (ARS), as described above, may be used.

[0182] 3. Vector Transfer Genetic modification or introduction of exogenous nucleic acids into cells may be carried out using any suitable method for nucleic acid delivery 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 connection with expression vectors, the vectors may be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method of the art. For example, expression vectors may be delivered to host cells by physical, chemical, or biological means.

[0183] These methods include direct DNA transfer, e.g., in vitro transfection (Wilson et al., 1989; Nabel et al., 1989); transduction; viral transduction; microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215 (included as reference herein)); and injection (U.S. Patents No. 5,994,624, 5,981,274, 5,945,100, 5,945,100). U.S. Patent Nos. 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each included as reference herein); electroporation (U.S. Patent No. 5,384,253, included as reference herein; 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); use of polyethylene glycol following DEAE dextran (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 containment-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); macroinjection or nanoparticle gun (PCT applications WO94 / 09699 and 95 / 06128; U.S. Patent No. 5,610,042; Nos. 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, as included herein by reference); stirring with silicon carbide fibers (Kaeppler et al.) This includes, but is not limited to, the following: 1990; U.S. Patents 5,302,523 and 5,464,765, respectively; Agrobacterium-mediated transformation (U.S. Patents 5,591,616 and 5,563,055, respectively); PEG-mediated transformation of protoplasm (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, respectively); desiccation / inhibition-mediated DNA absorption (Potrykus et al., 1985); thermal shock (Froger and Hall, 2007); and combinations thereof. Such techniques may be applied to stably or temporarily deform organelles, cells, tissues, or organisms.

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

[0185] Biological methods for introducing polynucleotides of interest into host cells may include the use of DNA and RNA vectors into which the polynucleotide or transgene of interest can be inserted. Viral vectors have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses (see, for example, U.S. Patents 5,350,674 and 5,585,362).

[0186] Chemical means for introducing polynucleotides into host cells include macromolecular 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 considered. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0187] Methods for gene therapy and, for example, methods for transferring genes to a target body using adeno-related viruses are described in Patent Documents US6,967,018, WO2014 / 093622, US2008 / 0175845, US2014 / 0100265, EP2432490, EP2352823, EP2384200, WO2014 / 127198, WO2005 / 122723, WO2008 / 137490, and WO2013 / 1421. 14, WO2006 / 128190, WO2009 / 134681, EP2341068, WO2008 / 027084, WO2009 / 054994, WO2014059031, US7,977,049 and WO2014 / 059029 (each of which is included in its entirety as a reference in this application).

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

[0189] In certain implementations, nucleic acids may be captured 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 the aqueous medium. The lipid layers are formed spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement before a closed structure is formed, capturing water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The amount of liposomes used may vary depending not only on the cells used but also on the characteristics of the liposomes; for example, about 5 to 20 μg of vector DNA per 1 million to 10 million cells may be considered.

[0190] Liposome-mediated nucleic acid transfer and in vitro expression of foreign DNA were highly successful (see references: Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The possibility of liposome-mediated transfer and foreign DNA expression in cultured chicken embryos, HeLa, and liver cancer cells was also demonstrated (Wong et al., 1980).

[0191] In certain embodiments, liposomes may form complexes with hemagglutinating virus (HVJ). This has been shown to promote fusion with the cell membrane and facilitate the entry of liposome-encapsulated DNA into the cell (Kaneda et al., 1989). In other embodiments, liposomes may be complexed with or used together with nuclear non-histone chromosome protein (HMG-1) (Kato et al., 1991). In yet another embodiment, liposomes may be complexed with or used together with both HVJ and HMG1. In yet another embodiment, the transmission vehicle may include ligands and liposomes.

[0192] Lipids suitable for use in a variety of applications can be obtained from commercial sources. For example, lipofectamine can be obtained from Thermo Fisher Scientific in Waltham, Massachusetts, USA; dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma in St. Louis, Missouri, USA; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories in Plainview, New York, USA; cholesterol ("Choi") can be obtained from Calbichem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. in Birmingham, Alabama, USA. The lipid stock solution may be stored at approximately -20°C in chloroform or chloroform / methanol. Chloroform may be used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a diverse range of single and multilayer lipid vehicles formed by the formation of a surrounding lipid bilayer or aggregate. Liposomes may also be characterized as having a porous structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Lipid layers are formed spontaneously when phospholipids are suspended in an excess 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 with structures different from the normal porous structure in solution are also included. For example, lipids may exist in micelle structures or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0193] b. Electroporation method In certain implementations, nucleic acids are introduced into cells via electroporation. Electroporation involves exposing the cell suspension and DNA to a high-voltage electrical discharge. Recipient cells can be made more sensitive to transformation by mechanical injury. The amount of vector used may vary depending on the characteristics of the cells used; for example, approximately 5 to 20 μg of vector DNA per 1 million to 10 million cells may be considered.

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

[0195] c. Calcium phosphate method In other implementations, nucleic acids are introduced into cells using calcium phosphate precipitation. Human KB cells were transfected with adenovirus 5DNA using this technique (Graham and Van Der Eb, 1973). Furthermore, neomycin marker genes were transfected into mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3, and HeLa cells using this method (Chen and Okayama, 1987), and various marker genes were transfected into rat hepatocytes (Rippe et al., 1990).

[0196] d. DEAE-dextran method In other implementations, nucleic acids are delivered into cells using DEAE-dextran followed by polyethylene glycol. Reporter plasmids were introduced into mouse myeloma and erythroleukemia cells using this method (Gopal, 1985).

[0197] 4. Selectable or screenable markers Regardless of the method of introducing exogenous nucleic acids into host cells or the method of exposing cells to the inhibitor of the present invention, a variety of tests can be performed to confirm the presence of recombinant DNA sequences within host cells. Such tests include “molecular biological” tests well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and “biochemical” tests such as detecting the presence or absence of specific peptides, such as immunological means (ELISA and Western blotting) or tests described in this application for confirming agonists that fall within the scope of this disclosure.

[0198] In certain applications, cells containing exogenous nucleic acids can be identified in vitro or in vivo by including a marker in the expression vector or the exogenous nucleic acid. Such markers will confer a identifiable change to the cells so that they can be easily identified. Generally, selection markers may confer attributes that allow selection. Positive selection markers are those whose presence allows selection, while negative selection markers are those whose presence inhibits selection. An example of a positive selection marker is a drug resistance marker.

[0199] In addition to markers that confer phenotypes enabling the differentiation of transformants based on the embodiment of conditions, other types of markers should be considered, including screenable markers such as GFP based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT) may be used as negative screening markers. Those skilled in the art will also be aware of methods that may use immunological markers in conjunction with FACS analysis. The markers used are considered irrelevant as long as they can be expressed simultaneously with the nucleic acid encoding the gene product. Examples of additional screening 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 procedures for introducing foreign DNA into cells. Selectable markers are often antibiotic resistance genes; cells that have undergone the procedure of introducing foreign DNA are grown in antibiotic-containing medium, and cells that are able to grow successfully absorb and express the introduced genetic material. Examples of selectable markers include the Abicr or Neo genes of Tn5 that confer antibiotic resistance to genethecin.

[0201] A screenable marker may include a reporter gene that allows researchers to distinguish between desired and undesired cells. A particular embodiment of the present invention utilizes a reporter gene to indicate a specific cell lineage. For example, the reporter gene may be located within an expression element under the control of a ventricular- or atrial-selective gene coding region for co-expression and a generally associated ventricular- or atrial-selective regulatory element. The reporter can place cells of a specific lineage under drug or other selective pressure or isolate them without jeopardizing their viability.

[0202] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitopes), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). Vector-containing cells may be separated by FACS using, for example, a substrate that can be converted to a fluorescent product by a fluorescently tagged antibody against a cell surface protein or by an enzyme encoded by the vector.

[0203] In certain applications, reporter genes are fluorescent proteins. Broad-spectrum fluorescent protein genetic variants have been developed, characterized by fluorescence emission spectral profiles spanning almost the entire visible light spectrum (see Table 1 for non-restrictive examples). Mutagenic efforts against the original Aequorea victoria jellyfish green fluorescent protein have resulted in novel fluorescent probes with diverse hues ranging from blue to yellow, which are among 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 marine anemones, Discosoma striata, and reef corals belonging to the Anthozoa species. Yet another species has been mined to produce similar proteins with blue-green, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins to enhance their overall utility.

[0204] TIFF0007867713000015.tif231168TIFF0007867713000016.tif98168

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

[0206] In certain embodiments, the disclosed method further includes a step of treating an object diagnosed with a neurodegenerative disease, disorder, or condition. In certain embodiments, the disclosed method further includes a step of treating an object diagnosed with symptoms of a neurodegenerative disease, disorder, or condition. In certain embodiments, the disclosed method further includes a step of treating an object identified as being at risk of having a neurodegenerative disease, disorder, or condition. Using the tests and diagnostic methods publicly known in the art and described herein, an object may be diagnosed with, or be diagnosed with, or at risk of having, a neurodegenerative disease, disorder, or condition.

[0207] In some embodiments, the method further comprises the step of determining a target body requiring treatment, comprising a therapeutically effective amount of a composition containing a vector encoding a wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof. In some embodiments, a vector encoding an Aβ peptide may encode a minigene that enables the expression of the Aβ peptide protein, and in some embodiments, the minigene may contain 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, or any derivable value within these ranges. In some embodiments, the method further includes providing a subject with a therapeutically effective amount of a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof, if it is determined that the subject requires such treatment. In some embodiments, the step of determining a subject that requires treatment comprising a therapeutically effective amount of a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof includes the step of diagnosing the subject with a neurodegenerative disease, disorder, or condition. In some embodiments, the step of determining a subject that requires treatment comprising a therapeutically effective amount of a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof includes the step of diagnosing the subject having symptoms of a neurodegenerative disease, disorder, or condition. In some embodiments, the step of determining a subject that requires treatment comprising a therapeutically effective amount of a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof includes the step of identifying a subject at risk of having a neurodegenerative disease, disorder, or condition.

[0208] In some embodiments, the disclosed methods include administering a therapeutically effective amount of a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof to a subject suffering from a neurodegenerative disease, disorder, or condition. As disclosed herein, neurodegenerative diseases may be associated with the aggregation or oligomerization of Aβ peptides, and it has been unexpectedly revealed that administration of a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof surprisingly prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils, or plaques. Furthermore, 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. Accordingly, in some embodiments, compositions and corresponding methods are disclosed for treating subjects suffering from neurodegenerative diseases, disorders, or conditions with a therapeutically effective amount of a composition comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof. In an additional embodiment, a method is disclosed for inhibiting the aggregation of endogenous Aβ peptides in vivo, comprising the step of contacting at least one endogenous Aβ peptide in vivo with a therapeutically effective amount of an Aβ peptide variant expressed from a vector encoding an Aβ peptide variant, wherein the vector is optionally included in the composition. In an additional embodiment, a method is disclosed for promoting the aggregation of endogenous Aβ peptides in vivo, comprising the step of contacting at least one endogenous Aβ peptide in vivo with a therapeutically effective amount of a wild-type Aβ peptide or Aβ peptide variant expressed from a vector encoding a wild-type-Aβ peptide or an Aβ peptide variant, wherein the vector is optionally included in the composition. In some embodiments, a 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 organism may have, for example, a medical condition having abnormal aggregation of Aβ peptides as a symptom and / or mechanism. The embodiments of this disclosure include the treatment or prevention of any medical condition in which the regulation of Aβ peptide aggregation is beneficial. In a particular embodiment, a therapeutically effective amount is provided of one or more compositions comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative for attenuating, or delaying or reversing, Aβ peptide aggregation in an organism. In a particular embodiment, a therapeutically effective amount is provided 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 for promoting Aβ peptide aggregation in an organism. In a particular embodiment, the medical conditions treated or prevented with a composition comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative include neurodegenerative diseases, disorders, or conditions characterized by Aβ peptide aggregation. In a particular embodiment, Aβ peptide aggregation is not treated with the compositions of this disclosure. In some cases, compositions comprising vectors encoding Aβ peptide variants or fragments or functional derivatives thereof can treat or prevent medical conditions in an individual by, for example, improving, inhibiting, delaying, or reversing Aβ peptide aggregation. In some cases, compositions comprising vectors encoding wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof can treat or prevent medical conditions in an individual by, for example, promoting Aβ peptide aggregation. In some embodiments, improving, inhibiting, delaying, or reversing Aβ peptide aggregation can weaken tau protein seeding.

[0210] Subjects may have conditions having, for example, abnormal amyloid plaque formation, protein misfolding, increased tau protein levels and / or phosphorylated tau protein levels, increased tau protein and / or Aβ peptide seeding, neuroinflammation, cognitive decline, neuronal degeneration, neuronal loss, and / or synaptic loss as symptoms and / or mechanisms. Modes of implementation of the present disclosure include the treatment or prevention of any medical condition in which the modulation of amyloid plaque formation, protein misfolding, tau protein levels, tau protein phosphorylation, seeding rate of tau protein and / or Aβ peptide seeding, neuroinflammation, cognitive decline, neuronal degeneration, neuronal loss, and / or synaptic loss is beneficial. In a particular mode of implementation, a therapeutically effective amount is provided to an individual of one or more compositions comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative for attenuation of 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 a particular mode of implementation, medical conditions treated or prevented with a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative include neurodegenerative diseases, disorders or conditions that may lead to protein misfolding, aggregation of endogenous Aβ peptides, 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 peptides, amyloid plaque formation, neuroinflammation, neuronal degeneration, 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, compositions comprising vectors encoding Aβ peptide variants or fragments or functional derivatives thereof may treat or prevent medical conditions in an individual by, for example, protein misfolding, aggregation of endogenous Aβ peptides, amyloid plaque formation, neuroinflammation, neuronal degeneration, neuronal loss or synaptic loss; abnormal tau levels, tau phosphorylation, or phosphorylated tau levels; tau seeding or endogenous Aβ peptide seeding; and / or improving or suppressing cognitive decline.

[0211] The embodiments of this disclosure include compositions and methods for treating or preventing cognitive decline as a result of protein misfolding, aggregation of endogenous Aβ peptides, 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 neurodegenerative disorders. In specific cases, transmission to an individual of a composition comprising a vector encoding an Aβ peptide variant or a fragment or functional derivative thereof prevents or reduces the formation of endogenous Aβ peptide oligomers, protofibrils, fibrils, or plaques or cytotoxicity of endogenous Aβ peptide aggregates. In specific cases, transmission to an individual of a composition comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof promotes the formation of plaques of endogenous Aβ peptide oligomers, protofibrils, fibrils, or endogenous Aβ peptide aggregates.

[0212] In some embodiments, a vector encoding the wild-type Aβ peptide contains 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, or any derivable value within these ranges. In some embodiments, the wild-type Aβ peptide of this disclosure contains, constitutes, or is essentially constituted by, SEQ ID NO: 2, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 2 contains 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 with KLVFFAE (SEQ ID NO: 39), or any derivable value within these ranges.

[0213] In some implementations, the vector encoding the Aβ peptide variant or its fragment or functional derivative contains the variant of SEQ ID NO: 2, which corresponds to the wild-type Aβ peptide amino acid sequence.

[0214] In some implementations, Aβ peptide variants are TIFF0007867713000017.tif13168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 3, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 3 contains 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 with KLVDFAE (SEQ ID NO: 10), or any derivable value within these ranges.

[0215] In some implementations, Aβ peptide variants are TIFF0007867713000018.tif11168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 4, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 4 contains 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 with KLVFPAE (SEQ ID NO: 11), or any derivable value within these ranges.

[0216] In some implementations, Aβ peptide variants are TIFF0007867713000019.tif11168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 5, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 5 contains 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 with KLVDFAE (SEQ ID NO: 10), or any derivable value within these ranges.

[0217] In some implementations, Aβ peptide variants are TIFF0007867713000020.tif11168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 6, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 6 contains 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 with KLVPFAE (SEQ ID NO: 12), or any derivable value within these ranges.

[0218] In some implementations, Aβ peptide variants are TIFF0007867713000021.tif11168, or a fragment or functional derivative thereof, 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, or any derivable value within these ranges. In some embodiments, the Aβ peptide variants of this disclosure comprise, constitute, or are essentially composed of, SEQ ID NO: 7, or a fragment or functional derivative thereof. In some embodiments, the fragment or functional derivative of SEQ ID NO: 7 contains 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 with KLPFFAE (SEQ ID NO: 65), or any derivable value within these ranges. In some embodiments, the Aβ peptide variant contains, is composed of, or is not essentially composed of SEQ ID NO: 7.

[0219] In some embodiments, wild-type Aβ peptides or Aβ peptide variants that include, or are composed of, or essentially constitute, 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 include an N-terminal cleavage. In some embodiments, the N-terminal cleavage includes 1 to 22 amino acid cleavages. In some embodiments, the N-terminal cleavage includes 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 cleavages.

[0220] In some embodiments, wild-type Aβ peptides or Aβ peptide variants that include, or are composed of, or essentially constitute, 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 include a C-terminal cleavage. In some embodiments, the C-terminal cleavage includes 1 to 27 amino acid cleavages. In some embodiments, the C-terminal cleavage includes 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 cleavages.

[0221] In some embodiments, wild-type Aβ peptides or Aβ peptide variants that include, or are composed of, or are essentially composed 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 include N-terminal and C-terminal cleavage. In some embodiments, the N-terminal cleavage includes 1 to 22 amino acid cleavages, and the C-terminal cleavage includes 1 to 27 amino acid cleavages. In some embodiments, the N-terminal cleavage includes 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 cleavages, and the C-terminal cleavage includes 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 cleavages.

[0222] Wild-type Aβ peptide or Aβ peptide variants may be encoded by a vector encoding a minigene that enables the expression of the Aβ peptide protein. In some embodiments, the minigene may contain 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, or any derivable value within these ranges.

[0223] In addition, in some embodiments, pharmaceutical compositions are disclosed that include a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof. In some cases, a composition including a vector encoding 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 the treatment or prevention of protein misfolding, aggregation of endogenous Aβ peptide, amyloid plaque formation, neuroinflammation, neurodegeneration, neuronal loss or synaptic loss; abnormal tau levels, phosphorylated tau levels, or phosphorylated tau levels; tau seeding or seeding of endogenous Aβ peptide; and / or cognitive decline; or in other cases, the individual is provided with one or more additional therapies to promote Aβ peptide aggregation, amyloid plaque formation and / or fibril formation.

[0224] One or more add-on therapies may be of any kind, but in certain cases, one or more add-on therapies are one or more additional neurodegenerative disease treatments, e.g., Alzheimer's disease drugs. In some practices, Alzheimer's disease drugs include aducanumab, donepezil, rivastigmine, galantamine, memantine, or tacrine. In some practices, Alzheimer's disease drugs are 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 formulations that reduce tau levels or prevent tau aggregation or reduce tau pathological phosphorylation (i.e., kinase inhibitors, anti-tau manual immunization, anti-tau active immunization, or antisense oligonucleotides); drugs that stabilize microtubules and Drugs and formulations; drugs and formulations that reduce neurogenic degeneration; drugs and formulations that block or alter inflammatory responses; drugs and formulations that reduce neuropsychiatric symptoms; drugs and formulations that improve cognition (i.e., neurotransmitter inhibitors, modulators, and activators); drugs and formulations that preserve or improve vascular function; or drugs and formulations that alter cellular metabolism (see, for example, KG Yiannopoulou & S.G. Papageorgiou, J. Cent. Nerv. Sys. Dis. 12:1179573520907397 (2020), and J. Cummings, as well as Alzheimer's Dement. (NY) 5:272-293 (2019)).

[0225] In some settings, one or more additional therapies may include one or more therapies to treat age-related disorders or associated conditions, such as cardiovascular disease, diabetes, atherosclerosis, obesity, cancer, infection, and neuropathy. All well-established indicators of the progression of aging may be used. In some settings, one or more therapies to treat age-related disorders or associated conditions may have the effect of reducing cancer incidence; delaying or mitigating cardiovascular disease such as atherosclerosis; delaying and / or mitigating osteoporosis; improving glucose tolerance or reducing the incidence of related conditions such as diabetes and obesity; improving or reducing the decline in memory and other cognitive functions; improving or reducing declining neuromuscular coordination; and improving or reducing immune function decline. Improvement in age-related disorders may also result from a reduction in symptoms in the affected subject or a reduction in the incidence of disease or disorder in the population compared to the untreated population. One or more therapies may have the effect of treating and / or preventing a variety of age-related conditions and diseases, as assessed by specific markers and age-related disorders. Accordingly, in an additional aspect, the present invention relates to the treatment or prevention of a disorder or aging marker selected from the group of at least reduced cardiovascular function, osteoporosis, arthritis, glucose intolerance, insulin resistance, memory impairment, loss of neuromuscular coordination, increased cardiovascular disease, reduced cardiac, circulatory, or pulmonary function and reduced lifespan, or a combination thereof.

[0226] IV. Administration of therapeutic compositions In certain embodiments, compositions comprising vectors encoding wild-type Aβ peptides or Aβ peptide variants or fragments or functional derivatives thereof are formulated as pharmaceutical compositions for therapeutic administration. In some embodiments, the disclosed methods include the step of administering a neurodegenerative disease therapy to a subject or patient. The compositions described herein may be used for in vivo, in vitro, or in vitro administration. In some implementations, neurodegenerative disease therapy includes protein-based therapies, which may be wild-type Aβ peptide or Aβ peptide mutation therapy. In some implementations, neurodegenerative disease therapy includes polynucleotide-based therapies, which may include therapies containing vectors encoding wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof. In some implementations, neurodegenerative disease therapy includes drugs for one or more neurodegenerative diseases. 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 Aβ peptide variant or a polynucleotide encoding a wild-type Aβ peptide or Aβ peptide variant) and one or more additional neurodegenerative disease therapies (e.g., neurodegenerative disease drugs). The therapies may be administered in any adapted manner known to the art. For example, the first and one or more additional neurodegenerative disease therapies may be administered sequentially (at different times) or simultaneously (simultaneously or nearly simultaneously; also, "simultaneously" or "substantially simultaneously"). In some embodiments, the first and one or more additional neurodegenerative disease therapies may be administered in separate compositions. In some embodiments, the first and one or more additional neurodegenerative disease therapies may be in the same composition. Different therapies may be administered in one or more compositions, such as two, three, or four compositions. A variety of combinations of formulations may be used.

[0228] In some implementations, a composition(s) containing a vector encoding wild-type Aβ peptide or an Aβ peptide variant or its fragment or functional derivative, and one or more additional neurodegenerative disease drugs are administered substantially simultaneously. In some implementations, a composition(s) containing a vector encoding wild-type Aβ peptide or an Aβ peptide variant or its fragment or functional derivative, and one or more additional neurodegenerative disease drugs are administered sequentially. In some implementations, a composition(s) containing a vector encoding wild-type Aβ peptide or an Aβ peptide variant or its fragment or functional derivative is administered before the administration of one or more additional neurodegenerative disease drugs. In some implementations, a composition(s) containing a vector encoding wild-type Aβ peptide or an Aβ peptide variant or its fragment or functional derivative is administered after the administration of one or more additional neurodegenerative disease drugs.

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

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

[0231] A. Carrier In certain embodiments, the pharmaceutical compositions of this disclosure include one or more compositions comprising an effective amount of polynucleotides encoding wild-type Aβ peptide or Aβ peptide variants or variants dissolved or dispersed in a pharmaceutically acceptable carrier. The terms “pharmaceutical” and “pharmaceutically acceptable” are used interchangeably herein and mean molecular entities and compositions that, when appropriately administered to a subject such as a human, do not interfere with the therapeutic methods of this disclosure without causing adverse reactions, allergies, or other undesirable responses. The manufacture of pharmaceutical compositions comprising Aβ peptide variants or fragments thereof, functional derivatives, or at least one vector encoding an additional active ingredient will be known to those skilled in the art in light of this disclosure, as exemplified in the literature [see: Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, specifically, the full text of which is included as a reference herein]. It will also be understood that formulations for administration to a subject must meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0232] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, analogs, and combinations thereof, as made public to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, specifically, the entire text of which is included herein by reference). Any conventional carrier is considered for use in a pharmaceutical composition unless it is incompatible with the active ingredient. Compositions containing wild-type Aβ peptide or Aβ peptide variants or polynucleotides encoding them may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether sterilization is required for administration routes such as infusion.

[0233] Furthermore, according to the present disclosure, the compositions of the present disclosure suitable for administration may be provided with or without an inert diluent on a pharmaceutically acceptable carrier. The carrier should be assimilated and may include 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 unless any common medium, activator, diluent or carrier is detrimental to the therapeutic effect of the recipient or the composition contained therein. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, alcohols, etc., or combinations thereof. The compositions may also contain a variety of antioxidants to delay the oxidation of one or more components. Furthermore, prevention of microbial activity may be induced by a variety of antimicrobial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0234] According to this disclosure, the compositions are combined with carriers by any convenient and substantial method, namely, by solution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such operations are routine to those skilled in the art. Compositions comprising wild-type Aβ peptide or Aβ peptide variants or polynucleotides encoding them may be lyophilized.

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

[0236] In further embodiments, the Disclosure may include the use of a pharmaceutical lipid vehicle composition comprising a composition comprising a wild-type Aβ peptide or an Aβ peptide variant or a polynucleotide encoding one or more such lipids, and an aqueous solvent. The term “lipid” as used herein will be defined as encompassing any broad range of substances that are characteristically insoluble in water and extractable with organic solvents. Such a broad category of compounds is well known to those skilled in the art, and the use of the term “lipid” herein is not limited to any particular structure. Examples of lipids include compounds comprising long-chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are generally biological substances. Biological lipids are well known in the art and include, for example, triglycerides, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, ethers, and lipids and polymerizable lipids having ester-linked fatty acids, and combinations thereof. Of course, compounds other than lipids specifically described herein, which are understood by those skilled in the art to be lipids, are also included in the compositions and methods of this disclosure.

[0237] Those skilled in the art will be familiar with the range of techniques that may be used to disperse compositions in lipid vehicles. For example, a composition (one or more) comprising wild-type Aβ peptide or Aβ peptide variant or polynucleotide encoding it may be dispersed in a lipid-containing solution, dissolved with lipids, liquefied with lipids, mixed with lipids, covalently bound to lipids, contained in a suspension with lipids, contained in or complexed in micelles or liposomes, or associated with lipids or lipid structures by any means known to those skilled in the art. The dispersion may or may not form liposomes.

[0238] A 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 into a free base, neutral, or salt form. Pharmacovigilantly acceptable salts include acid addition salts, e.g., salts formed with a free amino group of a proteinaceous composition, or salts formed with an inorganic acid such as hydrochloric acid or phosphoric acid, or an organic acid such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with a free carboxyl group may also be derived from an inorganic base such as sodium, potassium, ammonium, calcium, or iron hydroxide; or from an organic base such as isopropylamine, trimethylamine, histidine, or procaine. Upon formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The dosage forms are readily administered in a variety of dosage forms, such as those formulated for parenteral administration, such as an infusion or aerosol for delivery to the lungs, or for digestive administration, such as a drug-release capsule.

[0239] B. Route of administration The therapeutic agent of the present invention may be administered by the same or different routes of administration. The routes of administration of the composition may include, for example, intravenous, intracerebral, intracranial, intramuscular, subcutaneous, topical, oral, mucosal, intradermal, percutaneous, intraperitoneal, intraarterial, intraorbital, intravaginal, intraspinal, intraarticular, intraventricular, intralubricating membrane, or nasal cavity by implantation; inhalation, infusion, administration, continuous administration, via catheter, direct topical perfusion bath of target cells by lavage; cream or lipid composition (e.g., liposomes); other methods or any combination of the aforementioned methods known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, specifically the full text of which is included as a reference herein).

[0240] In some implementations, a composition(s) containing a vector encoding wild-type Aβ peptide, an Aβ peptide variant, or a fragment or functional derivative thereof is delivered systemically or locally. In some implementations, a composition containing a vector encoding wild-type Aβ peptide, an Aβ peptide variant, or a fragment or functional derivative thereof is delivered to the central nervous system via orbital-posterior injection. In some implementations, a composition containing a vector encoding wild-type Aβ peptide, an Aβ peptide variant, or a fragment or functional derivative thereof is delivered systemically to the central nervous system via peripheral injection. In some implementations, peripheral injection is intravenous injection. In some implementations, a composition containing a vector encoding wild-type Aβ peptide, an Aβ peptide variant, or a fragment or functional derivative thereof is delivered to the cerebrospinal fluid (CSF). In some implementations, the composition is delivered to the CSF by non-surgical injection. In some implementations, non-surgical injection into the CSF includes non-surgical intrathecal injection. In some implementations, the composition is delivered to the CSF by neurosurgery. In some implementations, neurosurgical infusion into the cerebrospinal fluid includes neurosurgical infusion into the cisterna magna. In some implementations, compositions containing vectors encoding wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives are delivered to the ventricular system. In some implementations, compositions are delivered to the ventricular system by neurosurgical infusion. The delivery of compositions to the cerebrospinal fluid and / or ventricular system is described, for example, in the literature [WALiguore et al., Molecular Therapy 27(11):2018~2037], which is included in full text in this application. In some implementations, compositions delivered to the central nervous system, CSF and / or ventricular system cross the blood-brain barrier.

[0241] In some embodiments, compositions comprising the wild-type Aβ peptide or Aβ peptide variant disclosed herein may be dosage-formed to improve the in vivo stability of the wild-type Aβ peptide or Aβ peptide variant and / or the uptake or absorption of the wild-type Aβ peptide or Aβ peptide variant by cells, as described in the reference [ALLewis and J. Richard, Therapeutic Delivery 6(2):149-163 (2015), specifically, the full text of which is included in this application by reference]. For example, the wild-type Aβ peptide or Aβ peptide variant may be dosage-formed with absorption enhancers, 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 variant by cells. In some implementations, the dosage form for improving the in vivo stability of wild-type Aβ peptides or Aβ peptide variants and / or absorption or absorption of wild-type Aβ peptides or Aβ peptide variants by cells will depend on the route of administration of the composition, e.g., orally or by infusion.

[0242] 1. Parenteral route Accordingly, in certain embodiments, compositions (one or more) comprising a vector encoding wild-type Aβ peptide or an Aβ peptide variant or a fragment or functional derivative thereof may be administered via parenteral routes. As used herein, the term “parenteral” includes routes that bypass the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example, orbitoposterior, intracerebral, intracranial, intravenous, intradermal, intramuscular, intraarterial, intrathecal, subcutaneous, or intraperitoneal (U.S. Patents 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363, each specifically incorporated herein by reference in its entirety).

[0243] Solutions of the active compound as a free base or a pharmacokinetically acceptable salt may be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and oils. Under general storage and use conditions, these formulations contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injectable applications include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions (e.g., U.S. Patent No. 5,466,468, specifically the entire text of which is included as a reference herein). In all cases, the forms must be sterile and fluid enough to be easily injected. They must be stable under manufacturing and storage conditions and preserved against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity may be maintained, for example, by the use of a coating such as lecithin, maintaining the particle size required for dispersion, and using surfactants. Prevention of microbial activity may be induced by a variety of antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it would be preferable to include an isotonic agent, such as sugar or sodium chloride. The injectable composition may be absorbed for a longer period by using a formulation that delays absorption, such as aluminum monostearate and gelatin in the composition.

[0244] For example, for parenteral administration in aqueous solutions, the solution must be appropriately buffered if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that may be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in an isotonic NaCl solution and injected into the proposed injection site (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dose will inevitably occur depending on the condition of the patient being treated. The person administering the drug will determine the appropriate dose for each individual patient in all cases. Also, for human administration, the formulation must meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Biotechnology Office.

[0245] Sterile injection solutions can be prepared by mixing the required amount of active compound with the various other components listed above in a suitable solvent, and then, if necessary, by sterilizing by filtration. Generally, dispersions are prepared by mixing various sterilizing active ingredients with a sterilizing vehicle containing a basic dispersion medium and other necessary components listed above. In the case of sterilizing powders for the preparation of sterilizing injection solutions, preferred preparation methods are vacuum drying and freeze-drying techniques to produce powder of the active ingredient and any additional desired components from a previously sterilized filtered solution. The powdered composition is combined with or without a stabilizer with a liquid carrier, such as water or saline solution.

[0246] 2. Digestive pathway In certain embodiments of this disclosure, compositions (one or more) comprising vectors encoding wild-type Aβ peptides or Aβ peptide variants or fragments or functional derivatives thereof are formulated for administration via the digestive pathway. The digestive pathway includes all possible routes of administration in which the composition comes into direct contact with the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. Thus, these compositions may be formulated with an inert diluent or an assimilated food carrier, encapsulated in hard- or soft-shell gelatin capsules, compressed into tablets, or directly integrated with menu foods.

[0247] In certain embodiments, the active compound may be mixed with excipients and may be used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Patents 5,641,515, 5,580,579 and 5,792,451, each included in full for reference). Tablets, lozenges, pills, capsules, etc. may also include the following: Good: For example, binders such as tragacanth gum, 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; for example Sweeteners such as sucrose, lactose, saccharin, or combinations thereof; flavorings such as peppermint, wintergreen oil, cherry flavor, orange flavor, etc. If the dosage unit is in the form of a capsule, a liquid carrier may be included in addition to the above types of substances. A variety of other substances may be present as coating agents, or otherwise alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. If the dosage unit is in the form of a capsule, a carrier such as a liquid carrier may be included in addition to the above types of substances. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coating prevents the 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. Upon reaching the small intestine, the internal basic pH dissolves the coating, releasing the composition into specialized cells, such as epithelial enterocytes and Peyer's cells. The patch is designed to be absorbed by M cells. The elixir syrup may contain sweeteners such as sucrose, which is the active compound, preservatives such as methyl and propylparabens, dyes, and flavorings (e.g., cherry or orange flavor).Of course, any substances used to manufacture any dosage unit form must be pharmacokinetically pure and substantially non-toxic in the amounts used. Furthermore, the active compound may be included in sustained-release formulations and dosage forms.

[0248] For oral administration, one or more compositions comprising vectors encoding the wild-type Aβ peptide or Aβ peptide variants or fragments or functional derivatives thereof as disclosed herein may alternatively be incorporated with one or more excipients in the form of mouthwashes, toothpastes, buccal tablets, oral sprays or sublingual oral-administration dosage forms. For example, a mouthwash can be prepared by incorporating the required amount of the active ingredient in a suitable solvent such as sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as a solution containing sodium borate, glycerin and potassium bicarbonate, dispersed in toothpaste (dentifrice), or added in a therapeutically effective amount to a composition which may contain water, binders, abrasives, flavorings, preservatives and wetting agents. Alternatively, the composition may be made in the form of tablets or solutions that can be placed under the tongue or dissolved in the mouth.

[0249] Additional dosage forms suitable for other gastrointestinal administration methods include suppositories. Suppositories are solid dosage forms of varying weights and shapes, commonly used medicinally for rectal insertion. After insertion, suppositories soften, dissolve, or melt in the co-fluid. Generally, in the case of suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In particular embodiments, suppositories may be formed from a mixture 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 this disclosure, compositions (one or more) 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, such as topical (i.e., transdermal) administration, mucosal administration (nasal cavity, vagina, etc.), and / or inhalation.

[0251] Pharmaceutical compositions for topical administration may contain active compounds in dosage forms for pharmaceutical application, such as ointments, pastes, creams, or powders. Ointments contain all retention, adsorption, emulsion, and water-soluble base compositions for topical application, while creams and lotions are compositions containing only emulsion bases. Topically administered drugs may contain penetration enhancers to facilitate the adsorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxide, pyrrolidone, 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 bases. Topical formulations may also optionally contain emulsifiers, gelling agents, and antimicrobial preservatives to preserve the active ingredient and provide a homogenized mixture. Transdermal administration of the contents of this disclosure may also include the use of "patches." For example, patches can deliver one or more active substances in a continuous manner at a predetermined rate over a fixed period of time.

[0252] In a particular embodiment, a pharmaceutical composition (one or more) comprising a vector encoding an 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 a composition directly to the lungs via a nasal aerosol spray are described, for example, in U.S. Patents 5,756,353 and 5,804,212 (each included herein by reference in its entirety). Similarly, drug delivery using intranasal microparticle resins (see, e.g., Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (see, e.g., U.S. Patent 5,725,871, which is included herein by reference in its entirety) is well known in the pharmaceutical field. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is ​​described, for example, in U.S. Patent 5,780,045 (which is included herein by reference in its entirety).

[0253] The term aerosol refers to a finely divided solid colloidal system of liquid particles dispersed in a liquefied or pressurized gaseous propellant. A typical aerosol of this disclosure for inhalation would consist of a suspension of the active ingredient from a liquid propellant or a mixture of a liquid propellant and a compatible solvent. Compatible propellants include hydrocarbons and hydrocarbon ethers. The appropriate container will vary depending on the pressure requirements of the propellant. The administration of the aerosol will vary depending on the age, weight, and severity and response of the subject.

[0254] C. Medication The appropriate dosage of the composition(s) of the present disclosure to be 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, the clinical condition of the individual, previous or concurrent therapeutic interventions, the individual's clinical history and response to treatment, the idiosyncrasy of the subject, the route of administration, and the discretion of the attending physician. Depending on the dosage and route of administration, the preferred dosage and / or the number of administrations of an effective amount may vary according to the response of the subject. The medical professional responsible for administration will in any case determine the concentration of the active ingredient(s) within the composition and the appropriate dosage(s) for the individual subject.

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

[0256] Treatment may involve various "unit dosages". A unit dosage is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, the particular route, and the dosage form are within the decision-making techniques of those skilled in the clinical art. A unit dosage need not be administered by a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dosage comprises a dosage that can be administered in a single administration.

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

[0258] In some embodiments, a single dose of wild-type Aβ peptide or an Aβ peptide variant is administered. In some embodiments, multiple doses of an Aβ peptide variant are administered. In some embodiments, a single dose of wild-type Aβ peptide or an 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 derivable range or value within these ranges. In certain embodiments, an effective dose of the Aβ peptide variant is a dose that can provide a blood level of 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 or 100 μM or any derivable range within these ranges. In certain embodiments, the wild-type Aβ peptide or Aβ peptide variant administered to a subject is metabolized in the body as a metabolic therapeutic agent, in which case the blood level can indicate the amount of the formulation. Alternatively, the blood levels discussed in the present application can refer to the unmetabolized wild-type or variant Aβ peptide as long as the wild-type Aβ peptide or Aβ peptide variant is not metabolized by the subject.

[0259] In some embodiments, a single dose of a polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant is administered. In some embodiments, multiple doses of a polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant are administered. In some embodiments, an effective dose of a polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant is administered. In some embodiments, the polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant is administered at at least, at most, or about 1×10 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 copies of the polynucleotide, or at any value or range of doses derivable within these ranges. In some embodiments, the polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant is administered at a dose of 1×10 8 to 1×10 18 copies of the polynucleotide per kg of the subject's body weight. In some embodiments, the polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant is administered at a dose of 1×10 11 to 1×10 14 copies of the polynucleotide per kg of the subject's body weight. In some embodiments, the polynucleotide encoding a wild-type Aβ peptide or an Aβ peptide variant is administered at a dose of 1×10 12 to 1×10 15 copies of the polynucleotide per kg of the subject's body weight.

[0260] In some implementations, the polynucleotide encoding the wild-type Aβ peptide or an Aβ peptide variant is included in the vector. In some implementations, the polynucleotide encoding the wild-type Aβ peptide or an Aβ peptide variant is included in the vector encoding a minigene that enables the expression of the mutant Aβ peptide protein. In some implementations, an effective dose of the vector containing the polynucleotide encoding the wild-type Aβ peptide or an Aβ peptide variant is administered. In some implementations, the vector is administered in an amount of at least, at most, or approximately 1 × 10⁶ kg per kilogram of body weight of the subject. 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 or 1 × 10 18 The vector genome, or a dose within any induceable range or value within these ranges, is administered. In some implementations, the vector is administered at a dose of 1 × 10⁶ per kg of body weight of the subject. 8 〜1×10 18 The vector genome is administered at a dose of 1 × 10⁶ per kg of body weight of the subject. In some implementations, the vector is administered at a dose of 1 × 10⁶ per kg of body weight of the subject. 11 〜1×10 14 The vector genome is administered at a dose of 1 × 10⁶ per kg of body weight of the subject. In some implementations, the vector is administered at a dose of 1 × 10⁶ per kg of body weight of the subject. 12 〜1×10 15 It is administered at the dose of the vector genome.

[0261] In some implementations, a single dose of one or more additional neurodegenerative disease drugs is administered. In some implementations, multiple doses of one or more additional neurodegenerative disease drugs are administered. In some implementations, an effective dose of one or more additional neurodegenerative disease drugs is administered. In some implementations, one or more additional neurodegenerative disease drugs are administered at doses of at least, up to, or approximately 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 induceable range or value within these ranges. In a particular mode of implementation, the effective dose of one or more additional neurodegenerative disease drugs is approximately, at least approximately, or up to approximately 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, 5 The dose is capable of providing blood levels 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 induced range within these ranges. In a particular implementation, one or more additional neurodegenerative disease drugs administered to a subject are provided as metabolized therapeutic agents that are metabolized in the body, in which case the blood level may represent the amount of the relevant preparation. Alternatively, unless one or more additional neurodegenerative disease drugs are metabolized by the subject, the blood levels discussed herein may represent unmetabolized wild-type or mutant Aβ peptides.

[0262] The precise amount of therapeutic composition depends on the judgment of the specialist and varies from individual to individual. Factors influencing the dosage include the patient's physical and clinical condition, route of administration, intended therapeutic objective (symptom relief versus treatment), efficacy of other therapies the specific therapeutic substance or subject may receive, stability, and toxicity.

[0263] Those skilled in the art will understand and recognize that dosage units of μg / kg or mg / kg of body weight can be converted and expressed in similar concentration units of μg / ml or mM (blood concentration). It will also be understood that intake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions in relation to absorption and concentration measurements are well known, enabling those skilled in the art to convert one concentration measurement to another and to draw reasonable comparisons and conclusions regarding the doses, efficacy, and results described herein.

[0264] V. Kit Certain aspects of this disclosure also relate to kits comprising compositions for embodying the compositions or methods of the disclosure. In some embodiments, the kit may be used to evaluate one or more biomarkers (e.g., Aβ peptide oligomers, protofibrils, fibrils, plaques, misfolding proteins, tau proteins, phosphorylated tau proteins), seeded tau proteins and / or seeded Aβ peptides, neuroinflammatory markers, markers of cognitive decline, markers of neurodegeneration, markers of neuronal loss, and / or markers of synaptic loss). In certain embodiments, the kit may be used to measure the expression of Aβ peptides or other proteins in vitro or in vivo. In certain applications, a kit may contain at least 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, or more probes, primers or primer sets, synthetic molecules or inhibitors, or any derivable values ​​or ranges and combinations within these ranges. In some applications, there are kits for evaluating the activity of biomarkers in cells.

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

[0266] Individual components may be provided in concentrated quantities in the kit; in some embodiments, components are provided individually at the same concentration as when they are in solution with other components. The concentrations of components may be 1×, 2×, 5×, 10×, or 20× or higher.

[0267] Kits for using probes, synthetic nucleic acids, non-synthetic nucleic acids, synthetic peptides, non-synthetic peptides, and / or inhibitors of the Disclosure are included as part of the Disclosure for prognostic or diagnostic applications. Any such molecule corresponding to any biomarker identified in this Application is specifically considered, including nucleic acid primers / primer sets and probes that are identical to or complementary to all or part of the biomarker, which may include not only the non-coding sequence of the biomarker but also the coding sequence of the biomarker.

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

[0269] Any embodiment of the disclosure that orders the inclusion of a specific biomarker is also considered to include an embodiment that includes a biomarker that is identical to the mature sequence of a specific nucleic acid by at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0270] Any method or composition described herein may be embodied in any other method or composition described herein, and different modes of implementation may be combined. The originally submitted claims are considered to include claims that are multiplely dependent on any submitted claim or combination of submitted claims. [Examples]

[0271] The following examples are included to illustrate embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples work well in carrying out the present invention and represent techniques discovered by the inventors. However, those skilled in the art should recognize that many modifications may be made in light of the present disclosure to obtain similar or similar results without deviating from the spirit and scope of the present invention.

[0272] Example 1 - Gene therapy using Aβ variants for amyloid reduction Peptide inhibitors meeting four therapeutic criteria were identified in vitro, and the top two peptide candidates were investigated in vivo in an animal model 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 an endogenous protease. Subsequently, the vectors were transmitted to neonatal mouse brains for broad-spectrum lifelong neuronal expression to test the efficacy of Aβ reduction in an APP / PS1 mouse model of Alzheimer's disease (AD) using AAV. This study yielded the counterintuitive result that peptides differing from pathogenic Aβ by simply one or two amino acids effectively reduced Aβ fibrillation, destabilized existing fibrils, and decreased oligomer toxicity during short-term in vitro use, while reducing amyloid aggregation and neuroinflammation when used chronically in vivo.

[0273] Furthermore, this study represents the first step in applying virus-mediated gene therapy to overcome past limitations in developing peptide Aβ inhibitors from the bench to the brain. The greater impact of this study lies in the potential application of this strategy to other protein misfolding diseases, including misfolding protein aggregation in toxic species that induce neurodegenerative disorders, which have been avoided for technical reasons due to current expression engineering and the ability to overcome them through viruses.

[0274] The A.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 monomer assembly rate and forms β-sheet hairpins in mature fibrils. Given its importance in fibril formation, in some implementations, targeted amino acid substitutions in this region can generate peptides that prevent aggregation of the wild-type Aβ42 peptide. Based on previous studies on this domain, five other Aβ42 mutant peptides—V18P, F19D, F19P, F19D / L34P, and F20P—were examined. For the first time, it was confirmed using thioflavin-T analysis that none of the five mutants aggregated in fibrils (Figure 1A). Next, we tested whether any of the five peptides could competitively inhibit the fibrilization of wild-type Aβ42 when the two peptides were mixed in equimolar ratios before the fibrilization reaction began (Figure 1B). Of the five mutant peptides, four reduced fibrillation of wild-type Aβ, while one (V18P) exacerbated aggregation. Two peptide mutants, F20P and F19D / L34P, selected for further study, were shown to completely prevent aggregation. The potential for degrading wild-type Aβ when these two peptides were added after fibril formation was tested. Both mutants degraded wild-type fibrils in a concentration-dependent manner, but F19D / L34P was even more effective than F20P in fibril degradation, achieving a ThT signal loss of >80% 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 equimolarly with the mutant peptide before oligomer assembly. After 24 hours, the peptide solution was added to the 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 did not significantly alter cell viability on their own. In particular, cell viability increased from 50% in the control group with wild-type Aβ42 alone to approximately 70% when the wild-type peptide was co-cultured with the mutant during oligomer assembly. Therefore, in some implementations, the F20P and F19D / L34P mutants meet the minimum in vitro criteria for candidate aggregation inhibitors.

[0276] B. Design of a vector for secreting Aβ mutants in mammalian cells Next, Aβ peptide variants were tested in the brain at levels high enough to be therapeutically effective. An expression strategy was designed to stably express mutant Aβ in 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 amyloid precursor protein (APP) to the APP C-terminal fragment (β-CTF), but this construct generated extracellular Aβ and caused Aβ-independent lysosomal-autophagy pathology due to overexpression of β-CTF. Thus, the inventors sought to avoid potential lysosomal complications by using the smallest β-CTF fragment necessary for γ-secretase cleavage. The cell membrane was targeted using the Gaussia luciferase signal peptide, and this signal peptide 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 and two intracellular lysine residues (Figure 2A). Four additional constructs were each cleaved by the transmembrane domain by two amino acids; the shortest construct contained only 45 residues (Aβ42 + 3 additional).

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

[0278] This minimal expression construct was cloned in an AAV transfer vector for subsequent in vivo expression under the control of a ubiquitous CAG promoter (Figure 2B). The woodchuck poliovirus reaction element was included at the 3' end to stabilize the mRNA, followed by a bovine growth hormone poly(A) sequence. Finally, mass spectrometry was used to confirm whether this vector truly produced 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 full-length peptides were produced. Through this analysis, the presence of peptides with appropriate masses for Aβ40 F20P (Figure 2C) and Aβ42 F20P (data not shown) was confirmed, along with the precise sequences for each peptide (Figure 2D).

[0279] Expression of in vivo Aβ mutants using C.AAV The peptides were injected into wild-type mice to confirm their in vivo expression and secretion. 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 expression levels and viral distribution. Viral diffusion was assessed by immunostaining with human-specific APP antibody (6E10) to detect mutant Aβ. Viral expression was biased towards the frontal lobe cortex, and subsequent analyses 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-transmitted precursor peptide was located on the cell membrane adjacent to endogenous APP (Figure 3B).

[0280] To test the in vivo expression of the full-length peptide, homogenates from WT mice transduced with Aβ F20P were subjected to IP followed by MS. Both Aβ40 and Aβ-42 F20P in brain homogenates showed identical elution times to Aβ isolated from cell culture; both peptides were reconfirmed at expected mass and their sequences were confirmed (Figures 3C, 3D; data for Aβ42 F20P are presented). In particular, partial cleavage products that may be immunoprecipitated at 6E10 (e.g., the complete TM domain without the signal peptide or γ-secretase-cleaved Aβ still attached to the signal peptide) were not shown in the spectrum.

[0281] Furthermore, the in vivo production of each mutant Aβ peptide was measured by ELISA. The human-specific capture antibody and terminal-specific detection antibody used in this assay ensured that the measured peptides were 1) human, and therefore, virus-transmitted mutant Aβ and 2) mature Aβ that had undergone γ-secretase cleavage. F20P was well expressed in vivo. Similar to natural APP, this construct produced Aβx-40 peptide at several times higher levels than Aβx-42 (Figure 3E). In contrast, F19D / L34P produced high levels of Aβx-42 but no detectable Aβx-40, indicating that in some implementations, the L34P substitution disrupts terminal-specific Aβ40 antibody binding. These results suggest that, in some implementations, viral transduction using minimal expression vectors achieves cell membrane localization and γ-secretase cleavage, releasing mutant Aβ peptides into the brain.

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

[0283] In APP / PS1 animals, plaque load was assessed by Aβ immunostaining in the frontal lobe cortex of one hemisphere and by Aβ ELISA in the other hemisphere. Compared to uninjected APP / PS1 control mice, F20P-treated mice showed nearly 75% reduction in Aβ deposition (Figure 4A, 4B). Consistent with immunostaining, insoluble Aβx-40 and Aβx-42 levels were also significantly reduced in F20P-treated mice (Figure 4C). The effect of F19D / L34P expression in APP / PS1 mice was even more variable than that of F20P. Insoluble Aβx-40 levels were significantly reduced in APP / PS1 mice expressing F19D / L34P, but neither Aβx-42 nor plaque load were significantly reduced (Figure 4B, 4C). Therefore, both F20P and F19D / L34P significantly reduced total Aβ accumulation, but F20P was even more effective in suppressing aggregation in APP / PS1 mice. Non-transgenic animals treated with F20P- or F19D / L34P- did not accumulate detectable levels of human Aβ for 7.5 months of expression (data not presented; total Aβ 40+42, soluble+insoluble, uninjected 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] Biophysical analysis suggested that at least one of the mutants, F19D / L34P, could not only prevent Aβ assembly but also promote the degradation of existing Aβ fibrils in vitro (Figure 1C). Therefore, we tested whether the mutant could support plaque removal when administered after amyloid initiation. We achieved viral transmission and expression in the adult brain using a newly evolved AAV serotype such as PHP.eB, enabling CNS transmission via peripheral infusion. Infusion of AAV PHP.eB was optimized using fluorescent proteins as readings for transduction efficiency and spatial distribution throughout the brain (Figure 5A). The AAV PHP.eB serotype was shown to be consistent with diffusion and approach the density achieved by neonatal AAV8 infusion, suggesting that it transmits mutant Aβ at levels sufficient for in vivo use. Viral transduction also allowed us to test whether alternative administration routes, which may be clinically applicable, are consistent with the efficacy of neuronal transduction. Viral transduction of the choroid plexus has been used in gene therapy in mouse models of lysosomal storage diseases, AD, and ALS. The inventors have found that ependymal cells of the choroidal plexus can be readily transduced by AAV1, which is used in this application to test the transduction potential of CSF by mutant Aβ (Figure 5B).

[0285] Expression of E. mutant Aβ reduces general neuroinflammation in APP / PS1 mice, but may induce mild astrocytosis. Amyloid plaques induce a clear neuroimmune response in which hypertrophic astrocytes and microglia cells migrate to surround the core deposit or divide. Under most conditions, the degree of glial induction is analogous to the degree of amyloid loading. Generally speaking, treatments that slow plaque accumulation also mitigate these changes in glial morphology and localization. Therefore, we investigated whether the clustering of hypertrophic astrocytes and microglia cells, which generally determines the extent of core Aβ deposits, decreases with plaque loading in mice treated with mutant Aβ. We focused our analysis on the F20P mutant because it has a more pronounced effect on plaque loading than F19D / L34P. Both collagen fiber protein (GFAP)-positive astrocytes and Iba1-positive microglia cells prominently outline amyloid plaques in the cortex of untreated APP / PS1 mice (Figures 5A, 5B). Indeed, the surface areas of both GFAP and Iba1 exceeded the surface area of ​​Aβ immunostaining in untreated transgenic mice (Figure 5C). Lifetime treatment with virus-transmitted AβF20P significantly reduced staining for all of these markers, suggesting that in some implementations, neuroinflammation is reduced along with amyloid loading.

[0286] Increased cortical GFAP staining was also observed in virus-injected non-transgenic mice compared to uninjected control mice (Figure 5A, 5C). GFAP-positive astrocytes were mainly restricted to bands adjacent to the corpus callosum, and the effect was specific to astrocytes: Iba1 levels in non-transgenic mice were not altered by viral exposure (Figure 5B, 5C). The same pattern of pericorpus callosum GFAP staining could be identified in F20P-treated APP / PS1 mice, although this region also contained plaque-associated astrocytosis, which confused quantification (Figure 5A).

[0287] Co-immunostaining for human Aβ(6E10) and GFAP was performed in 7.5-month-old NTG mice. While some astrocytes expressing human Aβ were detected, these co-labeled cells contained only small fragments of the GFAP+ population. These data suggest that, in some implementations, viral expression of mutant Aβ peptides may itself induce mild astrocyteopathy, but the net effect in amyloidosis models is a reduction in the severity of chronic neuroinflammation corresponding to a decrease in plaque load.

[0288] Example 2 - Deciphering the biophysical mechanism of the interaction between mutant and wild-type Aβ peptide In both isolation and the presence of wild-type Aβ, the form adopted by the mutant peptide in solution is determined to ensure that these properties are suitable for in vivo use.

[0289] A. Analysis of the structure adopted by Aβ peptide during long-term in vitro incubation. The structures formed by each mutant peptide are evaluated using four main analytical methods: circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), A11 / OC dot blot analysis, and transmission electron microscopy (TEM). Initial experiments determine how the secondary and tertiary structures formed by the separated F20P and F19D / L34P peptides change over time under diverse incubation conditions, and whether one of them assembles into a quaternary structure exhibiting aggregation. The secondary structure of each peptide is monitored by far-infrared CD spectroscopy, and the tertiary / quaternary structure by near-ultraviolet spectroscopy, using spectral and thermal scans at diverse protein concentrations and solvent conditions. Dynamical CD spectroscopy measurements complement equilibrium analysis to assess the temporal evolution of peptide folding and unfolding. Data collected by CD spectroscopy are used to guide the timing of the induction, algebraic growth, and stabilization phases for each peptide, and samples are removed for SEC to confirm whether monomeric peptides are converted into smaller oligomers and even larger structures. For A11 / OC dot blotting, separate over-timed samples are removed to test for the formation of non-fibrillary and pre-fibrillary oligomeric states. The final set of samples is collected at a stable reaction (>24 hours) for negative staining TEM imaging to confirm 1) the presence or absence of structured fibrils and, where appropriate, their periodicity and width, 2) the appearance of even smaller regular structures suggesting oligomers or protofibrils, or 3) irregular structures suggesting amorphous aggregation previously observed in proline substitution variants of amyloidogenic peptides.

[0290] B. Analysis of structures generated during co-incubation of wild-type Aβ monomers and Aβ mutants The structures formed by co-incubation of F20P or F19D / L34P with wild-type Aβ are evaluated using CD spectroscopy, SEC, A11 / OC dot blotting, and TEM.

[0291] C. Percentage of mutant peptide required to prevent aggregation of wild-type Aβ42 and the minimum concentration required to degrade pre-formed fibrils In some implementations, an equimolar mixture of mutant Aβ peptides prevents aggregation of wild-type Aβ42, but even lower mutant:wild-type ratios are sufficient. Most peptide inhibitors require concentrations above stoichiometric levels to prevent wild-type Aβ aggregation, but some candidates show low efficacy at inhibitor:wild-type ratios of around 1:50. The ThT test is used to determine whether F20P or F19D / L34P can reduce or prevent fibrillation of wild-type Aβ42 at substoichiometric concentrations down to a mutant:wild-type ratio of 1:50. The minimum concentration of mutant peptide required for the degradation of pre-formed Aβ42 fibrils can be confirmed through parallel experiments. In some applications, even small amounts of F19D / L34P, such as 5 μM, are sufficiently effective in reducing the ThT fluorescence of fibrils produced from 10 μM WT Aβ starting material; in some applications, even lower concentrations of F19D / L34P achieve the same effect, and F20P may be equally efficient in degrading fibrils.

[0292] D. Analysis of the structure revealed by degradation of wild-type Aβ fibrils by mutant Aβ peptides. In some implementations, wild-type Aβ fibrils lose ThT fluorescence over time upon addition of the F19D / L34P peptide, suggesting that in some implementations, this variant promotes fibril degradation. The structure resulting from the degradation of wild-type Aβ fibrils is determined in the presence of the mutant peptide. 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 concentration of F20P or F19D / L34P that can significantly reduce ThT fluorescence in the above degradation tests.

[0293] E. The effects of co-incubation of mutant peptides and 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 implementations, co-incubation of wild-type Aβ42 and one mutant during oligomer formation reduces subsequent cell death in N2a cultures. Studies were conducted in cell lines and primary neuron cultures, and the degradation products of Aβ fibrils by Aβ mutant peptides were tested in both cell systems. Reaction products for the assembly and degradation of Aβ42 were tested at equimolar and minimal effective peptide ratios of 0.1 to 10 μM for each mutant, and cell viability was measured by MTT testing.

[0294] Discussion These experiments provide a biophysical understanding of how mutant peptides prevent aggregation of wild-type Aβ and promote the degradation of pre-formed fibrils. By evaluating CD spectra for all equilibrium and kinetic reactions under different temperature, concentration, and solvent conditions, the physical structures that emerge from the interaction of wild-type and mutant peptides can be identified. In some implementations, isolated mutants remain predominantly in a random coil state compared to wild-type Aβ, and co-incubation with wild-type reduces the appearance of structured oligomeric and fibril forms. In some implementations, SEC analysis confirms the dominance of oligomeric or fibril-versus-monomer peptides for isolated Aβ mutants and for co-incubation with wild-type Aβ. In some implementations, TEM analysis of late-stage incubation detects fibrils only from wild-type Aβ42, while fibrils are not detected in mutants alone or in co-incubation with wild-type Aβ42.

[0295] Alternatively, in some implementations, SEC analysis shows the appearance of oligomeric species with a decrease in monomers, but without a substantial increase in larger species, and shows a larger proportion of β-sheet structures over time with shifts in the CD spectrum, but not to the extent observed at the time of fibril formation. In some implementations, cyclic or small fibrilary structures that can also react with A11 / OC are observed by TEM. In some implementations, such intermediates also arise after the degradation of pre-formed fibrils. In some implementations, these intermediates are less toxic than those formed by wild-type Aβ.

[0296] Example 3 - Determination of the effects of dosage, timing, and route of mutant Aβ administration on in vivo efficacy.

[0297] A. Analysis of lifetime mutant Aβ expression in APP mice In some implementations, lifetime expression of mutant Aβ peptide significantly affects plaque development in APP / PS1 mice. An additional group of APP / PS1 mice treated with AAV-encoding scrambled Aβ peptide is added. This additional control ensures that the effects observed in treatment with mutant Aβ are due to the peptide and not to side effects of viral transduction. It is also determined whether mutant Aβ expression affects APP expression or in vivo processing. In some implementations, the amyloid-reduction mechanism of mutant Aβ occurs after release from cells by binding to wild-type Aβ, and this is confirmed by measuring full-length APP, sAPP, and CTF by Western blotting. Finally, in some implementations, a second APP model is used. NL-F The knock-in mouse line confirms findings from APP / PS1 mice. Since KI mice develop plaque later than APP / PS1 mice, KI animals offer an opportunity to test whether prolonged expression of mutant Aβ at higher levels than endogenous peptides has unexpected effects on pathology or neuronal survival. APP / PS1 and APP NL-FBoth KI mice were intracerebral (intracerebroventricular:ICV) injected with mutant or scrambled AAV at P0 and retrieved at 7.5 months or 15 months, respectively, for analysis. Measures of results for this and subsequent experiments included histological plaque loading (Aβ and thioflavin-S), human Aβ ELISA, and human oligomeric Aβ ELISA (IBL, 82E11).

[0298] B. The ratio of mutant Aβ to wild-type Aβ required for the desired effect (dosage). In some implementations, an equimolar mixture of mutant and wild-type Aβ is sufficient to inhibit the aggregation of the wild-type peptide (Figure 1B). Therefore, in some implementations, the effective in vivo dose of mutant Aβ is quite low. The in vivo dose-response relationship between mutant Aβ production and total Aβ accumulation was tested. Viral expression in vivo is virtually a mosaic where all cells are not transduced, but because peptides are secreted, transducing only a portion of the cells is sufficient to expose the mutant peptide over a wide area. In some implementations, the viral expression density in the mouse brain is easily regulated by controlling the titer of the injected virus. Here, we determine whether there is a threshold level of mutant Aβ required to delay plaque formation in vivo. ELISA is used to empirically measure the F20P mutant:wild-type Aβ ratio using the levels of mutant Aβ at each titer in non-transgenic mice for comparison with 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 may also provide valuable information regarding the relative exposure of mutant Aβ required for efficacy in some implementations. APP / PS1 and non-transgenic mice were injected intraventricularly with F20P AAV at P0 and harvested for analysis after 7.5 months. Viral dilutions were known from previous test-tube studies and ranged from approximately 0.05 to 1× of previously used titers.

[0299] C. Ventricular administration expresses parenchymal delivery effect. In some implementations, mutant Aβ does not need to be expressed by neurons to suppress the aggregation of wild-type Aβ in the extracellular space. In some implementations, expression in any cell type that can access the interstitial fluid is sufficient to widely transmit mutant Aβ throughout the brain. In some implementations, the ventricular membrane lining of the lateral ventricles is ideal for transmitting secretory proteins to the brain via the CSF. We will test the sufficiency of the transmission of mutant Aβ to the CSF to reduce Aβ aggregation through the cortex, as observed in neuronal secretion. We will transduce the choroid plexus by injecting APP / PS1 and non-transgenic animals with AAV1-encoding F20P or scrambled Aβ. The injection will be performed between P3 and P7 after the ventricular membrane-brain barrier has matured. We will measure the concentration of mutant Aβ reaching the cortex after ependymal transduction using non-transgenic animals for comparison with what is achieved by neuronal transduction. APP / PS1 mice will be harvested at 7.5 months for analysis.

[0300] D. The Aβ variant maintains its advantages when introduced after plaque development. In vitro studies have shown that, in some implementations, mutant Aβ promotes degradation when introduced after fibril formation. We investigate the potential of mutant Aβ to remove existing aggregates in the brain. This experiment utilizes the AAV serotype PHP.eB to extensively transduce adult brain cells. APP / PS1 and non-transgenic siblings are injected orbital-posteriorly with PHP.eB virus carrying F20P mutant Aβ or scrambled peptide at 7.5 months of age. A human synapsin promoter is used for neuronal specific expression; the viral titer for injection is 1 × 10⁶. 11 〜1×10 12Empirically consistent for each production within the posted range of gc / mouse. Non-transgenic animals were used to measure the concentration of mutant Aβ in the cortex after injection into adults to compare with that achieved by treatment at P0. After treatment, animals were harvested at 3 and 6 months for analysis and compared with untreated mice harvested at 7.5 months of age.

[0301] Discussion This experiment investigates how dosage, timing, and route of administration affect the in vivo efficacy of mutant Aβ. In some implementations, the expression of F20P and, to a lesser extent, F19D / L34P reduces plaque and Aβ load in an expanded cohort of APP / PS1 mice compared to all non-injected APP / PS1 mice and newly added control groups expressing scrambled Aβ peptides. In some implementations, the same effect is observed with APP. NL-F While this is observed, these late onsets mean that mutant Aβ is overexpressed for an even longer period before harvesting, producing more mutants than non-transgenic mice compared to APP / PS1 mice.

[0302] In some implementations, there is a minimum ratio of mutant to wild-type Aβ required to delay plaque formation in vivo, which is roughly similar to the minimum ratio required for in vitro effects. Beyond this ratio, in some implementations, a gradual reduction in plaque is observed despite a less defined dose-response relationship than in vitro. Using non-transgenic animals, the concentration of mutant Aβ reaching the cortex via CSF is compared to the concentration achieved by neuronal expression to determine whether CSF transmission achieves cortical levels of mutant Aβ equal to or exceeding the minimum effective dose for neuronal expression. If not, transmission is improved using an alternative serotype for ventricular expression (i.e., AAV4) or an alternative promoter active in the choroid plexus (i.e., Prlr, Spint2, F5).

[0303] These experiments, which will be further developed in subsequent studies into cognitive tests, synaptic analysis, and electrophysiology, will establish a fundamental understanding of Aβ species from exposure to mutant peptides, leading to the identification of optimal therapeutic therapies to mitigate or reverse Aβ accumulation in vivo and to determine whether unintended neuroinflammation can interfere with physiological measurements.

[0304] Example 4 - Investigation of the neuroimmune response to variant Aβ as a possible accomplice to plaque reduction. In some implementations, neonatal viral transduction induces a mild, cell-specific neuroimmune response in the brain, but analysis is limited to morphological markers and does not reveal any underlying molecular changes. RNA profiling is used to evaluate a wide range of neuroimmune markers in the context of amyloid pathology or for modification by viral expression alone. Test the evolution of neuroimmune profiles with age to determine whether transduction in adult brains induces a more pronounced response than initially observed in mice injected as neonates. Complete histological analysis of astrocytes and microglial cells to determine how plaque loading, viral exposure, and cellular responses interact after treatment with the mutant Aβ at various stages of the disease. Effects in some implementations are attributable to astrocyte reactivity, while effects in others are attributable to the biophysical effects of the mutant Aβ compared to the wild-type peptide. In some implementations, the biophysical effects override the astrocyte response. Empirical testing is conducted to establish the usefulness of gene therapy as a platform for self-suppression in Alzheimer's disease and other protein aggregation disorders.

[0305] A. Effects of upward regulation of neuroinflammatory transcription markers and viral transmission age on viral transmission response in mutant Aβ. This experiment addresses whether the introduction of AAV expressing a non-native protein induces a neuroinflammatory response in the brain. Initially, the focus is on non-transformed animals to avoid confounding effects of plaque-associated gliomas in APP / PS1 animals. Animals for this and subsequent experiments are generated as in Example 3: a small portion of the frontal lobe cortex (approximately 20-40 mg) is collected from one hemisphere of each animal for whole RNA extraction, with the remaining cortex reserved for ELISA and Western blotting studies in Example 3. The contralateral hemisphere is fixed for histological studies. Here, the transcriptional profiles of non-transgenic control mice are 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 effect of extended viral expression on neuroinflammatory responses. Transcriptions are profiled using a Nanostring nCounter neuroinflammation panel for mice, dealing with 756 genes across a wide range of potential neuroimmune responses. Unlike RNA sequencing, this platform directly measures mRNA molecules digitally for linear quantification without template amplification. Expression data is analyzed using nSolver software, comparing gene expression in animals treated with F20P or scrambled peptides with that of an uninjected control group for each treatment / recovery age combination. Second-tier analysis is acquired from the Baylor College of Medicine Multi-Omics Data Analysis Core for enhancement of pathways and gene sets. RNA sequencing is also available, providing broader readings for potential variations.

[0306] B. Effects of virus-transmitted Aβ variants on neuroinflammatory responses This study addresses whether the introduction of AAV and its non-natural peptide cargo alters the neuroinflammatory response to amyloid deposits. It examines APP / PS1 and APP patients injected with F20P or scrambled Aβ at P0 or 7.5 months. NL-FAPP / PS1 and APP without injection of mouse cortical transcription profiles in each experiment. NL-F Compare this to sibling. Nanostring N-counter neuroinflammation panels are used for profiling neuroimmune responses in APP models.

[0307] Diverse morphologies and distribution of neuroinflammatory cells during viral expression of C. mutant Aβ We will examine the cellular profile of the neuroimmune response to viral transmission of mutant Aβ. Immunofluorescence will be used for non-transgenic, APP / PS1, and APP. NL-F It is used to detect microglia cells (Iba1 as a pan-microglia marker, P2RY12 for homeostatic microglia, CD68 for phagocytic microglia, and MHCII for inflammatory microglia) and astrocytes (Aldh1l1 as a pan-astrocytic marker, GFAP preferentially for reactive astrocytes) in mice. Mice were harvested after being injected with F20P or scrambled peptides at P0 or 7.5 months for comparison with uninjected mice. Measurements of results include 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 APP models.

[0308] discussion This work investigates the neuroinflammatory response to viral transmission of non-native proteins by transcription and cellular markers. These experiments also examine whether the amyloid reduction observed in pilot studies is due to a neuroinflammatory response to treatment. In some implementations, mild astrocytosis may be accompanied by viral transmission in non-transgenic mice, while both astrocytosis and microgliacytosis are reduced proportionally to plaque load in F20P-treated APP / PS1 animals. In some implementations, these molecular and histological experiments allow for the resolution of such discrepancies and support mechanisms based on Aβ stereorepression rather than neuroinflammatory phagocytic activity.

[0309] In some implementations, when 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 transmission limits viral exposure but does not limit the diffusion of mutant Aβ, the comparison with neuronal expression must accurately pinpoint whether the virus or peptide is the cause. Alternative in vitro studies directly test microglia / astrocytocyte responses to mutant Aβ peptides using purified primary cell cultures and recombinant proteins. To rule out the role of neuroinflammation in plaque reduction, therapeutic efficacy in the presence and absence of astrocytes / microglia is also compared.

[0310] Finally, in some implementations, mRNA panels demonstrate that additional cell types, such as circulating macrophages, T- or B- cells, are involved in any neuroinflammatory response, and that FACS classification of brain tissue can be used to identify cellular components and the degree of neuronal invasion. The transmission pathway (i.e., ventricle rather than neuron) or capsid (clinically used AAV9 vs. experimentally used AAV8 / PHP.eB) may be modified for future research to mitigate any treatment-associated neuroinflammation.

[0311] In some implementations, if mRNA marker panels or histological staining confirm increased responses in virus-treated animals, additional experiments may be performed, as needed, to map the time course of neuroinflammatory changes after viral transmission in P0 or adult animals.

[0312] Example 5 - Mutant Aβ slows down seeding of AD aggregation. This study investigated whether gene therapy using mutant Aβ could reduce the likelihood that pathogenic protein seeds accelerate disease progression in vivo. These experiments provide an opportunity to test whether mutant Aβ is limited to preventing seed formation from monomers or even inhibiting the growth of existing seeds. Furthermore, the experiments test pathogenic seeds in human Alzheimer's disease tissue, which presents a more diverse and realistic challenge than the gene model alone. The first experiment investigates whether prophylactic expression of mutant Aβ can slow plaque formation resulting from inoculation with Aβ seed-rich human Alzheimer's disease extracts. The final experiment tests the potential of Aβ reduction to attenuate tau secondary seeding in amyloid-containing mice.

[0313] A. Mechanism of the effect of mutant Aβ on the rate and extent of amyloid seeding in pre-deposited mice. This study investigates whether lifelong expression of mutant Aβ can be induced by inoculation into brain extracts from exogenous Alzheimer's disease patients and mitigate accelerated amyloidosis. Aβ extracts for in vivo seeding are prepared from two pathologically confirmed Alzheimer's disease subjects and one age-matched healthy control. Frozen frontal cortical samples are acquired. The protein extracts for injection are homogenized in PBS and clarified by sonication. The extracts are characterized by A11 / OC dot blotting for oligomeric Aβ, ELISA for total Aβ concentration, and testing of protein misfolding circulating amplification (PMCA) for seeding volume. At birth, APP / PS1 mice were administered AAV encoding either the F20P mutant or scrambled Aβ, and one month later, brain extracts from Alzheimer's disease or healthy control groups were bilaterally injected into the hippocampus and upper cortex. For Aβ immunohistochemistry and ELISA to assess the severity and extent of Aβ aggregation in animals expressing F20P vs. Scramble, animals in this model are harvested 3 to 5 months prior to the normal onset of plaque formation.

[0314] B. Mutant Aβ affects tau pathology seeded in amyloid-containing mice. The final experiment will test whether the presence of mutant Aβ can prevent or stunt the development of tau neuropathology in amyloid-containing mice. The tau extract for injection is prepared from cortical gray matter by fractional centrifugation of sarcosyl-insoluble brain homogenate, as originally described by the Lee group. The extract is characterized by PHF1 Western blot and ELISA against total tau concentration and tested for seeding potential using tau RD FRET biosensor cells (available from ATCC). Once the Alzheimer's disease-tau extract proceeds 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, the Alzheimer's disease (AD)-tau extract is bilaterally injected into the hippocampus and upper cortex to ensure sufficient amyloid loading. Animals are harvested at 3 or 6 months for tau immunohistochemistry (AT8, AT180, MC1) and amyloid immunostaining and histology (thioflavin-S) to measure the extent and total area of ​​phosphotau immunostaining against amyloid loading in animals expressing F20P vs. Scramble.

[0315] Discussion In some implementations, lifelong expression of mutant Aβ may reduce amyloid formation in APP / PS1 mice and decrease plaque and phosphotau neurite seeding by Alzheimer's disease extracts. In some implementations, mutant Aβ limits the expansion of injected AD-Aβ seeds as well as newly generated seeding. Similarly, in some implementations, mutant Aβ reduces the appearance of phosphotau neurites by attenuating the formation of amyloid plaques necessary to promote secondary tau seeding. Alternatively, in some implementations, mutant Aβ fails to attenuate seeding by exogenous aggregates, suggesting that in some implementations, mutant Aβ is sufficient to slow seed formation in APP mice, but once present, it either prevents seed growth or cannot interact with seeds present in AD extracts; this is less likely due to predicted mechanisms of steric hindrance during fibril expansion that must occur regardless of the starting material.

[0316] Such experiments provide an important foundation for future research testing how the timing of treatment related to inoculation controls efficacy. Tests will be conducted to determine whether seeding can be inhibited if the mutant Aβ is administered after plaque development. The effects of dose and administration route on amyloid-to-tau progression will also be investigated outside of normal time.

[0317] Example 6 - Exemplary Method Preparation of WT Aβ42 or mutant peptide stocks. Synthetic Aβ42 WT or mutant peptides were purchased from Biomatik (Wilmington, DE). To prepare a stock solution of Aβ42 WT or mutant peptides without aggregates, the powdered peptides were dissolved in 50% acetonitrile, frozen, and lyophilized overnight to remove residual trifluoroacetic acid. The 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 minutes and then divided into preparative portions. The HFIP was evaporated overnight in a fume hood and then transferred to a SpeedVac for 1 hour to remove any remaining traces of HFIP. Tubes containing the peptide films were stored on a desiccant at -20°C until use. Immediately before use in experiments, the lyophilized peptides were dissolved in DMSO to a final concentration of 5 mM and sonicated in an aqueduct for 10 minutes.

[0318] Preparation of oligomeric and fibrillary WT Aβ. Peptides were dissolved in Ham's F-12 medium (#30611040-1, Fisher Scientific, Pittsburgh, PA) with either 100 μM WT Aβ42 peptide or 100 μM WT + 100 μM mutant at a final concentration. These were then incubated at 4°C for 24 hours without shaking to produce oligomeric Aβ. Fibrillary Aβ was produced by dissolving the WT peptide in PBS at a final concentration of 100 μM and incubating at 37°C for 24 hours without shaking.

[0319] ThT assays were performed to test the kinetics of Aβ auto-aggregation, competition with WT peptides, and fibril degradation. Auto-aggregation of Aβ42 WT or mutant monomers was tested at an initial concentration of 10 μM in PBS containing 5 μM thioflavin T (ThT). 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. After incubation of the reaction mixture at 37°C without shaking, shaking was performed for 5 seconds before reading the fluorescence. Competition between mutants and WT Aβ was performed similarly, with each peptide in the mixture (1:1, WT:mutant) tested at a final concentration of 10 μM using an initial concentration of 10 μM, except for WT alone. Fibril degradation was evaluated by mixing 10 μl of WT Aβ42 fibrils (as described above) with 5, 10, or 20 μM monomer F19D / L34P or F20P peptide in a 1:1 ratio in PBS containing 5 μM ThT. Fluorescence was measured every 24 hours without shaking at 37°C for 48 hours.

[0320] Oligomer Aβ toxicity test. N2a cells were 1 × 10⁶ 4Cells were grown at 37°C in 5% CO2 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). Confluent cells were trypsinized and diluted in EMEM containing 1% N2 supplement to minimize cell growth, and then plated at 5,000 cells per well in clear flat-bed 96-well plates (#07-200-89, Fisher). 10 μl of each oligomer preparation (WT Aβ alone or WT+ variant, 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) as instructed by the manufacturer. A simplified method involved directly adding 20 μl of AQueous One Solution Reagent to culture wells, culturing at 37°C in a 5% CO2 atmosphere for 2 hours, and then recording absorbance at 490 nm using an Epoch 2 spectrophotometer (Biotek, Winooski, VT) for assay.

[0321] Plasmid construct. PCR was used to add a Gaussial cyperage signal peptide (GLSP, amino acids MGVKVLFALICIAVAEA, corresponding to SEQ ID NO: 20) to the N-terminus of Aβ-CTF. First, GLSP synthetic DNA with a 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). These oligonucleotides were incubated and annealed in a thermal cycler programmed to start at 95°C for 2 minutes and then gradually cool to 25°C. Secondly, a plasmid containing the human APP wild-type sequence (pBS-hAPPwt-IRES-GFP) was amplified using the forward primer 5'GTCGCAGAGGCAGATGCAGAATTCCGACATGAC 3' (SEQ ID NO: 42) and the reverse primer 5'GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG 3' (SEQ ID NO: 43) to generate Aβ-CTFs with a partial GLSP sequence. Subsequently, the GLSP-Aβ-CTFs were generated by Gibson assembly, which involved binding the GLSP+ partial Aβ to the Aβ-CTF+ partial GLSP using the forward and reverse primers 5'GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG 3' (SEQ ID NO: 45) to add the Kozak sequence 5'GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3' (SEQ ID NO: 44) from the two templates. The generated GLSP-Aβ-CTF fragments were cleaved with HindIII and EcoRV, and subcloned with pAAV containing the CAG promoter and WPRE to produce pAAV-GLSP-Aβ-CTF.

[0322] The GLSP-Aβ-CTF deletion series was constructed by cloning various GLSP-Aβ-CTF deletions in pAAV. The GLSP-Aβ-CTF deletion series was amplified by PCR from full-length pAAV-GLSP-Aβ-CTF using a series of reverse primers and a common forward primer, then cleaved with HindIII and EcoRV and ligated to pAAV.

[0323] Forward primer: Aβ-GLSP-F(KOZAK):5′ GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (Sequence ID: 46).

[0324] Reverse primer: Aβ-R(KK):5′ GCGCGgatatcTTACTACTTCTTCAGCATCACCAAGGTG 3′ (Sequence ID: 47); Aβ-R(ML):5′ GCGCGgatatcTTACTACAGCATCACCAAGGTGATGA 3′ (Sequence ID: 48); Aβ-R(LV):5′ GCGCGgatatcTTACTACACCAAGGTGATGACGATCA 3′ (Sequence ID: 49); Aβ-R(IT):5′ GCGCGgatatcTTACTAGGTGATGACGATCACTGTCG 3′(Sequence ID: 50); Aβ-R(IV):5′ GCGCGgatatcTTACTAGACGATCACTGTCGCTATGA 3′ (Sequence ID: 51); Aβ-R(IA):5′ GCGCGgatatcTTACTACGCTATGACAACACCGCCCA 3′ (Sequence ID: 52);

[0325] Two PCR reactions were performed to construct pAAV-GLSP-Aβ(F19D / L34P)-KK containing the F19D / L34P substitution. Fragments of GLSP-Aβ(F19D / L34P)-KK were amplified from pAAV-GLSP-Aβ-KK using forward primer 5′ GCGCGaagcttGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 53) and 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 inserts were cleaved with HindIII and EcoRV and subcloned into pAAV to produce 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 generated fragments were joined by Gibson assembly using forward primer 5′ GCGCGAAGCTTGCCACCATGGGCGTGAAGGTCCTGTT 3′ (SEQ ID NO: 63) and reverse primer 5′ GCGCGGATATCTTACTACTTCTTCAGCATCACCAAGGTG 3′ (SEQ ID NO: 64). The resulting inserts were cleaved with HindIII and EcoRV and subcloned into pAAV to produce pAAV-GLSP-Aβ(F20P)-KK. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).

[0327] Aβ peptide collection in N2a-regulated medium. N2a cells were 1 × 10⁶ 4Cells were grown in 6-well plates in DMEM (Dulbecco's modified Eagle's medium, #12-604F, VWR) medium supplemented with U / ml penicillin / streptomycin and 10% fetal bovine serum until approximately 90% fusion occurred. Subsequently, cells were 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 hours,...

Claims

1. A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, disorders, or conditions, comprising a vector encoding an Aβ peptide variant, wherein the Aβ peptide variant has the amino acid sequence: A pharmaceutical composition comprising, A pharmaceutical composition 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 dementia, vascular dementia, cerebral amyloid angiopathy, Lewy body dementia, chronic traumatic encephalopathy, Down syndrome, and pathological aging.

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

3. The pharmaceutical composition according to claim 2, wherein the vector encoding the Aβ peptide variant encodes a nucleotide sequence corresponding to an amino acid sequence containing the cleaved β-CTF fused to a signal peptide sequence at the N-terminus of the β-CTF, and the signal peptide sequence comprises a gaussial luciferase signal peptide or a mouse immunoglobulin heavy chain signal peptide.

4. The pharmaceutical composition according to claim 2, wherein the cleaved β-CTF comprises an Aβ peptide variant sequence, a transmembrane domain sequence, and a cytoplasmic sequence.

5. The pharmaceutical composition according to claim 4, wherein the cytoplasmic sequence comprises an amino acid sequence for membrane anchoring, promotion of gamma-secretase cleavage, and / or extracellular release of Aβ peptide variants.

6. The pharmaceutical composition according to claim 1, wherein the vector comprises a nucleotide sequence encoding an amino acid sequence 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.

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

8. The pharmaceutical composition according to claim 1, wherein the vector is an adenovirus, lentivirus, retrovirus, or adeno-associated virus vector.

9. The pharmaceutical composition according to claim 1, wherein the vector is an AAV vector.

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

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

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

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

14. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable carrier comprises liposomes, polymer micelles, microspheres, or nanoparticles.

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