Gene therapy for alzheimer's disease

Abstract

A gene therapy for Alzheimer's disease. Provided are compositions and methods for preventing, inhibiting, or treating a disease or disorder associated with APOE4 expression in a mammal.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080073402.8, filed on October 16, 2020, entitled "Gene Therapy for Alzheimer's Disease". Application No. 202080073402.8 is the national phase application of International Application No. PCT / US2020 / 056051, which claims priority to U.S. Provisional Patent Application No. 62 / 915,988, filed on October 16, 2019.

[0002] Cross-references to related applications This application claims the benefit of U.S. Application No. 62 / 915,988, filed October 16, 2019, the disclosure of which is incorporated herein by reference. Background Technology

[0003] Apolipoprotein E (APOE) is an important central nervous system (CNS) apolipoprotein closely associated with the pathogenesis of the most common late-onset familial and sporadic forms of Alzheimer's disease (AD) (Yu et al., 2014). In the general population, three common APOE alleles (ε4, ε3, and ε2) encode three APOE isotypes primarily expressed in the liver and brain. APOE4 Carriers have a significantly increased risk of developing AD (compared to) APOE3 Compared to homozygotes, heterozygotes and homozygotes are 3-15 times more likely to develop the disease and at a younger age (approximately 5 years for each ε4 allele; Corder et al., 1993; Farrer et al., 1997; Lambert et al., 2013; Saunders et al., 1993; Strittmatter et al., 1993). The fact that 45% of AD patients carry at least one ε4 allele (compared to only 15% of age-matched healthy controls) makes... APOE4 It has become the most common genetic risk factor for late-onset AD (the most common form of AD) to date. In contrast, APOE2 It is a protective allele that reduces the risk of AD by about 50% and significantly delays the age of onset (Corder et al., 1994; Farrer et al., 1997; Suri et al., 2013; Talbot et al., 1994; Yu et al., 2014).

[0004] The major physiological difference between APOE3 (the most common isoform) and APOE2 and APOE4 is due to an amino acid difference at one of the two positions, namely residues 112 (APOE4) and 158 (APOE2), which is a cysteine-arginine interchange (Hatters et al., 2006). This two-amino acid difference leads to differences in protein structure, as well as the binding affinity of these APOE isoforms to lipoproteins and lipoprotein receptors, and the regulation of Aβ aggregation, degradation, efflux, and phagocytosis (Castellano et al., 2011; Deane et al., 2008; Hashimoto et al., 2012; Hatters et al., 2006; Holtzman et al., 2012; Li et al., 2012; Manelli et al., 2004; Walker et al., 2000; Yu et al., 2014; Zhao et al., 2009). Summary of the Invention

[0005] In one embodiment, this disclosure provides a gene therapy vector for Alzheimer's disease. In one embodiment, the gene therapy vector comprises an AAV expression vector encoding a human APOE2 gene and a cis- or trans-artificial microRNA targeting endogenous APOE4. This vector system, combined with a beneficial APOE2 gene from a gene therapy vector (e.g., an AAV vector), silences the expression of harmful endogenous APOE4. Exemplary artificial microRNA sequences designed to target endogenous APOE4 mRNA for repression are disclosed herein. The microRNA (miRNA) may be incorporated into a sequence at the 5' end of the APOE2 coding sequence, such as in an intron (e.g., a CAG promoter intron), or into a sequence at the 3' end of the APOE2 coding sequence, such as at the 5' end of the polyA tail of a vector transgenic plasmid encoding the human APOE2 coding sequence. Alternatively, the microRNA may be inserted between a PolIII promoter (e.g., a U6 promoter) and a terminator following the polyA site of the APOE2 expression cassette. The vector-derived human APOE2 DNA sequence optionally includes silent nucleotide changes to reduce or suppress microRNA repression, and in one embodiment may include a tag for detection (e.g., for preclinical testing studies), such as an HA tag. In one embodiment, the expression construct is packaged into a serotype of AAV capsid that targets astrocytes and glial cells (e.g., AAV9), a prominent site of endogenous APOE expression in the CNS, but may be provided in other vectors, such as other viral vectors, plasmids, nanoparticles, or liposomes.

[0006] In one embodiment, a gene therapy vector is provided, the gene therapy vector comprising: a first promoter operatively linked to a nucleic acid sequence including an open reading frame encoding APOE2 and a 3' untranslated region, and providing a separated nucleotide sequence including one or more RNAi nucleic acid sequences for repressing APOE4 mRNA. In one embodiment, the vector includes the nucleotide sequence. In one embodiment, the nucleotide sequence is inserted at the 5' or 3' of the open reading frame. In one embodiment, the nucleotide sequence is inserted at both the 5' and 3' of the open reading frame. In one embodiment, the nucleotide sequence is located on a different vector. In one embodiment, the separated nucleotide sequence includes a second promoter operatively linked to the one or more RNAi nucleic acid sequences. In one embodiment, the gene therapy vector is a viral vector. In one embodiment, the different vector is a viral vector. In one embodiment, the viral vector is an AAV, adenovirus, lentivirus, herpesvirus, or retrovirus vector. In one embodiment, the AAV is AAV5, AAV9, or AAVrh10. In one embodiment, the APOE4 is human APOE4. In one embodiment, the APOE2 is human APOE2. In one embodiment, the first promoter is a PolI promoter (e.g., a constitutive promoter) or a modulating promoter (e.g., an inducible promoter). In one embodiment, the second promoter is a PolIII promoter. In one embodiment, the isolated nucleotide sequence comprises the nucleic acid of one or more miRNAs, the miRNA comprising two or more of the RNAi nucleic acid sequences, for example, one or more RNAi sequences are embedded in the miRNA sequence. In one embodiment, the RNAi comprises siRNA containing multiple siRNA sequences. In one embodiment, the RNAi comprises an shRNA sequence of about 15 to 25 nucleotides in length. In one embodiment, the open reading frame of APOE2 includes a plurality of silent nucleotide substitutions relative to SEQ ID NO:6. For example, the open reading frame includes a nucleotide sequence of SEQ ID NO:7 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 98% nucleic acid sequence identity with SEQ ID NO:7 and encodes APOE2, or the open reading frame encodes APOE2 and includes a nucleotide sequence of at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 98% nucleic acid sequence identity with GAAAGAACTCAAAGCTTATAAGAGCGAGCTGGAGG (SEQ ID NO:13), but the sequence is not SEQ ID NO:7.In one embodiment, the plurality of silent nucleotide substitutions in the open reading frame of APOE2 are not present in the RNAi nucleic acid sequence within the isolated nucleotide sequence; that is, the sequence having the nucleotide substitutions differs from the RNAi nucleotide sequence, such that the mRNA having the nucleotide substitutions does not bind to, for example, a double strand having the RNAi sequence, such as isolated RNAi or RNAi sequences expressed by a vector. In one embodiment, at least 50%, 60%, 70%, 80%, or 90% of the codons in the open reading frame of APOE2 have silent nucleotide substitutions. In one embodiment, at least 5%, 10%, 20%, 30%, or 40% of the codons in the open reading frame of APOE2 have silent nucleotide substitutions, for example, in a portion of the APOE2 sequence corresponding to the RNAi sequence. That is, the silent nucleotide substitutions in the human APOE2 coding sequence produce a sequence different from the endogenous human APOE4 sequence and different from the APOE4 RNAi sequence. In one embodiment, the inhibited APOE4 has a sequence having at least 80%, 85%, 90%, 95% or more amino acid sequence identity with the polypeptide encoded by SEQ ID NO:22. In one embodiment, the APOE2 has a sequence having at least 80%, 85%, 90%, 95% or more amino acid sequence identity with the polypeptide encoded by SEQ ID NO:9. In one embodiment, the one or more RNAi nucleic acid sequences have at least 60%, 70%, 80%, 90% or more nucleotide sequence identity with one of SEQ ID No. 1-4 or 20-22 or their complement. In one embodiment, the vector has a first PolI promoter operatively linked to a nucleic acid sequence comprising an open reading frame encoding human APOE2 and a separated nucleotide sequence having one or more RNAi nucleic acid sequences for inhibiting human APOE4 mRNA. In one embodiment, the nucleotide sequence is inserted at the 5' of the open reading frame. In one embodiment, the nucleotide sequence is inserted at the 3' of the open reading frame. In one embodiment, the nucleotide sequence is inserted at both the 5' and 3' of the open reading frame. In one embodiment, the isolated nucleotide sequence includes a second promoter operatively linked to the one or more RNAi nucleic acid sequences. In one embodiment, the RNAi nucleic acid sequence is about 125 to 500 nucleotides in length (e.g., about 150 to 175 nucleotides). In one embodiment, the gene therapy vector may have 2, 3, 4 or more copies of the RNAi nucleic acid sequence, which may contain a miRNA sequence, such as a miRNA sequence side-linked with an APOE4 repressor sequence.

[0007] In one embodiment, a method for preventing, inhibiting, or treating Alzheimer's disease in a mammal is provided, the method comprising: administering to the mammal an effective amount of a composition comprising the gene therapy vector. In one embodiment, the composition comprises nanoparticles comprising the gene therapy vector or the different vectors or both. In one embodiment, the gene therapy vector or the different vectors or both comprise a viral vector. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the composition is administered systemically. In one embodiment, the composition is administered orally. In one embodiment, the composition is administered intravenously. In one embodiment, the composition is administered locally. In one embodiment, the composition is injected. In one embodiment, the composition is administered to the central nervous system. In one embodiment, the composition is administered to the brain. In one embodiment, the composition is a sustained-release composition. In one embodiment, the mammal is a human. In one embodiment, the RNAi nucleic acid sequence comprises a plurality of miRNA sequences.

[0008] In one embodiment, a method for preventing, inhibiting, or treating diseases associated with APOE4 expression in mammals is provided, the method comprising: administering to the mammal an effective amount of a composition comprising the gene therapy vector. In one embodiment, the composition comprises liposomes comprising the gene therapy vector or the different vectors or both. In one embodiment, the composition comprises nanoparticles comprising the gene therapy vector or the different vectors or both. In one embodiment, the gene therapy vector or the different vectors or both comprise a viral vector. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the composition is administered systemically. In one embodiment, the composition is administered orally. In one embodiment, the composition is administered intravenously. In one embodiment, the composition is administered locally. In one embodiment, the composition is injected. In one embodiment, the composition is administered to the central nervous system. In one embodiment, the composition is administered to the brain. In one embodiment, the composition is a sustained-release composition. In one embodiment, the mammal is a human. In one embodiment, the RNAi sequence comprises a plurality of miRNA sequences. Attached Figure Description

[0009] Figure 1Repressive RNA was generated from an exemplary target transcript template (Boudreau and Davidson. 2012. Methods in Enzymology, Vol. 507).

[0010] Figure 2 Inhibition of mRNA pathways (Borel et al., 2014. Molecular Therapy) Mol Ther )》 22:692-701).

[0011] Figure 3 An exemplary construct for miRNA insertion.

[0012] Figure 4 Single-vector and dual-vector constructs.

[0013] Figure 5 A single-vector construct with two miRNA sequence sites.

[0014] Figure 6 APOE knockdown was performed in vitro using the expression of four different siRNAs.

[0015] Figure 7 The mir155 scaffold was used as an exemplary scaffold for miRNA expression.

[0016] Figure 8 Mouse experiments. Detailed Implementation

[0017] definition "Vector" refers to a macromolecule or macromolecular conjugate comprising or associated with polynucleotides and capable of mediating the delivery of polynucleotides to cells in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery agents. The polynucleotide to be delivered, sometimes referred to as the "target polynucleotide" or "transgenic," may include coding sequences of interest in gene therapy (such as genes encoding proteins of therapeutic interest), coding sequences of interest in vaccine development (such as polynucleotides expressing proteins, polypeptides, or peptides suitable for evoking an immune response in mammals), and / or selectable or detectable biomarkers.

[0018] As used herein, “transduction,” “transfection,” “transformation,” or “transducing” refers to the term used for the process of introducing exogenous polynucleotides into host cells to result in the expression of polynucleotides (e.g., transgenes in cells), and includes the introduction of exogenous polynucleotides into host cells using recombinant viruses. The transduction, transfection, or transformation of polynucleotides in cells can be determined by methods well known in the art, including but not limited to protein expression (including steady-state levels), such as by ELISA, flow cytometry, and Western blotting, and by measuring DNA and RNA through hybridization assays (e.g., Northern blotting, Southern blotting, and gel mobility assays). Methods for introducing exogenous polynucleotides include well-known techniques such as viral infection or transfection, lipid transfection, transformation, and electroporation, as well as other non-viral gene delivery techniques. The introduced polynucleotides can be maintained stably or transiently in the host cells.

[0019] "Gene delivery" refers to the introduction of exogenous polynucleotides into cells for gene transfer, and can encompass targeting, binding, uptake, transport, localization, replicon integration, and expression.

[0020] "Gene transfer" refers to the introduction of exogenous polynucleotides into cells. This introduction may include targeting, binding, uptake, transport, localization, and replicon integration, but is different from and does not imply subsequent gene expression.

[0021] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification.

[0022] An "infectious" virus or viral particle is a virus or viral particle that includes a polynucleotide component capable of delivering it into a trophic cell of the viral species. The term does not necessarily imply any replication capability of the virus.

[0023] The term "polynucleotide" refers to a polymeric form of nucleotides of any length or its analogues, comprising deoxyribonucleotides or ribonucleotides. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure may be made before or after polymer assembly, if present. As used herein, the term polynucleotide is used interchangeably to refer to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein is a polynucleotide, covering both double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0024] "Isolated" polynucleotides, such as plasmids, viruses, polypeptides, or other substances, refer to formulations lacking at least some of the other components, which may also be present in naturally occurring substances or substances originally prepared therefrom, or similar substances. Thus, for example, the isolated substance can be prepared by enriching it from a source mixture using purification techniques. The isolated nucleic acid, peptide, or polypeptide exists in a form or environment different from its natural occurrence. For example, a given DNA sequence (e.g., a gene) is found on the chromosome of a host cell near a neighboring gene; an RNA sequence, such as a specific mRNA sequence encoding a particular protein, is found in a cell as a mixture with many other mRNAs encoding multiple proteins. The isolated nucleic acid molecule can exist in single-stranded or double-stranded form. When the isolated nucleic acid molecule is used to express a protein, the molecule will contain at least a sense strand or a coding strand (i.e., the molecule may be single-stranded), but may contain both sense and antisense strands (i.e., the molecule may be double-stranded). Enrichment can be measured on an absolute basis, such as the weight per volume of solution, or relative to a second potential interfering substance present in the source mixture. Increased enrichment is contemplated in embodiments of the invention. Therefore, for example, 2x enrichment, 10x enrichment, 100x enrichment, or 1000x enrichment.

[0025] "Transcriptional regulatory sequence" refers to a genomic region that controls the transcription of a gene or coding sequence operatively linked to it. The transcriptional regulatory sequence used in this invention typically contains at least one transcription promoter and may also contain one or more transcriptional enhancers and / or terminators.

[0026] "Operationally linked" refers to an arrangement of two or more components in which the components described so far are in a relationship that allows them to function in a coordinated manner. By way of illustration, if a transcriptional regulatory sequence (TRS) or promoter promotes transcription of a coding sequence, then the transcriptional regulatory sequence or promoter is operationally linked to the coding sequence. Operationally linked TRSs are typically cis-linked to the coding sequence, but not necessarily directly adjacent to it.

[0027] "Heterologous" refers to an entity that is genotype different from the entity being compared with. For example, polynucleotides introduced into different cell types through genetic engineering are heterologous polynucleotides (and can encode heterologous polypeptides when expressed). Similarly, transcriptional regulatory elements, such as promoters that have been removed from their native coding sequences and are operatively linked to different coding sequences, are heterologous transcriptional regulatory elements.

[0028] A “terminator” is a polynucleotide sequence that tends to reduce or prevent readthrough transcription (i.e., it reduces or prevents transcription originating from one side of the terminator from continuing to the other side). The degree to which transcription is disrupted is usually a function of the base sequence and / or the length of the terminator sequence. Specifically, as is well known in many molecular biology systems, a particular DNA sequence (often called a “transcription termination sequence”) is a specific sequence that tends to disrupt readthrough transcription by RNA polymerase (possibly by causing the RNA polymerase molecule to stop and / or detach from the transcribed DNA). A typical example of such sequence-specific terminators contains polyadenylation (“polyA”) sequences, such as SV40 polyA. In addition to or instead of such sequence-specific terminators, inserting a relatively long DNA sequence between the promoter and the coding region also tends to disrupt transcription of the coding region, which is usually proportional to the length of the inserted sequence. This effect can occur because RNA polymerase molecules always have some tendency to detach from the transcribed DNA, and increasing the length of the sequence that traverses before reaching the coding region will generally increase the likelihood that detachment will occur before the coding region transcription is complete or even before it begins. Therefore, a terminator can prevent transcription from only one direction (“unidirectional” terminator) or from both directions (“bidirectional” terminator), and can contain a sequence-specific terminator sequence or a sequence-nonspecific terminator sequence or both. Various such terminator sequences are known in the art; and illustrative uses of such sequences are provided below in the context of this invention.

[0029] "Host cell," "cell line," "cell culture," "packaging cell line," and other such terms refer to higher eukaryotic cells, such as mammalian cells, including human cells, that can be used in this invention to produce, for example, recombinant viruses or recombinant fusion peptides. These cells contain progeny of the transduced original cell. It should be understood that the progeny of a single cell may not necessarily be identical to the original parent cell (in terms of morphology or genomic complement).

[0030] When applied to polynucleotides, "recombination" means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps and other procedures that produce constructs of polynucleotides different from those found in nature. A recombinant virus is a viral particle comprising recombinant polynucleotides. The terms respectively encompass copies of the original polynucleotide construct and progeny of the original viral construct.

[0031] "Control elements" or "control sequences" are nucleotide sequences involved in molecular interactions that facilitate the functional regulation of polynucleotides, including replication, duplication, transcription, splicing, translation, or degradation. Regulation can affect the frequency, rate, or specificity of a process and can be either enhancing or repressive in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, binds to RNA polymerase and initiates transcription of a coding region that is typically located downstream of the promoter (along the 3' direction). Promoters include AAV promoters (e.g., P5, P19, P40, and AAV ITR promoters) and heteropromoters.

[0032] An "expression vector" is a vector that includes a region encoding a gene product of interest and is used to achieve the expression of the gene product in intended target cells. An expression vector also includes control elements operatively linked to the coding region to facilitate protein expression in the target. The combination of the control element and one or more genes operatively linked thereto for expression is sometimes referred to as an "expression cassette," and many expression cassettes are known and available in the art or can be readily constructed from components available in the art.

[0033] The terms “peptide” and “protein” are used interchangeably herein to refer to amino acid polymers of any length. The terms also cover modified amino acid polymers; for example, those that have undergone disulfide bond formation, glycosylation, acetylation, phosphorylation, esterification, or conjugation with labeled components.

[0034] The term "exogenous" when used with respect to proteins, genes, nucleic acids, or polynucleotides in cells or organisms refers to proteins, genes, nucleic acids, or polynucleotides that have been introduced into cells or organisms by artificial or natural means. Exogenous nucleic acids can originate from different organisms or cells, or they can be one or more additional copies of nucleic acids that are naturally present in an organism or cell. As non-limiting examples, exogenous nucleic acids may be located at a different chromosomal location than in natural cells, or may otherwise be side-linked with nucleic acid sequences different from those found in nature, such as expression cassettes that link promoters from one gene to the open reading frame of a gene product from a different gene.

[0035] As used herein, “transformed” or “genetically modified” includes any host cell or cell line that has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells of this invention are typically generated by transfection with a DNA sequence from a plasmid expression vector as an isolated linear DNA sequence or by infection with a recombinant viral vector.

[0036] The term "sequence homology" refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, such as 50%, the percentage represents the proportion of matches at a selected sequence length compared to another sequence. Vacancies (in either of the two sequences) are allowed to maximize the match; typically, a vacancy length of 15 bases or less is used, such as 6 bases or less, 2 bases or less. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is typically no less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); no less than 9 matches out of 10 possible base pair matches (90%); or no less than 19 matches out of 20 possible base pair matches (95%).

[0037] Two amino acid sequences are homologous if they share partial or complete identity. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for a maximum match. Vacancies are allowed in a maximum match (in either of the matched sequences); the vacancy length can be 5 or fewer, or 2 or fewer. Alternatively, two protein sequences (or peptide sequences derived from peptides of at least 30 amino acids in length) are homologous, as used herein, if an alignment score greater than 5 (in standard deviation units) is obtained using the ALIGN procedure with a mutation data matrix and a vacancy penalty of 6 or greater. Two sequences or portions thereof are considered more homologous if 50% or more of their amino acids are present when optimally aligned using the ALIGN procedure.

[0038] The term "corresponds to" is used herein to mean that a polynucleotide sequence is structurally related to all or part of a reference polynucleotide sequence, or a polypeptide sequence is structurally related to all or part of a reference polypeptide sequence, for example, having at least 80%, 85%, 90%, 95% or more (e.g., 99% or 100%) sequence identity. In contrast, the term "complementary to" is used herein to mean that a complementary sequence is homologous to all or part of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0039] The term "sequence identity" means that two polynucleotide sequences are identical within a comparison window (i.e., on a nucleotide-by-nucleotide basis). The term "sequence identity percentage" means that two polynucleotide sequences are identical within a comparison window (i.e., on a nucleotide-by-nucleotide basis). The term "sequence identity percentage" is calculated by: comparing two optimally aligned sequences within a comparison window; determining the number of positions in both sequences where the same nucleic acid base appears (e.g., A, T, C, G, U, or I) to produce the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size); and multiplying the result by 100 to produce the sequence identity percentage. As used herein, the term “substantially identical” refers to the characteristic of a polynucleotide sequence, wherein the polynucleotide includes a sequence that has at least 85% sequence identity (e.g., at least 90% to 95% sequence identity) or at least 99% sequence identity compared to a reference sequence in a comparison window of at least 20 nucleotide positions (typically in a window of at least 20-50 nucleotides), wherein the percentage of sequence identity is calculated by comparing the reference sequence with the polynucleotide sequence, which may contain deletions or additions that comprise a total of 20% or less of the reference sequence in the comparison window.

[0040] "Conservative" amino acid substitutions include, for example, aspartic acid-glutamic acid as polar acidic amino acids; lysine / arginine / histidine as polar basic amino acids; leucine / isoleucine / methionine / valine / alanine / glycine / proline as nonpolar or hydrophobic amino acids; and serine / threonine as polar or uncharged hydrophilic amino acids. Conservative amino acid substitutions also include groupings based on side chains. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. For example, it is reasonable to expect that replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly with structurally related amino acids will not have a major impact on the properties of the resulting polypeptide. Whether an amino acid change produces a functional polypeptide can be readily determined by measuring the specific activity of the polypeptide. Based on common side chain properties, naturally occurring residues are classified into several categories: (1) hydrophobic: ortholeucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues affecting chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0041] This disclosure also envisions polypeptides with non-conserved substitutions. Non-conserved substitutions require exchanging one member of the above categories for another.

[0042] Exemplary human APOE sequences include, but are not limited to: mkvlwaallv tflagcqa kv eqavetepep elrqqtewqs gqrwelalgr fwdylrwvqt lseqvqeell ssqvtqelra lmdetmkelk aykseleeql tpvaeetrar lskelqaaqa rlgadmedvc grlvqyrgev qamlgqstee lrvrlashlr klrkrllrda ddlqkrlavy qagaregaer glsairerlg plveqgrvra atvgslagqp lqeraqawge rlrarmeemg srtrdrldev keqvaevrak leeqaqqirl qaeafqarlk swfeplvedm qrqwaglvek vqaavgtsaa pvpsdnh (containing the signal peptide, italicized above) (SEQ ID NO:8) and a sequence having at least 80%, 85%, 90%, 95% or more (e.g., 99% or 100%) sequence identity with it, including a sequence having Cys at residue 112 (mature polypeptide number; bolded above c) and Cys at residue 158 (bolded above r) (APOE2), corresponding to SEQ ID NO:9, or having Arg at residue 112 (mature polypeptide number) and Arg at residue 158 (APOE4), corresponding to SEQ ID NO:9. NO:10, wherein in one embodiment, APOE4 may have 31K, 46P, 79T, 130R, 163C, 292H and / or 314R, and APOE2 may have 43C, 152Q, 154C / S, 163C / P, 164Q, 172A, 176C, 242Q, 246C, 254E.

[0043] SEQ ID NO:9 contains kv eqavetepep elrqqtewqs gqrwelalgr fwdylrwvqt lseqvqeell ssqvtqelra lmdetmkelk aykseleeql tpvaeetrar lskelqaaqa rlgadmedvc grlvqyrgev qamlgqstee lrvrlashlr klrkrllrda ddlqkclavy qagaregaer glsairerlg plveqgrvra atvgslagqp lqeraqawge rlrarmeemg srtrdrldev keqvaevrak leeqaqqirl qaeafqarlk swfeplvedm qrqwaglvek vqaavgtsaa pvpsdnh.

[0044] SEQ ID NO:10 contains kv eqavetepep elrqqtewqs gqrwelalgr fwdylrwvqt lseqvqeell ssqvtqelra lmdetmkelk aykseleeql tpvaeetrar lskelqaaqa rlgadmedvr grlvqyrgev qamlgqstee lrvrlashlr klrkrllrda ddlqkrlavy qagaregaer glsairerlg plveqgrvra atvgslagqp lqeraqawge rlrarmeemg srtrdrldev keqvaevrak leeqaqqirl qaeafqarlk swfeplvedm qrqwaglvek vqaavgtsaa pvpsdnh.

[0045] Exemplary human APOE nucleic acid sequences (e.g., if the nucleic acid sequence encodes APOE2, then it is used for silencing nucleotide substitutions) include, but are not limited to: ggaacttgat gctcagagag gacaagtcat ttgcccaagg tcacacagct ggcaactggc agagccagga ttcacgccct ggcaatttga ctccagaatc ctaaccttaa cccagaagca cggcttcaag cccctggaaa ccacaatacc tgtggcagcc agggggaggt gctggaatct catttcacat gtggggaggg ggctcccctg tgctcaaggt cacaaccaaa gaggaagctg tgattaaaac ccaggtccca tttgcaaagc ctcgactttt agcaggtgca tcatactgtt cccacccctc ccatcccact tctgtccagc cgcctagccc cactttcttt tttttctttt tttgagacag tctccctctt gctgaggctg gagtgcagtg gcgagatctc ggctcactgt aacctccgcc tcccgggttc aagcgattct cctgcctcag cctcccaagt agctaggatt acaggcgccc gccaccacgc ctggctaact tttgtatttt tagtagagat ggggtttcac catgttggcc aggctggtct caaactcctg accttaagtg attcgcccac tgtggcctcc caaagtgctg ggattacagg cgtgagctac cgcccccagc ccctcccatc ccacttctgt ccagccccct agccctactt tctttctggg atccaggagt ccagatcccc agccccctct ccagattaca ttcatccagg cacaggaaag gacagggtca ggaaaggagg actctgggcg gcagcctcca cattcccctt ccacgcttgg cccccagaat ggaggagggt gtctggatta ctgggcgagg tgtcctccct tcctggggac tgtggggggt ggtcaaaaga cctctatgcc ccacctcctt cctccctctg ccctgctgtg cctggggcag ggggagaaca gcccacctcg tgactggggg ctggcccagc ccgccctatc cctgggggag ggggcgggac agggggagcc ctataattgg acaagtctgg gatccttgag tcctactcag ccccagcgga ggtgaaggac gtccttcccc aggagccg(SEQ ID NO:11) or ccccagcgga ggtgaaggac gtccttcccc aggagccgac tggccaatca caggcaggaa gatgaaggtt ctgtgggctg cgttgctggt cacattcctg gcaggatgcc aggccaaggt ggagcaagcg gtggagacag agccggagcc cgagctgcgc cagcagaccg agtggcagag cggccagcgc tgggaactgg cactgggtcg cttttgggat tacctgcgct gggtgcagac actgtctgag caggtgcagg aggagctgct cagctcccaa gtcacccaag aactgagggc gctgatggac gagaccatga aggagttgaa ggcctacaaa tcggaactgg aggaacaact gaccccggta gcggaggaga cgcgggcacg gctgtccaag gagctgcaga cggcgcaggc ccggctgggc gcggacatgg aggacgtgtg cggccgcctg gtgcagtacc gcggcgaggt gcaggccatg ctcggccaga gcaccgagga gctgcgggtg cgcctcgcct cccacctgcg caagctgcgt aagcggctcc tccgcgatcc cgatgacctg cagaagcgcc tggcagtgta ccaggccggg gcccgcgagg gcgccgagcg cggcctcagc gccatccgcg agcgcctggg gcccctggtg gaacagggcc gcgtgcgggc cgccactgtg ggctccctgg ccggccagcc gctacaggag cgggcccagg cctggggcga gcggctgcgc gcgcggatgg aggagatggg cagtcggacc cgcgaccgcc tggacgaggt gaaggagcag gtggcggagg tgcgcgccaa gctggaggag caggcccagc agatacgcct gcaggccgag gccttccagg cccgcctcaa gagctggttc gagcccctgg tggaagacat gcagcgccag tgggccgggc tggtggagaa ggtgcaggct gccgtgggca ccagcgccgc ccctgtgccc agcgacaatc actgaacgcc gaagcctgca gccatgcgac cccacgccac cccgtgcctc ctgcctccgc gcagcctgca gcgggagacc ctgtccccgc cccagccgtc ctcctggggt ggaccctagt ttaataaaga ttcaccaagt ttcacgc (SEQ ID NO: 12), And sequences that have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more (e.g., 99% or 100%) sequence identity with the sequence encoding APOE.

[0046] Compositions and methods Alzheimer's disease (AD) directly affects 5 million Americans, and its prevalence and economic impact are rapidly increasing. Existing medications have little effect on the underlying disease process, and there are currently no preventative treatments available. Inheritance of the variant APOE4 gene carries a high risk of developing AD, while inheritance of the APOE2 gene is protective, reducing the risk of AD by approximately 50% and delaying the age of onset. APOE4 is associated with increased amyloid-beta load in the brain and greater memory impairment in AD. Conversely, APOE2 attenuates these effects. In humans, the odds ratio for developing AD is 14.9 in the E4 / E4 homozygous genotype, while it drops to 2.6 in the E2 / E4 heterozygous genotype. In addition to its role in promoting amyloid-beta production, APOE4 may be associated with abnormal brain function.

[0047] This disclosure provides gene therapy vectors for expressing APOE2, sequences for inhibiting APOE4 expression, and methods for using APOE2 and APOE4 inhibitory sequences.

[0048] Exemplary gene therapy vector This disclosure provides a gene therapy vector comprising a nucleic acid sequence encoding APOE2 and may contain a repressive sequence for endogenous APOE4 expression, or in one embodiment may contain another vector for expressing the repressive sequence or a composition having a repressive RNA sequence. Various aspects of the gene therapy vector and methods are discussed below. Although each parameter is discussed individually, the gene therapy vector and methods include combinations of the parameters listed below, for example, to induce protection against APOE4-related pathologies. Therefore, any combination of parameters can be used according to the gene therapy vector and methods.

[0049] Therefore, a "gene therapy vector" is any molecule or composition capable of carrying a heterologous nucleic acid sequence into a suitable host cell where the synthesis of the encoded protein takes place. Typically, a gene therapy vector is a nucleic acid molecule that has been engineered using recombinant DNA techniques known in the art to incorporate a heterologous nucleic acid sequence (e.g., heterologous relative to other vector sequences, such as promoters or vector backbone sequences, like viral sequences). Desiredly, a gene therapy vector contains DNA. Examples of suitable DNA-based gene therapy vectors include plasmids and viral vectors. However, gene therapy vectors that are not solely based on nucleic acids, such as liposomes or nanoparticles, can also be used. Gene therapy vectors can be based on a single type of nucleic acid (e.g., plasmids) or contain non-nucleic acid molecules (e.g., lipids or polymers). Gene therapy vectors can be integrated into the host cell genome or can exist in the host cell in a free form.

[0050] Gene or siRNA delivery vectors within the scope of this disclosure include, but are not limited to, isolated nucleic acids (e.g., plasmid-based vectors that can be maintained extrachromosomally) and viral vectors, such as recombinant adenoviruses, retroviruses, lentiviruses, herpesviruses, poxviruses, papillomaviruses, or adeno-associated viruses; viral and nonviral vectors present in liposomes, such as neutral or cationic liposomes, such as DOSPA / DOPE, DOGS / DOPE, or DMRIE / DOPE liposomes; and / or viral and nonviral vectors associated with other molecules, such as DNA anti-DNA antibody cationic lipid (DOTMA / DOPE) complexes or natural or synthetic polymers. Exemplary viral gene delivery vectors are described below. Gene delivery vectors can be administered via any route, including but not limited to intracranial, intrathecal, intramuscular, intraoral, rectal, intravenous, or intracoronary administration, and can be enhanced to transfer to cells using electroporation and / or iontophoresis and / or scaffolds (such as extracellular matrix or hydrogels, such as hydrogel patches).

[0051] In one embodiment, the gene therapy vector or other vector is a viral vector. Suitable viral vectors include, for example, retroviral vectors, lentiviral vectors, herpes simplex virus (HSV)-based vectors, parvovirus-based vectors, such as adeno-associated virus (AAV)-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors. Examples of such vectors include those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*. Molecular Cloning, a Laboratory Manual ), 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Ausubel et al., *Experimental Guide to Contemporary Molecular Biology* Current Protocols in Molecular Biolog The standard recombinant DNA technique described in Greene Publishing Associates and John Wiley & Sons (1994) can be used to prepare these viral vectors.

[0052] Retroviral vector Retroviral vectors exhibit several unique characteristics, including their ability to stably and precisely integrate into the host genome, thereby providing long-term transgenic expression. These vectors can be manipulated in vitro to eliminate infectious gene particles, thus minimizing the risk of systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can alter host cell tropism.

[0053] Lentiviral Lentivirals originate from the retrovirus family, including human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV). However, unlike retroviruses that infect only dividing cells, lentiviruses can infect both dividing and non-dividing cells. Despite the specificity of lentiviruses, pseudotyping the viral envelope with herpestostomosis virus yields a broader range of viruses (Schnepp et al., *Meth. Mol. Med.*, 69:427 (2002)).

[0054] Adenovirus vector Adenoviral vectors can be rendered unable to replicate by deleting early (E1A and E1B) genes responsible for viral gene expression from the genome, and can be stably maintained in host cells in an extrachromosomal form. These vectors have the ability to transfect both replicating and non-replicating cells. It has been shown that adenoviral vectors result in transient expression of therapeutic genes in vivo, peaking at 7 days and lasting for approximately 4 weeks. Furthermore, adenoviral vectors can be produced at very high titers, allowing for effective gene therapy using small amounts of virus.

[0055] Adeno-associated virus vector Recombinant adeno-associated virus (rAAV) is derived from non-pathogenic parvoviruses, generally does not induce a cellular immune response, and produces transgene expression that persists for several months in most systems. Furthermore, like adenoviruses, adeno-associated virus vectors also have the ability to infect both replicating and non-replicating cells.

[0056] AAV vectors include, but are not limited to, AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh10, including chimeric viruses whose AAV genomes originate from a different source than the capsid.

[0057] plasmid DNA vector Plasmid DNA is often referred to as “naked DNA” to indicate the lack of more sophisticated packaging systems. Direct injection of plasmid DNA into cardiomyocytes in vivo has been achieved. Plasmid-based vectors are relatively non-immunogenic and non-pathogenic, with the potential for stable integration into the cellular genome, leading to long-term gene expression in cells post-mitotically in vivo. Furthermore, plasmid DNA degrades rapidly in the bloodstream; therefore, the chance of transgene expression in distant organ systems is negligible. Plasmid DNA can be delivered to cells as part of macromolecular complexes (e.g., liposomes or DNA-protein complexes), and delivery can be enhanced using techniques involving electroporation.

[0058] Exemplary AAV carrier In one embodiment, this disclosure provides an adeno-associated virus (AAV) vector comprising, substantially consisting of, or consisting of a nucleic acid sequence encoding APOE2. When the AAV vector consists substantially of a nucleic acid sequence encoding APOE2, it may contain additional components (e.g., genetic elements, such as poly(A) sequences or restriction enzyme sites that promote manipulation of the vector in vitro) that do not materially affect the AAV vector. When the AAV vector consists of a nucleic acid sequence encoding APOE2, the AAV vector does not include any additional components (i.e., components that are non-endogenous to AAV and not essential to affecting the expression of the nucleic acid sequence).

[0059] Adeno-associated viruses (AAVs) are members of the parvovirus family and consist of a linear, single-stranded DNA genome of fewer than approximately 5,000 nucleotides. AAVs require co-infection with a helper virus (i.e., adenovirus or herpesvirus) or expression of a helper gene to achieve efficient replication. AAV vectors used for administering therapeutic nucleic acids typically lack approximately 96% of the parental genome, leaving only terminal repeat sequences (ITRs) containing recognition signals for DNA replication and packaging. This eliminates immunological or toxic side effects caused by viral gene expression. Additionally, delivery of specific AAV proteins to production cells, if desired, allows for the integration of AAV vectors, including AAV ITRs, into specific regions of the cell genome (see, for example, U.S. Patents 6,342,390 and 6,821,511). Host cells containing the integrated AAV genome show no changes in cell growth or morphology (see, for example, U.S. Patent 4,797,368).

[0060] The AAV ITR is flanked by unique nucleotide sequences encoding the non-structural replication (Rep) protein and the structural capsid (Cap) protein (also known as the viral particle protein (VP)). The terminal 145 nucleotides are self-complementary and organized into an energy-stable intramolecular double helix that allows the formation of T-shaped hairpins. These hairpin structures serve as the starting point for viral DNA replication by acting as primers for the cellular DNA polymerase complex. The Rep gene encodes Rep proteins, namely Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed by the p5 promoter, and Rep52 and Rep40 are transcribed by the p19 promoter. Rep78 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication to allow the AAV terminal to be broken down (see, for example, Im et al., Cell, 61:447 (1990)). These proteins also regulate transcription of the endogenous AAV promoter and helper viral promoters (see, for example, Pereira et al., *Journal of Virology*, 71:1079 (1997)). Other Rep proteins modify the function of Rep78 and Rep68. The cap gene encodes capsid proteins VP1, VP2, and VP3. The cap gene is transcribed by the p40 promoter.

[0061] AAV vectors can be generated using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stockpile or from human or nonhuman primate tissues (e.g., reviewed in Wu et al., Molecular Therapy, 14(3): 316 (2006)). Generally, AAV serotypes have significantly homologous genomic sequences at both the nucleic acid and amino acid sequence levels, resulting in different serotypes having the same set of genetic functions, producing essentially physically and functionally equivalent viral particles, and replicating and assembling through nearly identical mechanisms. AAV serotypes 1–5 and 7–9 are defined as “true” serotypes because they do not readily cross-react with neutralizing sera that are specific to all other existing and characterized serotypes. In contrast, AAV serotypes 6, 10 (also known as Rh10), and 11 are considered “variant” serotypes because they do not meet the definition of a “true” serotype. AAV serotype 2 (AAV2) has been widely used in gene therapy applications because of its lack of pathogenicity, widespread infectivity and ability to establish long-term transgenic expression (see, for example, Carter, Human Gene Therapy, 16:541 (2005); and Wu et al., ibid.). The genome sequences of various AAV serotypes and their comparisons are disclosed in, for example, GenBank accessions U89790, J01901, AF043303 and AF085716; Chiorini et al., Journal of Virology, 71:6823 (1997); Srivastava et al., Journal of Virology, 45:555 (1983); Chiorini et al., Journal of Virology, 73:1309 (1999); Rutledge et al., Journal of Virology, 72:309 (1998); and Wu et al., Journal of Virology, 74:8635 (2000).

[0062] AAV rep and ITR sequences are particularly conserved in most AAV serotypes. For example, the Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4, and AAV6 are reported to be approximately 89–93% identical (see Bantel-Schaal et al., Journal of Virology, 73(2):939 (1999)). AAV serotypes 2, 3A, 3B, and 6 are reported to share approximately 82% total nucleotide sequence identity at the genomic level (Bantel-Schaal et al., ibid.). Furthermore, the rep sequences and ITRs of many AAV serotypes are known to be effectively cross-complementary (e.g., functionally substituted) from corresponding sequences of other serotypes during the production of AAV particles in mammalian cells.

[0063] Typically, compared to the Rep gene, the cap protein, which determines AAV granule tropism, and the associated cap protein coding sequence are significantly less conserved across different AAV serotypes. Given the ability of Rep and ITR sequences to cross-complement corresponding sequences in other serotypes, AAV vectors can comprise mixtures of serotypes, and are thus “chimeric” or “pseudotyped” AAV vectors. Chimeric AAV vectors typically comprise AAV capsid proteins derived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes. In contrast, pseudotyped AAV vectors comprise one or more ITRs from one AAV serotype packaged into the capsid of another AAV serotype. Chimeric and pseudotyped AAV vectors are further described in, for example, U.S. Patent No. 6,723,551; Flotte, Molecular Therapy, 13(1):1 (2006); Gao et al., Journal of Virology, 78:6381 (2004); Gao et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA), 99:11854 (2002); De et al., Molecular Therapy, 13:67 (2006); and Gao et al., Molecular Therapy, 13:77 (2006).

[0064] In one embodiment, the AAV vector is generated using an AAV that infects humans (e.g., AAV2). Alternatively, the AAV vector is generated using AAV that infects non-human primates (e.g., great apes, chimpanzees, Old World monkeys, rhesus macaques, and New World monkeys, e.g., marmosets). In one embodiment, the AAV vector is generated using AAV that infects non-human primates pseudotyped with AAV that infects humans. Examples of such pseudotyped AAV vectors are disclosed, for example, in Cearley et al., Molecular Therapy, 13:528 (2006). In one embodiment, an AAV vector comprising a capsid protein from an AAV can be generated, which infects rhesus monkeys pseudotyped with an AAV2 inverted terminal repeat (ITR). In a particular embodiment, the AAV vector includes a capsid protein from AAV10 (also known as “AAVrh.10”), which infects rhesus monkeys pseudotyped with AAV2 ITR (see, for example, Watanabe et al., Gene Ther., 17(8):1042 (2010); and Mao et al., Human Gene Ther., 22:1525 (2011)).

[0065] In addition to the nucleic acid sequence encoding APOE2, the AAV vector may include expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc., that provide expression of the nucleic acid sequence in the host cell, and, in one embodiment, the sequence of APOE4 RNAi. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, *Gene Expression Technology: Methods in Enzymology*, Vol. 185, Academic Press, San Diego, CA (1990).

[0066] A wide variety of promoters, including constitutive, inducible, and repressive promoters, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available or can be synthesized based on sequences publicly available, for example, from depositories such as ATCC and other commercial or personal sources. Promoters can be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating transcription in the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (US Patents 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proceedings of the National Academy of Sciences, 93:3346 (1996)), the T-REXTM system (Invitrogen Carlsbad, CA), the LACSWITCH™ system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nucleic Acid Res., 27:4324 (1999); Nucleic Acid Res., 28:e99 (2000); US Patent 7,112,715; and Kramer and Fussenegger, Methods in Molecular Biology, 308:123). (2005)).

[0067] As used herein, the term "enhancer" refers to a DNA sequence that adds transcription to, for example, a nucleic acid sequence operatively linked thereto. Enhancers can be located many kilobases from the coding region of a nucleic acid sequence and can mediate changes in regulatory factors, DNA methylation patterns, or DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as clonal polynucleotides or within clonal polynucleotides (e.g., from depositories such as ATCC and other commercial or personal sources). Many polynucleotides that include promoters (such as the commonly used CMV promoter) also include enhancer sequences. Enhancers can be located upstream, inside, or downstream of the coding sequence. In one embodiment, the nucleic acid sequence encoding APOE2 is operatively linked to the CMV enhancer / chicken β-actin promoter (also known as the “CAG promoter”) (see, for example, Niwa et al., Gene, 108:193 (1991); Daly et al., Proceedings of the National Academy of Sciences, 96:2296 (1999); and Sondhi et al., Molecular Therapy, 15:481 (2007)).

[0068] Typically, AAV vectors are generated using well-characterized plasmids. For example, human embryonic kidney 293T cells are transfected with a transgene-specific plasmid and one of two plasmids containing an adenovirus helper gene and AAV rep and cap genes (specific to AAVrh.10, 8, or 9 as needed). After 72 hours, cells are harvested and the vector is released from the cells via five freeze-thaw cycles. Subsequent centrifugation and benzonase treatment remove cell debris and uncoated DNA. Iodixanol gradients and ion-exchange columns can be used for further purification of each AAV vector. Next, the purified vector is concentrated to the desired concentration by rotating the column in a size-exclusion centrifuge. Finally, the buffer is exchanged to produce the final vector product prepared in, for example, 1× phosphate-buffered saline. This can be done via TaqMan. ® Viral titers can be measured by real-time PCR, and viral purity can be assessed by SDS-PAGE.

[0069] Delivery of pharmaceutical compositions and carriers This disclosure provides a composition comprising, substantially comprising, or consisting of the gene therapy vector described above, and a pharmaceutically acceptable (e.g., physiologically acceptable) vector or a vector for expressing RNAi. When the composition consists substantially of a gene therapy vector and a pharmaceutically acceptable vector, additional components (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.) that do not materially affect the composition may be included. When the composition consists of a gene therapy vector and a pharmaceutically acceptable vector, the composition does not include any additional components. Any suitable vector may be used within the context of this disclosure, and such vectors are well known in the art. The choice of vector will be determined in part by the specific site at which the composition can be administered and the specific method for administering the composition. In addition to the gene therapy vectors described herein, the composition may optionally be sterile. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile vector prior to use. It may be based on, for example, Remington: Pharmaceutical Science and Practice (… Remington: The Science and Practice of Pharmacy The conventional techniques described in the composition produced in Lippincott Williams & Wilkins, 21st edition, Philadelphia, Pennsylvania, (2001) are as follows.

[0070] Suitable formulations of the composition include aqueous and non-aqueous solutions, isotonic sterile solutions that may contain antioxidants, buffers, and antibacterial agents, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be present in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions requiring only the addition of a sterile liquid carrier (e.g., water) before immediate use. Temporary solutions and suspensions can be prepared from sterile powders, granules, and tablets of the aforementioned types. In one embodiment, the carrier is a buffered saline solution. In one embodiment, the gene therapy carrier is administered in the form of a composition formulated to protect the gene therapy carrier from damage prior to administration. For example, the composition may be formulated to reduce loss of the gene therapy carrier on devices (such as glassware, syringes, or needles) used for preparing, storing, or administering the gene therapy carrier. The composition may be formulated to reduce the photosensitivity and / or temperature sensitivity of the gene therapy carrier. Therefore, the composition may include a pharmaceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of: polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Using such compositions will extend the shelf life of gene therapy vectors, facilitate administration, and improve the efficiency of the method. For example, Wright et al., *New Insights in Drug Development* (… Curr. Opin. Drug Discov. Devel .)》, 6(2): 174-178 (2003) and Wright et al., Molecular Therapy, 12 Formulations for use in compositions containing gene therapy vectors are further described in 171-178 (2005).

[0071] The composition can also be formulated to enhance transduction efficiency. Additionally, those skilled in the art will understand that the gene therapy vector can be present in the composition with other therapeutic agents or bioactive agents. For example, anti-inflammatory factors (such as ibuprofen or steroids) may be part of the composition to reduce swelling and inflammation associated with in vivo administration of the gene therapy vector. Immune system stimulants or adjuvants (e.g., interleukins, lipopolysaccharides, and double-stranded RNA) may be administered to enhance or alter the immune response. Antibiotics, i.e., antimicrobial and antifungal agents, may be present to treat existing infections and / or reduce the risk of future infections, such as those associated with gene therapy procedures.

[0072] Injectable depot formulations are prepared by forming a microcapsule matrix of the subject compound within a biodegradable polymer, such as poly(lactide-polyglycolic acid). The drug release rate can be controlled based on the drug-to-polymer ratio and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot-type injectable formulations are also prepared by trapping the drug within liposomes or microemulsions that are compatible with body tissues.

[0073] In some embodiments, the formulation includes biocompatible polymers selected from the group consisting of: polyamides, polycarbonates, polyolefins, polymers of acrylic acid and methacrylates, polyethylene polymers, polyglycolic acid, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(orthocyanidates), poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures or copolymers thereof.

[0074] The composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible mesh, mechanical reservoir, or mechanical implant. Implants (see, for example, U.S. Patent No. 5,443,505) and devices (see, for example, U.S. Patent No. 4,863,457), such as implantable devices (e.g., mechanical reservoirs, implants, or devices comprising the polymer composition), are particularly useful for the administration of gene therapy vectors. The composition can also be administered in the form of a sustained-release formulation (see, for example, U.S. Patent No. 5,378,475), which includes, for example, gel foams, hyaluronic acid, gelatin, chondroitin sulfate, polyphosphates such as bis-2-hydroxyethyl terephthalate (BHET), and / or polylactic-co-glycolic acid.

[0075] Delivery of compositions including gene therapy vectors using devices known in the art can be intracerebral (including but not limited to intraparenchymal, intravenous, or intracisional), intrathecal (including but not limited to the lumbar vertebral or cerebellomedullary cistern), or systemic (including but not limited to intravenous), or any combination thereof. Delivery can also be performed by surgical implantation of the implantation device.

[0076] The dosage of the gene therapy vector in a composition administered to mammals will depend on many factors, including the size (mass) of the mammal, the extent of any side effects, the specific route of administration, etc. In one embodiment, the method includes administering a “therapeutic effective amount” of a composition comprising the gene therapy vector described herein. A “therapeutic effective amount” refers to an effective amount, measured in doses and sustained for the required period of time, to achieve the desired therapeutic outcome. Therapeutic effective amounts can vary depending on factors such as the individual’s pathological condition, age, sex, and weight, as well as the gene therapy vector’s ability to elicit the desired response in the individual. The dosage of the gene therapy vector in the composition required to achieve a specific therapeutic effect is typically administered in units of vector genome copies per cell (gc / cell) or vector genome copies per kilogram of body weight (gc / kg). Based on these and other factors well known in the art, those skilled in the art can readily determine the appropriate range of gene therapy vector dosages for treating patients with a specific disease or condition. Therapeutic effective amounts can range from 1 × 10⁻⁶. 10 One genome copy to 1 × 10 13 Between one genome copy. The therapeutically effective dose can be between 1 × 10⁻⁶. 11 One genome copy to 1 × 10 14 Between one genome copy. The therapeutically effective dose can be between 1 × 10⁻⁶. 12 One genome copy to 1 × 10 15 Between 1000 genomic copies. The therapeutically effective dose can be 1 × 10⁻⁶. 13 One genome copy (gc) to 1 × 1016 gc, for example, 1 × 10 13 gc to 1 × 10 14 gc, 1 × 10 14 gc to 1 × 10 15 gc or 1 × 10 15 gc to 1 × 10 14 gc. Assuming a 70 kg person, the dosage range could be 1.4 × 10⁻⁶. 8 gc / kg to 1.4 × 10 11 gc / kg, 1.4 × 10 9 gc / kg to 1.4 × 10 12 gc / kg, 1.4 × 10 10 gc / kg to 1.4 × 10 13 gc / kg or 1.4 × 10 11 gc / kg to 1.4 × 10 14 gc / kg.

[0077] In one embodiment, the composition is administered to a mammal once. It is believed that a single administration of the composition will result in inhibition of APOE2 and APOE4 expression in the mammal with minimal side effects. However, in some cases, it may be appropriate to administer the composition multiple times during the treatment period to ensure adequate cell exposure. For example, the composition may be administered to the mammal two or more times during the treatment period (e.g., 2, 3, 4, 5, 6, 6, 8, 9, or 10 or more times).

[0078] Therefore, this disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of a gene therapy vector, the gene therapy vector comprising a nucleic acid sequence encoding APOE2 and a sequence inhibiting APOE4 expression.

[0079] Subjects Subjects can be any animal, including both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, but mammals such as non-human primates, sheep, dogs, cats, cows, and horses are envisioned as subjects. Subjects can also be livestock, such as cattle, pigs, sheep, poultry, and horses, or pets, such as dogs and cats.

[0080] In one embodiment, subjects include human subjects who have or are at risk of having the medical diseases and conditions described herein. Subjects are typically diagnosed with their conditions by a skilled technician, such as a licensed physician.

[0081] The methods described herein can be used for subjects of any species, sex, age, ethnic group, or genotype. Therefore, the term "subject" includes both males and females, and includes older adults, older-to-adult transitional subjects, adults, adults-to-pre-adult transitional subjects, and pre-adults, including adolescents, children, and infants.

[0082] Examples of human racial groups include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semitic peoples, and Pacific Islanders. This approach may be more suitable for certain racial groups, such as Caucasians, particularly those from Northern Europe, and Asian populations.

[0083] As described above, the term "subject" also includes subjects with any genotype or phenotype, provided they require treatment. Additionally, subjects may have any genotype or phenotype of hair color, eye color, skin color, or any combination thereof. The term "subject" includes subjects with any height, weight, or the size or shape of any organ or body part.

[0084] Exemplary nanoparticle formulations Biodegradable nanoparticles (e.g., gene therapy vectors or isolated nucleic acids or vectors for RNAi expression) may comprise or be formed from biodegradable polymer molecules, said biodegradable polymer molecules may comprise, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA (i.e., polylactic-co-glycolic acid (PLGA)), poly-ε-caprolactone (PCL), polyethylene glycol (PEG), poly(3-hydroxybutyrate), poly(p-dioxanone), and polypropylene glycol. Alcohol fumarate, poly(orthoester), polyol / dienone acetal addition polymers, polyalkyl-cyanoacrylate (PAC), poly(sebacic anhydride) (PSA), poly(carboxylated dicarboxyphenoxyphenoxyhexanone) (PCPP), poly[bis(p-carboxyphenoxy)methane] (PCPM), copolymers of PSA, PCPP and PCPM, poly(amino acids), poly(pseudoamino acids), polyphosphazenes, poly[(dichloro)phosphazenes] and poly[(organo)phosphazenes] derivatives, poly-hydroxybutyric acid or S-hexanoic acid, elastin or gelatin. (See, for example, Kumari et al., Colloids and Surfaces B: Biointerfaces, 75 (2010)) 1-18; and U.S. Patent Nos. 6,913,767; 6,884,435; 6,565,777; 6,534,092; 6,528,087; 6,379,704; 6,309,569; 6,264,987; 6,210,707; 6,090,925; 6,022,564; 5,981,719; 5,871,747; 5,723,269; 5,603,960; and 5,578,709; and U.S. Publication No. 2007 / 0081972; and International Application Publication No. WO2012 / 115806; and WO Document No. 2012 / 054425; the contents of the aforementioned document are incorporated herein by reference in their entirety.

[0085] Biodegradable nanoparticles can be prepared by methods known in the art. (See, for example, Nagavarma et al., *Asian J. of Pharma. And Clin. Res.*, Vol. 5, Supplement 3, 2012, pp. 16-23; Cismaru et al., *Rev. Roum. Chim.*, 2010, 55(8), 433-442; and International Application Publication No. WO 2012 / 115806; and No. WO 2012 / 054425; the contents of which are incorporated herein by reference in their entirety). Suitable methods for preparing nanoparticles may include methods utilizing pre-formed polymer dispersions, which may include, but are not limited to, solvent evaporation, nanoprecipitation, emulsification / solvent diffusion, salting out, dialysis, and supercritical fluid techniques. In some embodiments, nanoparticles may be prepared by forming a double emulsion (e.g., oil-in-water / water-in-oil) followed by solvent evaporation. The nanoparticles obtained by the disclosed method may be subjected to further processing steps, such as washing and freeze-drying, as needed. Optionally, the nanoparticles may be combined with a preservative (e.g., trehalose).

[0086] Typically, the average effective diameter of nanoparticles is less than 1 micrometer. For example, the average effective diameter of nanoparticles is between about 25 nm and about 500 nm, such as between about 50 nm and about 250 nm, about 100 nm to about 150 nm, or about 450 nm to 650 nm. The size of the particles (e.g., the average effective diameter) can be assessed by methods known in the art, which may include, but are not limited to, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), photon correlation spectroscopy (PCS), nanoparticle surface area monitor (NSAM), condensed particle counter (CPC), differential mobility analyzer (DMA), scanning mobility particle size analyzer (SMPS), nanoparticle tracking analysis (NTA), X-ray diffraction (XRD), aerosol time-of-flight mass spectrometry (ATFMS), and aerosol particle mass analyzer (APM).

[0087] Biodegradable nanoparticles can possess a zeta potential that promotes uptake by target cells. Typically, the zeta potential of nanoparticles is greater than 0. In some embodiments, the zeta potential of nanoparticles is between about 5 mV and about 45 mV, between about 15 mV and about 35 mV, or between about 20 mV and about 40 mV. The zeta potential can be determined by properties including electrophoretic mobility or dynamic electrophoretic mobility. Electrokinetic and electroacoustic phenomena can be used to calculate the zeta potential.

[0088] In one embodiment, the non-viral delivery medium includes: polymers, including but not limited to poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), linear and / or branched PEIs with different molecular weights (e.g., 2, 22, and 25 kDa), dendritic polymers such as polyamidoamine (PAMAM), and polymethacrylates; lipids, including but not limited to cationic liposomes, cationic emulsions, DOTAP, DOTMA, DMRIE, DOSPA, distearate phosphatidylcholine (DSPC), DOPE, or DC-cholesterol; peptide-based carriers, including but not limited to poly-L-lysine or protamine; or poly(β-amino esters), chitosan, PEI-polyethylene glycol, PEI-mannose-glucan, DOTAP-cholesterol, or RNAiMAX.

[0089] In one embodiment, the delivery medium is a sugar-based polymer delivery medium, namely poly(glycosaminoglycan) (PGAA), which has the ability to complex with various polynucleotide types and form nanoparticles. These materials are produced by polymerizing various carbohydrate methyl esters or lactone derivatives (D-gluconic acid (D), meso-galactosidate (G), D-mannitate (M), and L-tartrate (T)) with a series of oligomeric ethylene amine monomers (containing 1-4 ethylene amines (Liu and Reineke, 2006)). The subset consisting of these carbohydrates and four ethylene amines in the polymer repeating unit produces excellent delivery efficiency.

[0090] In one embodiment, the delivery medium comprises polyethyleneimine (PEI), polyamidoamine (PAMAM), PEI-PEG, PEI-PEG-mannose, dextran-PEI, OVA conjugates, PLGA microparticles, or PLGA microparticles coated with PAMAM, or any combination thereof. The disclosed cationic polymer may comprise, but is not limited to, polyamidoamine (PAMAM) dendritic polymers. Polyamidoamine dendritic polymers suitable for preparing the currently disclosed nanoparticles may comprise third-generation, fourth-generation, fifth-generation, or at least sixth-generation dendritic polymers.

[0091] In one embodiment, the delivery medium comprises lipids, for example... N -[1-(2,3-dioleoyloxy)propyl]- N , N , N -Trimethylammonium (DOTMA), 2,3-diolenyloxy- N -[2-Sperminecarbamate]ethyl- N , N-Dimethyl-1-trifluoroacetic acid propane (DOSPA, Lipofectamine); 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP). N -[1-(2,3-dimyristoyloxy)propyl]; N , N -dimethyl- N -(2-hydroxyethyl)ammonium bromide (DMRIE), 3-β-[ N -( N , N [-Dimethylaminoethane]carbamoyl]cholesterol (DC-Chol); bis(octadecylaminoglycerol)spermine (DOGS, Transfectam); or methyloctadecylammonium bromide (DDAB). The positively charged hydrophilic headgroups of cationic lipids are typically composed of monoamines (such as tertiary and quaternary amines), polyamines, amidines, or guanidines. A series of pyridinium lipids have been developed (Zhu et al., 2008; van der Woude et al., 1997; Ilies et al., 2004). Besides pyridinium cationic lipids, other types of heterocyclic headgroups include imidazoles, piperazines, and amino acids. The primary function of cationic headgroups is to enhance cellular uptake and endosome escape by condensing negatively charged nucleic acids into slightly positively charged nanoparticles through electrostatic interactions.

[0092] Lipids with two straight-chain fatty acid chains (such as DOTMA, DOTAP, and SAINT-2 or DODAC) and tetraalkyl lipid chain surfactants, i.e. N , N -dioleenyl- N , N Dimers of dimethylammonium chloride (DODAC) can be used as delivery mediators. Compared to the cis-oriented counterparts of trans-oriented lipids, regardless of their hydrophobic chain length (C... 16:1 C 18:1 and C 20:1 However, all trans-oriented lipids appear to enhance transfection efficiency.

[0093] The structures of cationic polymers that can be used as delivery media include, but are not limited to, linear polymers (such as chitosan and linear poly(ethyleneimine)), branched polymers (such as branched poly(ethyleneimine) (PEI)), cyclic polymers (such as cyclodextrin), network (crosslinked) polymers (such as crosslinked poly(amino acid) (PAA)), and dendritic polymers. Dendritic polymers consist of a central core molecule from which several highly branched arms “grow” to form a tree-like structure with symmetrical or asymmetrical arrangements. Examples of dendritic polymers include polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendritic polymers.

[0094] DOPE and cholesterol are commonly used neutral accessory lipids in the preparation of cationic liposomes. Branched PEI-cholesterol water-soluble lipid polymer conjugates self-assemble into cationic micelles. Alternatively, nonionic polymers Pluronic and SP1017, a combination of Pluronics L61 and F127, can be used.

[0095] In one embodiment, PLGA particles are used to increase the encapsulation frequency, although using PLL to form a composite can also increase encapsulation efficiency. Other cationic materials, such as PEI, DOTMA, DC-Chol, or CTAB, can be used to fabricate nanospheres.

[0096] In one embodiment, the complex is embedded in or applied to a material comprising, but not limited to, poloxamer, polyacrylamide, poly(2-hydroxyethyl methacrylate), carboxyvinyl polymers (e.g., Carbopol 934, Goodrich Chemical Co.), cellulose derivatives (e.g., methylcellulose, cellulose acetate, and hydroxypropylcellulose), polyvinylpyrrolidone, or polyvinyl alcohol, or a combination thereof, as a hydrogel.

[0097] In some embodiments, the biocompatible polymer material is derived from a biodegradable polymer, such as collagen, for example hydroxylated collagen, fibrous protein, polylactic acid-polyglycolic acid, or polyanhydride. Other examples include, but are not limited to, any biocompatible polymer, whether hydrophilic, hydrophobic, or amphiphilic, such as ethylene vinyl acetate copolymer (EVA), polymethyl methacrylate, polyamide, polycarbonate, polyester, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, N-isopropylacrylamide copolymer, poly(ethylene oxide) / poly(propylene oxide) block copolymer, poly(ethylene glycol) / poly(D,L-lactide-co-glycolic acid) block copolymer, polyglycolic acid, polylactide (PLLA or PDLA), poly(caprolactone) (PCL), or poly(dioxanone) (PPS).

[0098] In another embodiment, the biocompatible material comprises polyethylene terephthalate, polytetrafluoroethylene, copolymers of polyethylene oxide and polypropylene oxide, combinations of polyglycolic acid and polyhydroxyalkanoates, gelatin, alginate, poly-3-hydroxybutyrate, poly-4-hydroxybutyrate and polyhydroxyoctanoate, and polyacrylonitrile polyvinyl chloride.

[0099] In one embodiment, polymers such as natural polymers like starch, chitin, glycosaminoglycans like hyaluronic acid, dermatan sulfate, and chondroitin sulfate, and microbial polyesters like hydroxyalkyl esters, such as copolymers of hydroxyvalerate and hydroxybutyrate, and synthetic polymers like poly(orthoesters) and polyanhydrides, and homopolymers and copolymers comprising glycolide and lactide (e.g., poly(L-lactide), poly(L-lactide-co-D,L-lactide), poly(L-lactide-co-glycolide), polyglycolide and poly(D,L-lactide), poly(D,L-lactide-co-glycolide), poly(lactic acid co-lysine), and polycaprolactone may be used.

[0100] In one embodiment, the biocompatible material is derived from isolated extracellular matrix (ECM). ECM can be isolated from the endothelial layer of various cell populations, tissues, and / or organs, such as any organ or tissue source, including the dermis of the skin, liver, digestive tract, respiratory tract, intestine, urinary tract, or reproductive tract of warm-blooded vertebrates. The ECM used in this invention can be derived from a combination of sources. Isolated ECM can be prepared in sheet, microparticle, gel, or other forms.

[0101] Biocompatible scaffold polymers may include filaments, elastin, chitin, chitosan, poly(d-hydroxy acid), poly(acid anhydride), or poly(orthoester). More specifically, biocompatible polymers can be formed from: polyethylene glycol, poly(lactic acid), poly(glycolic acid), copolymers of lactic acid and glycolic acid, copolymers of lactic acid and glycolic acid with polyethylene glycol, poly(E-caprolactone), poly(3-hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate, poly(orthoester), polyol / dienone acetal addition polymers, poly(sebacic anhydride) (PSA), poly(carboxylated dicarboxyphenoxyphenoxyhexanone) (PCPP), poly[bis(p-carboxyphenoxy)methane] (PCPM), copolymers of SA, CPP and CPM, poly(amino acids), poly(pseudoamino acids), polyphosphazenes, poly[(dichloro)phosphazenes] or poly[(organo)phosphazenes] derivatives, poly-hydroxybutyric acid or S-hexanoic acid, polylactide-co-glycolic acid, polylactic acid, polyethylene glycol, cellulose, oxidized cellulose, alginate, gelatin or derivatives thereof.

[0102] Therefore, the polymer can be formed from any of a variety of materials, including polymers, including naturally occurring polymers, synthetic polymers, or combinations thereof. In one embodiment, the scaffold comprises a biodegradable polymer. In one embodiment, the naturally occurring biodegradable polymer can be modified to provide a synthetic biodegradable polymer derived from the naturally occurring polymer. In one embodiment, the polymer is poly(lactic acid) (“PLA”) or poly(lactic-co-glycolic acid) (“PLGA”). In one embodiment, the scaffold polymer comprises, but is not limited to, alginate, chitosan, poly(2-hydroxyethyl methacrylate), xyloglucan, copolymer of 2-methacryloyloxyethyl phosphocholine, poly(vinyl alcohol), silicone, hydrophobic and hydrophilic polyesters, poly(lactide-co-glycolic acid), N-isopropylacrylamide copolymer, poly(ethylene oxide) / poly(propylene oxide), polylactic acid, poly(orthoester), polyanhydride, polyurethane, copolymer of 2-hydroxyethyl methacrylate and sodium methacrylate, phosphorylcholine, cyclodextrin, polysulfone and polyvinylpyrrolidone, starch, poly-D,L-lactic acid-p-dioxanone-polyethylene glycol block copolymer, polypropylene, poly(ethylene terephthalate), poly(tetrafluoroethylene), poly-ε-caprolactone, or cross-linked chitosan hydrogel.

[0103] Alternatively, the nucleic acid or vector may be administered at a dose of at least about 0.0001 mg / kg to about 1 mg / kg, at least about 0.001 mg / kg to about 0.5 mg / kg, at least about 0.01 mg / kg to about 0.25 mg / kg, or at least about 0.01 mg / kg to about 0.25 mg / kg body weight, although other doses may provide beneficial results.

[0104] Alternatively, the nucleic acid or vector may be administered at a dose of at least about 0.0001 mg / kg to about 1 mg / kg, at least about 0.001 mg / kg to about 0.5 mg / kg, at least about 0.01 mg / kg to about 0.25 mg / kg, or at least about 0.01 mg / kg to about 0.25 mg / kg body weight, although other doses may provide beneficial results.

[0105] Exemplary embodiments In one embodiment, a gene therapy vector is provided, the gene therapy vector comprising: a promoter operatively linked to a nucleic acid sequence including an open reading frame encoding APOE2 and a 3' untranslated region (3' UTR); and a nucleotide sequence having an RNAi sequence corresponding to APOE4 for repressing APOE4 mRNA. In one embodiment, the vector includes the nucleotide sequence. In one embodiment, the nucleotide sequence is the 5' or 3' of the open reading frame. In one embodiment, the nucleotide sequence is the 5' and 3' of the open reading frame. In one embodiment, the nucleotide sequence is located on a different vector. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is an AAV, adenovirus, lentivirus, herpesvirus, or retrovirus vector. In one embodiment, the AAV is AAV5, AAV9, or AAVrh10. In one embodiment, the APOE4 is human APOE4. In one embodiment, the APOE2 is human APOE2. In one embodiment, the nucleotide sequence is linked to a second promoter. In one embodiment, the second promoter is a PolIII promoter. In one embodiment, the RNAi comprises a miRNA containing a plurality of miRNA sequences. In one embodiment, the RNAi comprises siRNA containing multiple siRNA sequences. In one embodiment, the open reading frame comprises multiple silent nucleotide substitutions. In one embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the codons have silent nucleotide substitutions. In one embodiment, the open reading frame further comprises a peptide tag. In one embodiment, the tag comprises HA, histidine tag, AviTag, maltose-binding tag, Strep-tag, FLAG-tag, V5-tag, Myc-tag, Spot-tag, T7-tag, or NE-tag.

[0106] A host cell or mammal comprising the vector is also provided. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the mammal is a non-human primate. In one embodiment, the mammal is a human.

[0107] A further method for preventing, inhibiting, or treating Alzheimer's disease in mammals is provided, the method comprising administering to the mammal an effective amount of a composition including the gene therapy vector.

[0108] A method is provided for preventing, inhibiting, or treating diseases associated with APOE4 expression in mammals, the method comprising administering an effective amount of a composition including the gene therapy vector to the mammal.

[0109] In one embodiment, the composition comprises liposomes, the liposomes comprising the carrier. In one embodiment, the composition comprises nanoparticles, the nanoparticles comprising the nucleic acid. In one embodiment, the gene therapy vector comprises a viral vector. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the composition is administered systemically. In one embodiment, the composition is administered orally. In one embodiment, the composition is administered intravenously. In one embodiment, the composition is administered locally. In one embodiment, the composition is injected. In one embodiment, the composition is administered to the central nervous system. In one embodiment, the composition is administered to the brain. In one embodiment, the composition is a sustained-release composition. In one embodiment, the mammal is a human. In one embodiment, the RNAi sequence comprises multiple miRNA sequences, such as the same miRNA sequence.

[0110] The invention will be described by way of the following non-limiting examples.

[0111] Example 1 Alzheimer's disease (AD) affects 5 million Americans, and its prevalence is rapidly increasing. Existing drugs have little effect on the underlying disease process, and there are currently no available preventative therapies. Inheritance of the APOE4 allele represents a high risk of disease development, while inheritance of the APOE2 allele is protective, reducing the risk of developing AD by ≥50% and delaying the age of onset. Delivery of adeno-associated virus (AAV) of the human APOE2 gene into an AD mouse model expressing human APOE4 (homozygous expression) showed a reduction in amyloid-β peptide and amyloid burden. The odds ratio of developing AD was reduced in E2 / E4 heterozygotes compared to E4 / E4 homozygotes (2.6 vs. 14.9). Inhibition of APOE4, for example by delivery of an AAV vector, while simultaneously expressing human APOE2, can further reduce the risk of AD. In one embodiment, gene therapies, such as AAV therapies, are engineered to deliver the human APOE2 gene coding sequence and artificial RNA (such as microRNAs (miRNAs) targeting endogenous APOE4). The combination of knockdown of harmful endogenous APOE4 expression and expression of beneficial APOE2 alleles can provide enhanced protection against AD in individuals who are homozygous for the APOE4 allele.

[0112] In one embodiment, siRNA interacts with mRNA to silence translation. To express siRNA from a DNA sequence (such as a gene therapy expression vector), the target sequence must be embedded in a small hairpin RNA (shRNA) or miRNA scaffold. The artificial miRNA expressed via the vector is similar to endogenous RNAi and undergoes two processing steps. Because the miRNA is expressed at a low level, it is less likely to induce liver and CNS toxicity when delivered via a gene therapy vector.

[0113] In one embodiment, multiple miRNAs targeting different segments of APOE mRNA can be used to knock down all isotypes of miRNAs targeting endogenous APOE, thereby enhancing silencing. In one embodiment, vector-derived human APOE2 may contain a silencing mutation in its coding sequence to prevent silencing.

[0114] like Figure 3 As shown, miRNAs with RNAi sequences for inhibiting APOE4 expression can be inserted into 5' non-coding sequences (e.g., introns) and / or 3' non-coding sequences. Multiple miRNAs can be placed in tandem to enhance silencing, for example, APOE4. hAPOE2-HA and miRNA expression levels were found to be similar. Lower miRNA expression levels (compared to the U6 promoter) resulted in fewer off-target effects and lower toxicity potential.

[0115] In one embodiment (see Figure 4 Constitutive promoters (such as CAG) drive hAPOE2-HA, and the U6 promoter (exemplary Pol III promoter) drives the miRNA. In one embodiment, multiple miRNAs are placed in tandem to enhance APOE4 silencing, for example, two, three, four, or more miRNAs. In one embodiment, the Pol III promoter is used for transcription of rRNA, tRNA, and / or miRNA. In one embodiment, the vector may have a defined terminator, such as poly A is not required, because Pol III transcription terminates via an oligonucleotide (dT) extension in the non-template strand (dA in the template strand). In one embodiment, a dual-vector system may be used, wherein the second vector contains a stuffer sequence, for example for a reporter gene, to maintain length and track expression.

[0116] Therefore, this disclosure provides a vector, such as a viral vector (e.g., an AAV vector), that delivers both the human APOE2 gene and an artificial miRNA targeting human APOE4. These gene therapy vectors can be used to mitigate the risk of AD in APOE4 homozygous individuals (and E2 / E4 heterozygous individuals) by tilting the balance toward the expression of the beneficial APOE2 allele.

[0117] In one embodiment, the vector can be used for conditions or diseases that may benefit from increased APOE2 and / or decreased APOE4. In one embodiment, the vector is delivered to a mammal, such as a person at risk of developing AD. AD currently affects 5 million people in the United States and is projected to rise to 65 million worldwide by 2030. The global prevalence of the APOE4 allele is 15%, and approximately 50% of AD patients carry at least one APOE4 allele. Compared to gene therapies that deliver only APOE2, targeting the harmful APOE4 gene to reduce its expression provides protective APOE2 expression while further reducing the risks associated with APOE4.

[0118] Example 2 Figure 5 A system is illustrated in which the miRNA knocks down expression of all APOE isotypes, and the vector-derived APOE2 is resistant to the miRNA. For example, by using the CAG promoter, the expression levels of hApoE2-HA and the miRNA are similar, and lower levels of miRNA expression (compared to the U6 promoter) may mean less silencing, but also less off-target effects and toxicity. In one embodiment, the miRNA may be inserted into a CAG intron or the 3' untranslated region.

[0119] Figure 6 This study describes the testing of APOE knockdown efficiency in U87 cells using siRNA. Based on a comparison of various siRNA design algorithms, four different siRNAs targeting APOE coding sequences were generated. The siRNAs were transfected into U87 cells (an astrocytoma cell line), and APOE mRNA copies were quantified by RT-qPCR. The identified sequences are as follows: 1.GGUGGAGCAAGCGGUGGAGuu (SEQ ID NO: 1) 2.GGAGUUGAAGGCCUACAAAuu (SEQ ID NO: 2) 3.GGAAGACAUGCAGCGCCAGuu (SEQ ID NO:3) 4.GCGCCGGAUGGAGGAGAUuu (SEQ ID NO: 4) The non-targeting siRNA is GTAGCGACTAAACACATCAuu (SEQ ID NO:5). Other sequences of siRNAs include: GCCGATGACCTGCAGAAGCuu (SEQ ID NO:20) GCGCGCGGATGGAGGAGATuu (SEQ ID NO:21) GTAAGCGGCTCCTCCGCGAuu (SEQ ID NO:22) The sequence from a siRNA (#2 above) was converted into a miRNA. A modified version of the scaffold based on mir155 was used (Fowler et al., Nucleic Acid Research, 2015, 44:e48, the contents of which are incorporated herein by reference). However, any miRNA backbone, such as mir21, mir30, or mir33, can be used.

[0120] For example, for miRs from siRNA#2, the following can be used: CTGGAGGCTTGCTGAAGGCTGTATGCTGATTTGTAGGCCTTCAACTCCT GTTTTGGCCACTGACTGACAGGAGTGAGGCCTACAAATCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 23); or CTGGAGGCTTGCTTTGGGCTGTATGCTGTTCCGATTTGTAGGCCTTCAAGTTTTGGCCACTGACTGACTTGAAGTCACAAATCGGAACAGGACACAAGGCCCTTTATCAGCACTCACATGGAACAAATGGCCACCGTGGGAGGATGACAA (SEQ ID NO: 25) In one embodiment, the miRNA has one or more of the following: U or A at guide position 1 relative to the 5' microprocessor cleavage site, U or A at positions 2-7, 10-14, and 17, and G or C at positions 19-21, and / or a G / C content of 36.4% to 45.5%, and / or a guide strand 2 nucleotides longer than the lagging strand and / or mismatches, wherein the mismatches are 1) loop mismatches, wherein 3 to 5 adjacent nucleotides of the guide strand are not paired with bases of the target strand, 2) 3 bp spacer mismatches, wherein 2 single guide strand nucleotide mismatches are separated by 3 guide / lagging base pairs, and / or 3) 4 bp spacer mismatches, wherein 2 single guide strand nucleotide mismatches are separated by 4 guide / lagging base pairs. Mismatches in the lagging strand are selected to obtain optimal GC content and position. Mfold is used to predict the hairpin secondary structure of the miRNA. Two tandem copies of the miRNA are cloned into the 3' untranslated region of the CAG intron or pAAV expression cassette. Within the AAV size limit, up to four copies of miRNA (of the stated length) can be inserted.

[0121] The vector-derived APOE2 was modified to be resistant to silencing of the aforementioned target miRNAs (see the underlined sequence below). The nucleotide sequence of the miRNA target region (red / bold) underwent silencing changes.

[0122] APOE2 from the vector: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCT GGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAA GGAGTTGAAGGCCTACAAATCGGAACTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:6) Modified APOE2: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTCAAAGCTTATAAGAGCGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC(SEQ ID NO:7) The above sequence silently alters all possible nucleotides, taking into account the codon usage of the three miRNAs derived from siRNA#2 within the composition recognition site. However, other examples are shown below: Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTTAAAGCATATAAGAGTGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:26); Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTCAAAGCATATAAGAGTGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:27); Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTTAAAGCTTATAAGAGTGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:28); Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTTAAAGCATATAAGAGCGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:29); Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTCAAAGCTTATAAGAGTGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:30) Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTCAAAGCATATAAGAGCGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC (SEQ ID NO:31); or Modified APOE: ATGAAGGTTCTGTGGGCTGCGTTGCTGGTCACATTCCTGGCAGGATGCCAGGCCAAGGTGGAGCAAGCGGTGGAGACAGAGCCGGAGCCCGAGCTGCGCCAGCAGACCGAGTGGCAGAGCGGCCAGCGCTGGGAACTGGCACTGGGTCGCTTTTGGGATTACCTGCGCTGGGTGCAGACACTGTCTGAGCAGGTGCAGGAGGAGCTGCTCAGCTCCCAGGTCACCCAGGAACTGAGGGCGCTGATGGACGAGACCATGAAAGAACTTAAAGCTTATAAGAGCGAGCTGGAGGAACAACTGACCCCGGTGGCGGAGGAGACGCGGGCACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGTGTGCGGCCGCCTGGTGCAGTACCGCGGCGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGTGGGCTCCCTGGCCGGCCAGCCGCTACAGGAGCGGGCCCAGGCCTGGGGCGAGCGGCTGCGCGCGCGGATGGAGGAGATGGGCAGCCGGACCCGCGACCGCCTGGACGAGGTGAAGGAGCAGGTGGCGGAGGTGCGCGCCAAGCTGGAGGAGCAGGCCCAGCAGATACGCCTGCAGGCCGAGGCCTTCCAGGCCCGCCTCAAGAGCTGGTTCGAGCCCCTGGTGGAAGACATGCAGCGCCAGTGGGCCGGGCTGGTGGAGAAGGTGCAGGCTGCCGTGGGCACCAGCGCCGCCCCTGTGCCCAGCGACAATCAC(SEQ ID NO:32)。

[0123] The vector can be tested in non-human animals, such as mice. In one embodiment, the vector comprises a sequence from the AAV9-CAG-APOE2 vector (AAV9-APOE2), i.e., an adeno-associated virus vector serotype 9 expressing APOE2 after the chicken β-actin promoter, or a sequence from the AAVrh.10-CAG-APOE2 vector (AAVrh.10-APOE2), i.e., a rhesus monkey adeno-associated virus vector serotype 10 expressing APOE2 transgene after the chicken β-actin promoter.

[0124] The AAVrh.10 and AAV9 vectors can be generated and purified as previously described (Sondhi et al., 2007, 2012; Zolotukhin et al., 2002). Briefly, the vectors are generated by co-transfecting HEK293T cells with expression cassette plasmids and adenovirus helper plasmids. The packaging cell line HEK293T was maintained in Dulbecco's modified Eagles medium supplemented with 5% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin at 37°C and 5% CO2. Cells were plated in CellSTACKS (Corning, Tewksbury, MA) at 30%–40% confluence for 24 hours (or at 70%–80% confluence) and then transfected with plasmids using the PEIpro program. Cells were incubated at 37°C for 3 days, followed by harvesting and lysis after 5 freeze-thaw cycles. Cell lysates were treated with 50 U / mL Benzonase at 37°C for 30 min. For the AAVrh.10 vector, cell lysates were purified by iodixanol density gradient followed by Q-HP ion exchange chromatography. For the AAV9 vector, cell lysates were precipitated overnight in PEG (final PEG concentration: 8%). After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 15 mL of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.5). Samples were purified by centrifugation at 24,000 rpm (182,000 g) at 20°C for 24 h using an SW28 rotor with 1.37 g / mL CsCl added to 38.5 mL isomorphous polymer tubes. 1 mL fractions were collected by inserting a 21-gauge needle (Hamilton, Reno, NV) into the bottom of the centrifuge tube. The fraction containing the vector construct sequence was then used. 32P-labeled probes were used to determine the fractions containing the vector via dot blot analysis. Positive fractions were then pooled and diluted with 1.37 g / mL CsCl, and the samples were loaded into 13.5 mL Quick-Seal tubes and centrifuged at 67,000 rpm (384,000 g) for 16–20 h at 20 °C in a 90 Ti ultracentrifuge (Beckman LE-80K; Beckman Coulter, Fullerton, CA). Fractions (0.5 mL) were collected, and positive fractions were pooled. The purified AAVrh.10 or AAV9 vector was concentrated in phosphate-buffered saline (PBS). The vector genomic titer was determined by Taq-Man quantitative polymerase chain reaction. The purified vector was sterile filtered; tested for growth for 14 days on media supporting aerobic, anaerobic, or fungal growth; tested for endotoxins; and confirmed to be free of mycoplasma. The AAV formulation (2 mL, 1.0 × 10⁻⁶) can be injected at a rate of 0.2 mL / min using, for example, a 33-gauge needle (Hamilton, Inc.) and an infusion pump (KD Scientific, Holliston, MA). 10 (vg or another dose).

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[0183] All publications, patents, and patent applications are incorporated herein by reference. Although the invention has been described in conjunction with certain embodiments thereof in the foregoing specification, and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the invention is susceptible to other embodiments, and that certain details described herein may be significantly varied without departing from the basic principles of the invention.

Claims

1. A gene therapy vector comprising a first promoter operatively linked to a nucleic acid sequence encoding an open reading frame and a 3' untranslated region of APOE2, and a separated nucleotide sequence comprising one or more RNAi nucleic acid sequences for repressing APOE4 mRNA.

2. The vector according to claim 1, wherein the vector comprises the nucleotide sequence.

3. The vector according to claim 2, wherein the nucleotide sequence is inserted into the 5' or 3' of the open reading frame.

4. The vector according to claim 2, wherein the nucleotide sequence is inserted into the 5' and 3' of the open reading frame.

5. The vector according to claim 1, wherein the nucleotide sequence is located on a different vector.

6. The vector according to any one of claims 1 to 5, wherein the isolated nucleotide sequence comprises a second promoter operatively linked to the one or more RNAi nucleic acid sequences.

7. The vector according to any one of claims 1 to 6, wherein the gene therapy vector is a viral vector.

8. The vector according to claim 5, wherein the different vector is a viral vector.

9. The vector according to claim 7 or 8, wherein the viral vector is an AAV, adenovirus, lentivirus, herpesvirus, or retrovirus vector.

10. The carrier according to claim 9, wherein the AAV is AAV5, AAV9, or AAVrh10.

11. The carrier according to any one of claims 1 to 10, wherein the APOE4 is human APOE4.

12. The carrier according to any one of claims 1 to 10, wherein the APOE2 is human APOE2.

13. The vector according to any one of claims 1 to 13, wherein the first promoter is the PoL1 promoter.

14. The vector according to claim 6, wherein the second promoter is the PolIII promoter.

15. The vector according to any one of claims 1 to 14, wherein the isolated nucleotide sequence comprises nucleic acids of one or more miRNAs, wherein the miRNAs comprise two or more of the RNAi nucleic acid sequences.

16. The vector according to any one of claims 1 to 14, wherein the RNAi comprises siRNA containing a plurality of siRNA sequences.

17. The vector according to any one of claims 1 to 16, wherein the open reading frame of APOE2 comprises a plurality of silent nucleotide substitutions relative to SEQ ID NO:

6.

18. The vector of claim 17, wherein the plurality of silent nucleotides in the APOE2 open reading frame replace the RNAi nucleic acid sequence not present in the separated nucleotide sequence.

19. The vector according to claim 16, 17 or 18, wherein at least 50%, 60%, 70%, 80% or 90% of the codons in the open reading frame have silent nucleotide substitutions.

20. The vector according to claim 16, 17 or 18, wherein at least 5%, 10%, 20%, 30% or 40% of the codons in the open reading frame have silent nucleotide substitutions.

21. The vector according to any one of claims 1 to 20, wherein the inhibited APOE4 has a sequence having at least 80%, 85%, 90%, 95% or more amino acid sequence identity with the polypeptide comprising SEQ ID NO:

10.

22. The vector according to any one of claims 1 to 21, wherein the APOE2 has a sequence having at least 80%, 85%, 90%, 95% or more amino acid sequence identity with the polypeptide encoded by SEQ ID NO:

11.

23. The vector according to any one of claims 1 to 22, wherein the one or more RNAi nucleic acid sequences have at least 60%, 70%, 80%, 90% or more nucleotide sequence identity with one of SEQ ID No. 1-4 or its complement.

24. The vector of claim 1, comprising a first promoter operatively linked to a nucleic acid sequence encoding an open reading frame of human APOE2 and a separated nucleotide sequence having one or more RNAi nucleic acid sequences for repressing human APOE4 mRNA.

25. The vector of claim 24, wherein the nucleotide sequence is inserted at the 5' of the open reading frame.

26. The vector of claim 24, wherein the nucleotide sequence is inserted at the 3' of the open reading frame.

27. The vector of claim 24, wherein the nucleotide sequence is inserted into the 5' and 3' of the open reading frame.

28. The vector according to any one of claims 24 to 27, wherein the isolated nucleotide sequence comprises a second promoter operatively linked to the one or more RNAi nucleic acid sequences.

29. A composition comprising a gene therapy vector according to any one of claims 1 to 28 and optionally a pharmaceutically acceptable vector.

30. A method for preventing, inhibiting, or treating Alzheimer's disease in mammals, the method comprising: The effective amount of the composition comprising the gene therapy vector according to any one of claims 1 to 28 or the composition according to claim 29 is administered to the mammal.

31. A method for preventing, inhibiting, or treating diseases associated with APOE4 expression in mammals, the method comprising: The effective amount of the composition comprising the gene therapy vector according to any one of claims 1 to 28 or the composition according to claim 29 is administered to the mammal.

32. The method of claim 30 or 31, wherein the composition comprises liposomes, the liposomes comprising the gene therapy vector or the different vectors or both.

33. The method of claim 30 or 31, wherein the composition comprises nanoparticles, the nanoparticles comprising the gene therapy carrier or the different carriers or both.

34. The method of claim 30 or 31, wherein the gene therapy vector or the different vectors or both comprise a viral vector.

35. The method according to any one of claims 30 to 34, wherein the mammal is an E2 / E4 heterozygote.

36. The method according to any one of claims 30 to 34, wherein the mammal is an E4 / E4 homozygote.

37. The method according to any one of claims 30 to 36, wherein the composition is administered systemically.

38. The method according to any one of claims 30 to 37, wherein the composition is administered orally.

39. The method according to any one of claims 30 to 37, wherein the composition is administered intravenously.

40. The method according to any one of claims 30 to 37, wherein the composition is applied topically.

41. The method according to any one of claims 30 to 37, wherein the composition is injected.

42. The method according to any one of claims 30 to 37, wherein the composition is applied to the central nervous system.

43. The method according to any one of claims 30 to 37, wherein the composition is applied to the brain.

44. The method according to any one of claims 30 to 43, wherein the composition is a sustained-release composition.

45. The method according to any one of claims 30 to 44, wherein the mammal is a human.

46. ​​The method according to any one of claims 30 to 45, wherein the RNAi sequence comprises a plurality of miRNA sequences, each of the plurality of miRNA sequences comprising one or more RNAi nucleic acid sequences for inhibiting APOE4 mRNA.

47. The method of claim 46, wherein one of the miRNA sequences in the vector is inserted into the 5' of the open reading frame and the other is inserted into the 3' of the open reading frame.

48. The method according to any one of claims 30 to 45, wherein the RNAi sequence comprises a miRNA sequence, the miRNA sequence comprising one or more RNAi nucleic acid sequences for repressing APOE4 mRNA.

49. The method of claim 48, wherein the miRNA sequence in the vector is inserted into the 5' of the open reading frame.

50. The method of claim 48, wherein the miRNA sequence in the vector is inserted into the 3' of the open reading frame.

51. The method according to any one of claims 30 to 45, wherein the vector comprises a PolIII promoter operatively linked to the RNAi sequence.

52. The method according to any one of claims 30 to 45, wherein the second vector comprises a PolIII promoter operatively linked to the RNAi sequence.

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