Gene therapy methods for Alzheimer's disease

Abstract

Kind Code: A1 Compositions and methods are provided for preventing, inhibiting, or treating in a mammal a disease or disorder associated with expression of APOE4, an APOE allele that encodes apolipoprotein E (APOE). [Solution] A gene therapy vector is provided, comprising a first promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding APOE2 and a 3' untranslated region, and an isolated nucleotide sequence comprising one or more RNAi nucleic acid sequences for inhibiting APOE4 mRNA, as well as a composition comprising the vector and, optionally, a pharmaceutically acceptable carrier.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Patent Application No. 62 / 915,988, filed October 16, 2019, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] background Apolipoprotein E (APOE) is a key central nervous system (CNS) apolipoprotein closely involved in 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, there are three common APOE alleles (ε4, ε3, and ε2) that encode three APOE isoforms expressed primarily in the liver and brain. APOE4 carriers have a significantly increased risk of developing AD (3-15 times higher for heterozygotes and homozygotes, respectively, compared with APOE3 homozygotes) and an earlier age of onset of disease (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 by far the most common genetic risk factor for late-onset AD, the most common form of AD. In contrast, APOE2 is a protective allele that reduces AD risk by approximately 50% and significantly delays age at onset (Corder et al., 1994; Farrer et al., 1997; Suri et al., 2013; Talbot et al., 1994; Yu et al., 2014).

[0003] The major physiological difference between the most common isoform, APOE3, and APOE2 and APOE4, results from an amino acid difference at one of two positions, residues 112 (APOE4) and 158 (APOE2), which is a cysteine-arginine interchange (Hatters et al., 2006). These two amino acid differences result in differences in protein structure and the corresponding binding affinities of these APOE isoforms to lipoproteins and lipoprotein receptors, as well as in their regulation of Aβ aggregation, degradation, excretion, 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

[0004] overview In one embodiment, the present disclosure provides a gene therapy vector for Alzheimer's disease. In one embodiment, the gene therapy vector includes an AAV expression vector encoding the human APOE2 gene and an artificial microRNA targeting endogenous APOE4 in either cis or trans. This vector system silences harmful endogenous APOE4 expression in combination with supplementation of the beneficial APOE2 gene from a gene therapy vector, e.g., an AAV vector. As disclosed herein, exemplary artificial microRNA sequences have been designed that target endogenous APOE4 mRNA for suppression. The microRNA (miRNA) can be incorporated into a sequence 5' to the APOE2 coding sequence, e.g., an intron such as the CAG promoter intron, or into a sequence 3' to the APOE2 coding sequence, e.g., a sequence 5' to the polyA tail of a vector transgene plasmid encoding the human APOE2 coding sequence. Alternatively, the microRNA can be inserted between the Pol III promoter, e.g., a U6 promoter, following the polyA site of the APOE2 expression cassette and the terminator. The human APOE2 DNA sequence from the vector optionally includes silent nucleotide changes to reduce or inhibit microRNA-mediated repression, and in one embodiment may include a tag, such as an HA tag, for detection, e.g., for preclinical detection studies. In one embodiment, the expression construct is packaged in an AAV capsid of a serotype (e.g., AAV9) that targets astrocytes and glial cells, the predominant sites of endogenous APOE expression in the CNS, but may be provided in other vectors, e.g., other viral vectors, plasmids, nanoparticles, or liposomes.

[0005] In one embodiment, a gene therapy vector is provided, comprising a first promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding APOE2 and a 3' untranslated region, and an isolated nucleotide sequence is provided comprising one or more RNAi nucleic acid sequences for inhibiting APOE4 mRNA. In one embodiment, the vector comprises the nucleotide sequence. In one embodiment, the nucleotide sequence is inserted 5' or 3' relative to the open reading frame. In one embodiment, the nucleotide sequence is inserted 5' and 3' relative to the open reading frame. In one embodiment, the nucleotide sequence is on a different vector. In one embodiment, the isolated nucleotide sequence comprises a second promoter operably linked to 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 AAVrhlO. In one embodiment, the APOE4 is human APOE4. In one embodiment, the APOE2 is human APOE2. In one embodiment, the first promoter is a Pol I promoter, e.g., a constitutive promoter, or a regulatable promoter, e.g., an inducible promoter. In one embodiment, the second promoter is a Pol III promoter. In one embodiment, the isolated nucleotide sequence comprises nucleic acids for one or more miRNAs comprising two or more RNAi nucleic acid sequences, e.g., one or more RNAi sequences embedded in an miRNA sequence. In one embodiment, the RNAi comprises an siRNA comprising multiple siRNA sequences. In one embodiment, the RNAi comprises an shRNA sequence approximately 15-25 nucleotides in length.In one embodiment, the open reading frame for APOE2 contains multiple silent nucleotide substitutions relative to SEQ ID NO:6, for example, the open reading frame comprises SEQ ID NO:7, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 98% nucleic acid sequence identity to SEQ ID NO:7, and encodes APOE2, or the open reading frame encodes APOE2. The nucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 98% nucleic acid sequence identity to TIFF2026041903000001.tif4128, but the sequence is not SEQ ID NO: 7. In one embodiment, the multiple silent nucleotide substitutions in the open reading frame of APOE2 are not within the RNAi nucleic acid sequence in the isolated nucleotide sequence, i.e., the sequence with nucleotide substitutions is different from the RNAi nucleotide sequence, so that the mRNA with nucleotide substitutions does not bind to (e.g., duplex with) the RNAi sequence, for example, the isolated RNAi or the RNAi sequence expressed from a vector. In one embodiment, at least 50%, 60%, 70%, 80%, or 90% of the codons in the open reading frame for APOE2 have silent nucleotide substitutions. In one embodiment, at least 5%, 10%, 20%, 30% or 40% of the codons in the open reading frame for APOE2 have silent nucleotide substitutions, for example, in the portion of the APOE2 sequence corresponding to the RNAi sequence. That is, the silent nucleotide substitutions in the human APOE2 coding sequence result in a sequence that is different from the endogenous human APOE4 sequence and different from the APOE4 RNAi sequence. In one embodiment, the APOE4 to be inhibited has a sequence that has 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 that has at least 80%, 85%, 90%, 95% or more amino acid sequence identity with the polypeptide encoded by SEQ ID NO: 9. In one embodiment, one or more RNAi nucleic acid sequences have at least 60%, 70%, 80%, 90%, or more nucleotide sequence identity to one of SEQ ID NOs: 1-4 or 20-22 or their complements.In one embodiment, the vector comprises a first Pol I promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding human APOE2 and an isolated nucleotide sequence comprising one or more RNAi nucleic acid sequences for inhibiting human APOE4 mRNA. In one embodiment, the nucleotide sequence is inserted 5' to the open reading frame. In one embodiment, the nucleotide sequence is inserted 3' to the open reading frame. In one embodiment, the nucleotide sequence is inserted 5' and 3' to the open reading frame. In one embodiment, the isolated nucleotide sequence comprises a second promoter operably linked to one or more RNAi nucleic acid sequences. In one embodiment, the RNAi nucleic acid sequence is about 125-500, e.g., about 150-175, nucleotides in length. In one embodiment, the gene therapy vector may comprise two, three, four, or more copies of the RNAi nucleic acid sequence, which may include an miRNA sequence, e.g., an miRNA sequence flanking an APOE4 inhibitory sequence.

[0006] In one embodiment, a method is provided for preventing, inhibiting, or treating Alzheimer's disease in a mammal, comprising administering to the mammal an effective amount of a composition comprising a gene therapy vector. In one embodiment, the composition comprises nanoparticles comprising the gene therapy vector, a different vector, or both. In one embodiment, the gene therapy vector, a different vector, 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 topically. 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 human. In one embodiment, the RNAi nucleic acid sequence comprises multiple miRNA sequences.

[0007] In one embodiment, a method is provided for preventing, inhibiting, or treating a disease associated with APOE4 expression in a mammal, comprising administering to the mammal an effective amount of a composition comprising a gene therapy vector. In one embodiment, the composition comprises a liposome comprising the gene therapy vector or a different vector, or both. In one embodiment, the composition comprises a nanoparticle comprising the gene therapy vector or a different vector, or both. In one embodiment, the gene therapy vector or the different vector, or both, comprises a viral vector. In one embodiment, the mammal is E2 / E4 heterozygote. In one embodiment, the mammal is 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 human. In one embodiment, the RNAi sequence comprises multiple miRNA sequences. [The present invention 1001] a first promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding APOE2 and a 3' untranslated region; an isolated nucleotide sequence comprising one or more RNAi nucleic acid sequences for the inhibition of APOE4 mRNA; A gene therapy vector comprising: [The present invention 1002] 1001. A vector of the present invention, comprising the nucleotide sequence. [The present invention 1003] 1002. The vector of claim 10, wherein said nucleotide sequence is inserted 5' or 3' to said open reading frame. [The present invention 1004] 1002. The vector of claim 10, wherein said nucleotide sequences are inserted 5' and 3' to said open reading frame. [The present invention 1005] 1001. The vector of claim 10, wherein said nucleotide sequences are on different vectors. [The present invention 1006] The vector of any one of claims 1001 to 1005, wherein said isolated nucleotide sequence comprises a second promoter operably linked to said one or more RNAi nucleic acid sequences. [The present invention 1007] The vector of any one of claims 1001 to 1006, wherein the gene therapy vector is a viral vector. [The present invention 1008] 1005. The vector of the present invention, wherein the different vector is a viral vector. [The present invention 1009] The vector of claim 1007 or 1008, wherein the viral vector is an AAV, adenovirus, lentivirus, herpesvirus, or retrovirus vector. [The present invention 1010] The vector of the present invention 1009, wherein the AAV is AAV5, AAV9, or AAVrhlO. [The present invention 1011] The vector of any one of claims 1001 to 1010, wherein the APOE4 is human APOE4. [The present invention 1012] The vector of any one of claims 1001 to 1010, wherein the APOE2 is human APOE2. [The present invention 1013] The vector of any one of claims 1001 to 1013, wherein the first promoter is a PolI promoter. [The present invention 1014] 1006. The vector of the present invention, wherein the second promoter is a PolIII promoter. [The present invention 1015] The vector of any one of claims 1001 to 1014, wherein the isolated nucleotide sequence comprises a nucleic acid for one or more miRNAs comprising two or more of the RNAi nucleic acid sequences. [The present invention 1016] The vector of any one of claims 1001 to 1014, wherein the RNAi comprises an siRNA comprising a plurality of siRNA sequences. [The present invention 1017] The vector of any of claims 1001 to 1016, wherein the open reading frame for APOE2 comprises multiple silent nucleotide substitutions relative to SEQ ID NO:6. [The present invention 1018] The vector of the present invention 1017, wherein the multiple silent nucleotide substitutions in the APOE2 open reading frame are not in the RNAi nucleic acid sequence in the isolated nucleotide sequence. [The present invention 1019] The vector of claim 1016, 1017, or 1018, wherein at least 50%, 60%, 70%, 80%, or 90% of the codons in said open reading frame have silent nucleotide substitutions. [The present invention 1020] The vector of claim 1016, 1017, or 1018, wherein at least 5%, 10%, 20%, 30%, or 40% of the codons in said open reading frame have silent nucleotide substitutions. [The present invention 1021] Any of the vectors of the present inventions 1001 to 1020, wherein the APOE4 to be inhibited has a sequence having at least 80%, 85%, 90%, 95%, or more amino acid sequence identity to a polypeptide comprising SEQ ID NO: 10. [The present invention 1022] Any of the vectors of present inventions 1001 to 1021, wherein the APOE2 has a sequence having at least 80%, 85%, 90%, 95%, or more amino acid sequence identity to the polypeptide encoded by SEQ ID NO: 11. [The present invention 1023] The vector of any of claims 1001 to 1022, wherein the one or more RNAi nucleic acid sequences have at least 60%, 70%, 80%, 90%, or more nucleotide sequence identity to one of SEQ ID NOs: 1 to 4 or their complements. [The present invention 1024] a first promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding human APOE2; An isolated nucleotide sequence having one or more RNAi nucleic acid sequences for the inhibition of human APOE4 mRNA. 1001. A vector of the present invention comprising: [The present invention 1025] 1024. The vector of claim 10, wherein said nucleotide sequence is inserted 5' to said open reading frame. [The present invention 1026] 1024. The vector of the present invention, wherein the nucleotide sequence is inserted 3' to the open reading frame. [The present invention 1027] 1024. The vector of the present invention, wherein the nucleotide sequences are inserted 5' and 3' to the open reading frame. [The present invention 1028] The vector of any one of claims 1024 to 1027, wherein the isolated nucleotide sequence comprises a second promoter operably linked to the one or more RNAi nucleic acid sequences. [The present invention 1029] A gene therapy vector according to any one of claims 1001 to 1028 of the present invention; Optionally, a pharmaceutically acceptable carrier A composition comprising: [The present invention 1030] 1. A method for preventing, inhibiting, or treating Alzheimer's disease in a mammal, comprising: administering to the mammal an effective amount of a composition comprising any one of the gene therapy vectors of the present inventions 1001 to 1028 or the composition of the present invention 1029. A method comprising: [The present invention 1031] 1. A method for preventing, inhibiting, or treating a disease associated with APOE4 expression in a mammal, comprising: administering to the mammal an effective amount of a composition comprising any one of the gene therapy vectors of the present inventions 1001 to 1028 or the composition of the present invention 1029. A method comprising: [The present invention 1032] 1032. The method of any one of claims 1030 to 1031, wherein said composition comprises a liposome comprising said gene therapy vector or said different vector, or both. [The present invention 1033] 1032. The method of any one of claims 1030 to 1031, wherein said composition comprises nanoparticles comprising said gene therapy vector or said different vector, or both. [The present invention 1034] 1032. The method of any one of claims 1030 to 1031, wherein said gene therapy vector or said different vector, or both, comprises a viral vector. [This invention 1035] The method according to any one of claims 1030 to 1034, wherein the mammal is an E2 / E4 heterozygote. [The present invention 1036] The method of any one of claims 1030 to 1034, wherein the mammal is an E4 / E4 homozygote. [This invention 1037] 1037. The method of any one of claims 1030 to 1036, wherein the composition is administered systemically. [The present invention 1038] 8. The method of any one of claims 1030 to 1037, wherein the composition is administered orally. [This invention 1039] 8. The method of any one of claims 1030 to 1037, wherein the composition is administered intravenously. [The present invention 1040] 8. The method of any one of claims 1030 to 1037, wherein the composition is administered topically. [This invention 1041] 8. The method of any one of claims 1030 to 1037, wherein the composition is injected. [The present invention 1042] The method of any one of claims 1030 to 1037, wherein the composition is administered to the central nervous system. [This invention 1043] The method of any one of claims 1030 to 1037, wherein the composition is administered to the brain. [This invention 1044] The method of any one of claims 1030 to 1043, wherein the composition is a sustained-release composition. [This invention 1045] The method of any one of claims 1030 to 1044, wherein the mammal is a human. [The present invention 1046] 1046. The method of any of claims 1030 to 1045, wherein said RNAi sequence comprises a plurality of miRNA sequences, each comprising said one or more RNAi nucleic acid sequences for inhibition of APOE4 mRNA. [This invention 1047] 1046. The method of claim 1046, wherein one of the miRNA sequences in the vector is inserted 5' to the open reading frame and another miRNA sequence is inserted 3' to the open reading frame. [This invention 1048] 1046. The method of any of claims 1030 to 1045, wherein said RNAi sequence comprises a miRNA sequence comprising said one or more RNAi nucleic acid sequences for inhibition of APOE4 mRNA. [This invention 1049] 1048. The method of claim 1048, wherein the miRNA sequence in the vector is inserted 5' to the open reading frame. [The present invention 1050] 1048. The method of claim 1048, wherein the miRNA sequence in the vector is inserted 3' to the open reading frame. [This invention 1051] 1046. The method of any of claims 1030 to 1045, wherein said vector comprises a PolIII promoter operably linked to said RNAi sequence. [This invention 1052] 1046. The method of any of claims 1030 to 1045, wherein the second vector comprises a PolIII promoter operably linked to said RNAi sequence. [Brief explanation of the drawings]

[0008] [Figure 1] Production of inhibitory RNA from an exemplary target transcript template (Boudreau and Davidson. 2012. Methods in Enzymology, Volume 507). [Figure 2] pathway to inhibit mRNA (Borel et al., 2014. Mol Ther 22:692-701). [Figure 3] Exemplary constructs for miRNA insertion. [Figure 4] Single-vector and double-vector constructs. [Figure 5] A single vector construct with two sites for miRNA sequences. [Figure 6] Knockdown of APOE expression by four different siRNAs in vitro. [Figure 7] Use of the mir155 scaffold as an exemplary scaffold for miRNA expression. [Figure 8] Mouse experiments. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description definition A "vector" refers to a macromolecule or association of macromolecules that contains or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotide to be delivered, sometimes referred to as a "target polynucleotide" or "transgene," may contain a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest in vaccine development (such as a polynucleotide that expresses a protein, polypeptide, or peptide suitable for eliciting an immune response in a mammal), and / or a selectable or detectable marker.

[0010] As used herein, "transduction," "transfection," "transformation," or "transducing" refers to a process for introducing an exogenous polynucleotide into a host cell, resulting in expression of the polynucleotide, e.g., a transgene, in the cell, including the use of recombinant viruses to introduce the exogenous polynucleotide into the host cell. Transduction, transfection, or transformation of a polynucleotide in a cell can be determined by methods well known in the art, including, but not limited to, measuring protein expression (including steady-state levels) by ELISA, flow cytometry, and Western blot, DNA and RNA by hybridization assays, e.g., Northern blot, Southern blot, and gel shift mobility assay. Methods used for introducing an exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation, and electroporation, as well as other non-viral gene delivery techniques. The introduced polynucleotide can be maintained stably or transiently in the host cell.

[0011] "Gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer and can encompass targeting, binding, uptake, transport, localization, replicon integration, and expression.

[0012] "Gene transfer" refers to the introduction of an exogenous polynucleotide into a cell, which may include targeting, binding, uptake, transport, localization, and replicon integration, but is distinct from or does not imply subsequent expression of a gene.

[0013] "Gene expression" or "expression" refers to the processes of transcription, translation, and post-translational modification of a gene.

[0014] An "infectious" virus or viral particle is one that contains a polynucleotide component that it can deliver into a cell for which the viral species is trophic. This term does not necessarily imply any replicative capacity of the virus.

[0015] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may contain modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0016] An "isolated" polynucleotide, such as a plasmid, virus, polypeptide, or other substance, refers to a preparation of the substance that lacks at least some of the other components that may also be present in the location in which the substance or similar substance naturally occurs or from which it was originally prepared. Thus, for example, an isolated substance can be prepared by concentrating it from a source mixture using purification techniques. An isolated nucleic acid, peptide, or polypeptide exists in a form or context that is different from that in which it is found in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome in close proximity to neighboring genes, and an RNA sequence, such as a particular mRNA sequence encoding a particular protein, is found in the cell as a mixture with many other mRNAs that encode many other proteins. Isolated nucleic acid molecules can exist in single-stranded or double-stranded form. When an isolated nucleic acid molecule is used to express a protein, the molecule contains at least the sense or coding strand (i.e., the molecule can be single-stranded), but can contain both the sense and antisense strands (i.e., the molecule can be double-stranded). Concentration can be measured in absolute terms, such as weight per volume of solution, or relative to a second potential interfering substance present in the source mixture. Incremental concentrations of embodiments of the present invention are contemplated. Thus, for example, 2-fold concentration, 10-fold concentration, 100-fold concentration, or 1000-fold concentration.

[0017] "Transcriptional regulatory sequence" refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences for use in the present invention generally include at least one transcriptional promoter and may also include one or more transcriptional enhancers and / or terminators.

[0018] "Operably linked" refers to an arrangement of two or more components wherein the components so described are in a relationship permitting them to function cooperatively. Illustratively, a transcriptional regulatory sequence or promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence. An operably linked TRS is generally linked in cis with the coding sequence, but is not necessarily directly adjacent to it.

[0019] "Heterologous" means derived from a genotypically different entity from the entity to which it is being compared. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and, when expressed, may encode a heterologous polypeptide). Similarly, a transcriptional regulatory element, such as a promoter, that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.

[0020] "Terminator" refers to a polynucleotide sequence that tends to reduce or prevent read-through transcription (i.e., reduces or prevents transcription originating on one side of the terminator from continuing to the other side of the terminator). The degree to which transcription is interrupted typically depends on the base sequence and / or the length of the terminator sequence. In particular, as is well known in many molecular biological systems, certain DNA sequences commonly referred to as "transcription termination sequences" are specific sequences that tend to interrupt read-through transcription by RNA polymerase, presumably by terminating the RNA polymerase molecule and / or disengaging it from the DNA being transcribed. Typical examples of such sequence-specific terminators include polyadenylation ("polyA") sequences, such as SV40 polyA. In addition to or instead of such sequence-specific terminators, the insertion of a relatively long DNA sequence between the promoter and the coding region also generally tends to interrupt transcription of the coding region in proportion to the length of the intervening sequence. This effect probably arises because there is always some tendency for RNA polymerase molecules to break away from the DNA being transcribed, and increasing the length of the sequence traversed before reaching the coding region generally increases the likelihood that breakaway will occur before transcription of the coding region is completed, or perhaps even initiated. Thus, terminators can prevent transcription from only one direction ("unidirectional" terminators) or from both directions ("bidirectional" terminators), and can be composed of sequence-specific termination sequences or sequence-nonspecific terminators, or both. A variety of such terminator sequences are known in the art, and exemplary uses of such sequences within the context of the present invention are provided below.

[0021] "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 are useful in the present invention, for example, to produce recombinant viruses or recombinant fusion polypeptides. These cells include the progeny of the original transduced cell. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or genomic complement) to the original parent cell.

[0022] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps, and other procedures that result in a construct that differs from polynucleotides found in nature. A recombinant virus is a viral particle that contains a recombinant polynucleotide. The term includes copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0023] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including polynucleotide replication, duplication, transcription, splicing, translation, or degradation. Regulation can affect the frequency, speed, or specificity of the process and can be enhancing or inhibitory 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 can bind to RNA polymerase under certain conditions and initiate transcription of a coding region typically located downstream (3' direction) of the promoter. Promoters include AAV promoters, such as P5, P19, P40, and AAV ITR promoters, as well as heterologous promoters.

[0024] An "expression vector" is a vector containing a region encoding a gene product of interest and is used to effect expression of the gene product in an intended target cell. Expression vectors also contain control elements operably linked to the coding region to promote expression of the protein in the target. The combination of control elements and one or more genes operably linked thereto for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0025] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The term also encompasses amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, acetylation, phosphonylation, lipid addition, or conjugation with a labeling component).

[0026] The term "exogenous," when used in reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid can be from a different organism or cell, or can be one or more additional copies of a nucleic acid that naturally occurs in the organism or cell. As a non-limiting example, an exogenous nucleic acid is at a different chromosomal location than that of the native cell, or is otherwise flanked by different nucleic acid sequences than those found in nature (e.g., an expression cassette linking a promoter from one gene to the open reading frame of a gene product from a different gene).

[0027] "Transformed" or "transgenic" are used herein to include any host cell or cell line that has been altered or enhanced by the presence of at least one recombinant DNA sequence. The host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or by infection with a recombinant viral vector.

[0028] The term "sequence homology" refers to the percentage of base matches between two nucleic acid sequences, or the percentage of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage (e.g., 50%), the percentage indicates the percentage of matches over the length of the selected sequence compared to some other sequence. Gaps (in either of the two sequences) are allowed to maximize matching, and gap lengths of 15 bases or less are usually used (6 bases or less, e.g., 2 bases or less). When oligonucleotides are used as probes or therapeutics, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally 17 or more target base pair matches (85%) out of 20 possible oligonucleotide base pair matches, 9 or more matches (90%) out of 10 possible base pair matches, or 19 or more matches (95%) out of 20 possible base pair matches.

[0029] Two amino acid sequences are homologous if there is partial or complete identity between them. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed to maximize matching (gap length of 5 or less or 2 or less). Alternatively, two protein sequences (or polypeptide sequences derived from them that are at least 30 amino acids long) are homologous, as this term is used herein, if they have an alignment score of more than 5 (standard deviation units) using the program ALIGN with a mutation data matrix and a gap penalty of 6 or more. Two sequences or portions thereof are more homologous if their amino acids are 50% or more identical when optimally aligned using the ALIGN program.

[0030] The term "corresponding" is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 95%, or more, e.g., 99% or 100%, sequence identity. In contrast, the term "complementary" is used herein to mean that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0031] The term "sequence identity" means that two polynucleotide sequences are identical over a comparison window (i.e., nucleotide-by-nucleotide). The term "sequence identity percentage" means that two polynucleotide sequences are identical over a comparison window (i.e., nucleotide-by-nucleotide). The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. As used herein, the term "substantial identity" refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85 percent sequence identity, e.g., at least 90-95 percent sequence identity, or at least 99 percent sequence identity, relative to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, where the percentage of sequence identity is calculated by comparing the reference sequence to a polynucleotide sequence that may contain deletions or additions totaling no more than 20 percent of the reference sequence over the comparison window.

[0032] "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, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not have a significant effect on the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can be readily determined by assaying the specific activity of the polypeptide. Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0033] The present disclosure also contemplates polypeptides with non-conservative substitutions, which entail exchanging a member of one of the above classes for another.

[0034] Exemplary human APOE sequences include the following: TIFF2026041903000002.tif31128 (including the signal peptide, shown in italics above) (SEQ ID NO: 8), as well as sequences having at least 80%, 85%, 90%, 95%, or more, e.g., 99% or 100%, sequence identity thereto (e.g., having a Cys at residue 112 (mature polypeptide numbering, shown in bold above), and a Cys at residue 158 (shown in bold above), corresponding to SEQ ID NO: 9 (APOE2), and and those having an Arg at residue 112 (mature polypeptide numbering), and an Arg at residue 158 (APOE4), which corresponds to SEQ ID NO: 10, where in one embodiment, APOE4 can have 31K, 46P, 79T, 130R, 163C, 292H, and / or 314R, and APOE2 can have 43C, 152Q, 154C / S, 163C / P, 164Q, 172A, 176C, 242Q, 246C, 254E.

[0035] SEQ ID NO: 9 comprises the following: TIFF2026041903000003.tif31128

[0036] SEQ ID NO: 10 comprises the following: TIFF2026041903000004.tif30128

[0037] Exemplary human APOE nucleic acid sequences, such as those for silent nucleotide substitution sequences when they encode APOE2, include the following: TIFF2026041903000005.tif101128 or TIFF2026041903000006.tif107128, as well as sequences encoding APOE having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more, e.g., 99% or 100% sequence identity thereto.

[0038] Compositions and Methods Alzheimer's disease (AD) directly affects 5 million Americans and is rapidly increasing in prevalence and economic impact. Existing drugs have little effect on the underlying disease process, and no preventive therapies are currently available. Inheritance of a variant APOE4 gene confers a high risk of developing AD, while inheritance of the APOE2 gene is protective, reducing the risk of developing AD by approximately 50% and delaying the age of onset. APOE4 is associated with increased cerebral amyloid burden and greater memory impairment in AD. Conversely, APOE2 attenuates these effects. In humans, the odds ratio for developing AD for the E4 / E4 homozygous genotype is 14.9, reduced to 2.6 in E2 / E4 heterozygotes. In addition to its role in promoting amyloid production, APOE4 may be associated with abnormal brain function.

[0039] The present disclosure provides gene therapy vectors for expression of APOE2, sequences for inhibiting APOE4 expression, and methods of using the APOE2 and APOE4 inhibitory sequences.

[0040] Exemplary Gene Therapy Vectors The present disclosure provides a gene therapy vector comprising a nucleic acid sequence that encodes APOE2 and can comprise an inhibitory sequence for endogenous APOE4 expression, or in one embodiment, can comprise another vector for the expression of inhibitory sequence or a composition with inhibitory RNA sequence.Various aspects of gene therapy vectors and methods are discussed below.Although each parameter is discussed separately, gene therapy vectors and methods include the combination of the parameters described below, for example, to induce protection against APOE4-related pathology.Therefore, any combination of parameters can be used according to gene therapy vectors and methods.

[0041] Thus, 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 occurs. Typically, a gene therapy vector is a nucleic acid molecule engineered using recombinant DNA techniques known in the art to incorporate a heterologous nucleic acid sequence (e.g., heterologous with respect to other vector sequences, such as a promoter, or a vector backbone sequence, such as a viral sequence). Desirably, the gene therapy vector is composed of 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., a plasmid) or can 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 the form of an episome.

[0042] Gene or siRNA delivery vectors within the scope of the present disclosure include, but are not limited to, isolated nucleic acids, such as plasmid-based vectors that can be maintained extrachromosomally, and viral vectors, such as recombinant adenoviruses, retroviruses, lentiviruses, herpesviruses, poxviruses, papillomaviruses, or adeno-associated viruses, including viral and non-viral vectors present in neutral or cationic liposomes, such as liposomes, for example, DOSPA / DOPE, DOGS / DOPE, or DMRIE / DOPE liposomes, and / or 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 may be administered via any route, including, but not limited to, intracranial, intrathecal, intramuscular, buccal, rectal, intravenous, or intracoronary administration, and cellular transfer may be enhanced using electroporation and / or iontophoresis, and / or scaffolds such as extracellular matrices or hydrogels, e.g., hydrogel patches.

[0043] In one embodiment, gene therapy vector or other vector is a viral vector.Suitable viral vectors include, for example, retroviral vector, lentiviral vector, herpes simplex virus (HSV)-based vector, parvovirus-based vector, for example, adeno-associated virus (AAV)-based vector, AAV-adenovirus chimeric vector, and adenovirus-based vector.These viral vectors can be prepared using standard recombinant DNA technology, for example, as described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0044] Retroviral vectors Retroviral vectors exhibit several unique characteristics, including their ability to stably and precisely integrate into the host genome and provide long-term transgene expression. These vectors can be engineered ex vivo to remove infectious gene particles to minimize the risk of systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can change the tropism of host cells.

[0045] Lentivirus Lentivirus is derived from the retrovirus family, which includes human immunodeficiency virus and feline immunodeficiency virus.However, unlike retroviruses that only infect dividing cells, lentiviruses can infect both dividing and non-dividing cells.Although lentiviruses have specific tropism, pseudotyping of the viral envelope with vesicular stomatitis virus results in a more widespread virus (Schnepp et al.Meth.Mol.Med.,69:427(2002)).

[0046] Adenovirus vectors Adenoviral vectors can be made replication-defective by deleting the early (E1A and E1B) genes responsible for viral gene expression from the genome, and are stably maintained in host cells in an extrachromosomal form. These vectors have the ability to transfect both replicating and non-replicating cells. Adenoviral vectors have been shown to produce transient expression of therapeutic genes in vivo, peaking at 7 days and lasting for approximately 4 weeks. In addition, adenoviral vectors can be produced at very high titers, allowing for efficient gene therapy using small amounts of virus.

[0047] Adeno-associated virus vector Recombinant adeno-associated viruses (rAAVs) are derived from nonpathogenic parvoviruses, elicit essentially no cellular immune responses, and result in transgene expression that persists for several months in most systems. Furthermore, like adenoviruses, adeno-associated virus vectors also have the ability to infect replicating and non-replicating cells.

[0048] AAV vectors include, but are not limited to, AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrhlO (including chimeric viruses in which the AAV genome is from a different source than the capsid).

[0049] Plasmid DNA vectors Plasmid DNA is often referred to as "naked DNA" due to the lack of more elaborate packaging systems. Direct injection of plasmid DNA into cardiomyocytes in vivo has been achieved. Plasmid-based vectors are relatively non-immunogenic and non-pathogenic, and have the potential to stably integrate into the cellular genome, resulting in long-term gene expression in postmitotic cells in vivo. Furthermore, plasmid DNA is rapidly degraded in the bloodstream, thus negligible potential for transgene expression in distant organ systems. Plasmid DNA can be delivered to cells as part of macromolecular complexes, such as liposomes or DNA-protein complexes, and delivery can be enhanced using techniques including electroporation.

[0050] Exemplary AAV Vectors In one embodiment, the present disclosure provides an adeno-associated virus (AAV) vector that comprises, essentially consists of, or consists of the nucleic acid sequence encoding APOE2.When AAV vector essentially consists of the nucleic acid sequence encoding APOE2, it can contain additional components that do not substantially affect the AAV vector (for example, genetic elements such as poly(A) sequence or restriction enzyme site that facilitates the operation of the vector in vitro).When AAV vector consists of the nucleic acid sequence encoding APOE2, it does not contain any additional components (i.e., components that are not endogenous to AAV and are not required for the expression of the nucleic acid sequence).

[0051] Adeno-associated viruses are members of the Parvoviridae family and contain a linear, single-stranded DNA genome of less than approximately 5,000 nucleotides. For efficient replication, AAV requires co-infection with a helper virus (i.e., adenovirus or herpesvirus) or the expression of helper genes. AAV vectors used for administering therapeutic nucleic acids typically lack approximately 96% of the parent genome, leaving only the terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging. This eliminates immunological or toxic side effects resulting from viral gene expression. Furthermore, delivery of specific AAV proteins to producer cells allows for the integration of AAV vectors containing AAV ITRs into specific regions of the cellular genome, if desired (see, e.g., U.S. Patent Nos. 6,342,390 and 6,821,511). Host cells containing the integrated AAV genome do not exhibit changes in cell growth or morphology (see, e.g., U.S. Patent No. 4,797,368).

[0052] The AAV ITRs are flanked by unique coding nucleotide sequences for the nonstructural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)). The terminal 145 nucleotides are self-complementary and organized to allow the formation of energetically stable intramolecular duplexes that form T-shaped hairpins. These hairpin structures serve as origins for viral DNA replication by acting as primers for cellular DNA polymerase complexes. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. The Rep78 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication, allowing for AAV terminus resolution (see, e.g., Im et al., Cell, 61:447 (1990)). These proteins also regulate transcription from the endogenous AAV promoter and promoters in the helper virus (see, e.g., Pereira et al., J. Virol., 71:1079 (1997)). Other Rep proteins modify the function of Rep78 and Rep68. The cap gene encodes the capsid proteins VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter.

[0053] AAV vectors can be produced using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or non-human primate tissues (e.g., as reviewed in Wu et al., Molecular Therapy, 14(3):316 (2006)). Generally, AAV serotypes share significant genomic sequence homology at the nucleic acid and amino acid sequence levels, allowing different serotypes to share the same set of gene functions, produce essentially physically and functionally equivalent virions, and replicate and assemble via essentially the same mechanisms. AAV serotypes 1-5 and 7-9 are defined as "true" serotypes in that they do not cross-react effectively with neutralizing sera specific for any other existing characterized serotypes. In contrast, AAV serotypes 6, 10 (also referred to as Rh10), and 11 are considered "variant" serotypes because they do not adhere to the definition of a "true" serotype. AAV serotype 2 (AAV2) is widely used for gene therapy applications due to its lack of pathogenicity, broad range of infectivity, and ability to establish long-term transgene expression (see, e.g., Carter, Hum. Gene Ther., 16:541 (2005), and Wu et al., supra). The genomic sequences of various AAV serotypes and comparisons thereof are disclosed, for example, in GenBank accession numbers U89790, J01901, AF043303, and AF085716, Chiorini et al., J. Virol., 71:6823 (1997), Srivastava et al., J. Virol., 45:555 (1983), Chiorini et al., J. Virol., 73:1309 (1999), Rutledge et al., J. Virol., 72:309 (1998), and Wu et al., J. Virol., 74:8635 (2000)).

[0054] AAV rep and ITR sequences are particularly conserved across most AAV serotypes. For example, the Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4, and AAV6 are reportedly approximately 89–93% identical (see Bantel-Schaal et al., J. Virol., 73(2):939 (1999)). AAV serotypes 2, 3A, 3B, and 6 are reported to share approximately 82% overall nucleotide sequence identity at the genomic level (Bantel-Schaal et al., supra). Furthermore, the rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (e.g., functionally substitute for) corresponding sequences from other serotypes during AAV particle production in mammalian cells.

[0055] Generally, the cap protein, which determines the cell tropism of AAV particles, and the associated cap protein coding sequence are significantly less conserved across different AAV serotypes than the Rep gene. Considering the ability of Rep and ITR sequences to cross-complement the corresponding sequences of other serotypes, AAV vectors can contain a mixture of serotypes, thereby becoming "chimeric" or "pseudotyped" AAV vectors. Chimeric AAV vectors typically contain AAV capsid proteins from two or more (e.g., two, three, four, etc.) different AAV serotypes. In contrast, pseudotyped AAV vectors contain one or more ITRs from one AAV serotype packaged in 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, Mol. Ther., 13(1):1 (2006), Gao et al., J. Virol., 78:6381 (2004), Gao et al., Proc. Natl. Acad. Sci. USA, 99:11854 (2002), De et al., Mol. Ther., 13:67 (2006), and Gao et al., Mol. Ther., 13:77 (2006).

[0056] In one embodiment, the AAV vector is generated using an AAV that infects humans (e.g., AAV2). Alternatively, the AAV vector is generated using an AAV that infects non-human primates, such as great apes (e.g., chimpanzees), Old World monkeys (e.g., macaques), and New World monkeys (e.g., marmosets). In one embodiment, the AAV vector is generated using an AAV that infects non-human primates and is pseudotyped with an 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 containing a capsid protein from an AAV that infects rhesus monkeys and is pseudotyped with AAV2 inverted terminal repeats (ITRs) can be generated. In certain embodiments, the AAV vector comprises capsid proteins from AAV10 (also referred to as "AAVrh.10") that infect rhesus macaques pseudotyped with AAV2 ITRs (see, e.g., Watanabe et al., Gene Ther., 17(8):1042 (2010), and Mao et al., Hum. Gene Therapy, 22:1525 (2011)).

[0057] In addition to the nucleic acid sequence encoding APOE2, the AAV vector can include expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc., that provide for the expression of the nucleic acid sequence in host cells, as well as, 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).

[0058] Numerous promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Typical promoter sources include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences from, for example, repositories such as ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either 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 (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al. Proc. Natl. Acad. Sci., 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., Nuc. Acid. Res., 27:4324 (1999), Nuc. Acid. Res., 28:e99 (2000), U.S. Pat. No. 7,112,715, and Kramer & Fussenegger, Methods Mol. Biol., 308:123 (2005)).

[0059] The term "enhancer" as used herein refers to a DNA sequence that increases the transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from repositories such as ATCC, as well as other commercial or individual sources). Some polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream of, within, or downstream of the coding sequence. In one embodiment, the nucleic acid sequence encoding APOE2 is operably linked to a CMV enhancer / chicken beta-actin promoter (also referred to as a "CAG promoter") (see, e.g., Niwa et al., Gene, 108:193 (1991); Daly et al., Proc. Natl. Acad. Sci. USA, 96:2296 (1999); and Sondhi et al., Mol. Ther., 15:481 (2007)).

[0060] Typically, AAV vectors are produced using well-characterized plasmids. For example, human embryonic kidney 293T cells are transfected with one of the transgene-specific plasmids and another plasmid containing an adenovirus helper and the AAV rep and cap genes (specific for AAVrh.10, 8, or 9, as needed). After 72 hours, the cells are harvested, and the vector is released from the cells by five freeze / thaw cycles. Subsequent centrifugation and benzonase treatment remove cellular debris and unencapsidated DNA. An iodixanol gradient and ion exchange column can be used to further purify each AAV vector. The purified vector is then concentrated to the required concentration using a size-exclusion centrifugal spin column. Finally, the buffer is exchanged to produce the final vector product, which is formulated (for example) in 1x phosphate-buffered saline. Viral titer can be measured by TaqMan® real-time PCR, and viral purity can be assessed by SDS-PAGE.

[0061] Pharmaceutical Compositions and Vector Delivery The present disclosure provides compositions comprising, consisting essentially of, or consisting of the above-described gene therapy vector and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, or a vector for expression of RNAi. When a composition consists essentially of a gene therapy vector and a pharmaceutically acceptable carrier, additional components that do not substantially affect the composition (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.) may be included. When a composition consists of a gene therapy vector and a pharmaceutically acceptable carrier, the composition does not contain any additional components. Any suitable carrier may be used within the context of the present disclosure, and such carriers are well known in the art. The choice of carrier will depend, in part, on the particular site to which the composition will be administered and the particular method used to administer the composition. The composition may optionally be sterile, with the exception of the gene therapy vectors described herein. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions may be produced according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0062] Suitable formulations for the compositions include aqueous and non-aqueous solutions, isotonic sterile solutions (which may contain antioxidants, buffers, and bacteriostats), and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described. In one embodiment, the carrier is a buffered saline solution. In one embodiment, the gene therapy vector is administered in a composition formulated to protect the gene therapy vector from damage prior to administration. For example, the composition may be formulated to reduce loss of the gene therapy vector on devices used to prepare, store, or administer the gene therapy vector, such as glassware, syringes, or needles. The composition may be formulated to reduce the light and / or temperature sensitivity of the gene therapy vector. To this end, the composition may comprise, for example, 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. The use of such a composition may extend the shelf life of the gene therapy vector, facilitate administration, and increase the efficiency of the method. Formulations for gene therapy vector-containing compositions are further described, for example, in Wright et al., Curr. Opin. Drug Discov. Devel., 6(2):174-178 (2003) and Wright et al., Molecular Therapy, 12:171-178 (2005).

[0063] The composition may also be formulated to enhance transduction efficiency. Additionally, those skilled in the art will appreciate that the gene therapy vector may be present in the composition with other therapeutic or bioactive agents. For example, factors that control inflammation, 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, such as interleukins, lipopolysaccharides, and double-stranded RNA, may be administered to enhance or modify the immune response. Antibiotics, i.e., bactericides and fungicides, may be present to treat existing infections and / or reduce the risk of future infections, such as those associated with gene therapy procedures.

[0064] Injectable depot forms are prepared by forming microencapsule matrices of the target compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0065] In certain embodiments, the formulation comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic acid esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0066] The composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, a biocompatible meshwork, a mechanical reservoir, or a mechanical implant. Implants (see, e.g., U.S. Pat. No. 5,443,505), devices (see, e.g., U.S. Pat. No. 4,863,457), for example, implantable devices, such as mechanical reservoirs, or implants or devices composed of polymeric compositions, are particularly useful for administering gene therapy vectors. The composition can also be administered in the form of sustained-release formulations, including, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, polyphosphoesters, such as bis-2-hydroxyethyl-terephthalate (BHET), and / or polylactic-glycolic acid (see, e.g., U.S. Pat. No. 5,378,475).

[0067] Delivery of compositions containing gene therapy vectors can be intracerebral (including, but not limited to, intraparenchymal, intraventricular, or intracisternal), intrathecal (including, but not limited to, lumbar or cisterna magna), or systemic (including, but not limited to, intravenous), or any combination thereof, using devices known in the art. Delivery can also be via surgical implantation of an implanted device.

[0068] The dose of the gene therapy vector in the composition administered to the mammal depends on several factors, including the size (mass) of the mammal, the extent of any side effects, the particular route of administration, etc. In one embodiment, the method involves administering a "therapeutically effective amount" of a composition comprising a gene therapy vector described herein. A "therapeutically effective amount" refers to an amount effective, for the duration and dosage necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the extent of the condition, the age, sex, and weight of the individual, and the ability of the gene therapy vector to elicit the desired response in the individual. The dose of the gene therapy vector in the composition required to achieve a particular therapeutic effect is typically administered in units of vector genome copies per cell (gc / cell) or vector genome copies per kilogram body weight (gc / kg). One skilled in the art can easily determine the appropriate gene therapy vector dosage range for treating a patient with a particular disease or disorder based on these and other factors well known in the art. A therapeutically effective amount is defined as an amount of 1×10 10 Genome copies ~1 x 10 13 A therapeutically effective dose may be 1×10 genome copies. 11 Genome copies ~1 x 10 14 A therapeutically effective dose may be 1×10 genome copies. 12 Genome copies ~1 x 10 15 A therapeutically effective dose may be 1x10 genome copies. 13 Genome copies (gc) ~ 1x10 16 gc, e.g., 1x10 13 gc~1x10 14 gc, 1x10 14 gc~1x10 15 gc, or 1x10 15 gc~1x10 14 Assuming a 70 kg human, the dose range is 1.4 x 10 8 gc / kg~1.4×10 11 gc / kg, 1.4 × 10 9 gc / kg~1.4×10 12 gc / kg, 1.4 × 10 10 gc / kg~1.4×10 13 gc / kg, or 1.4 × 1011 gc / kg~1.4×10 14 It can be gc / kg.

[0069] In one embodiment, the composition is administered to mammal once.It is believed that a single administration of the composition will cause the expression of APOE2 and the suppression of APOE4 expression in mammal with minimal side effects.However, in certain cases, it may be appropriate to administer the composition multiple times during the treatment period to ensure sufficient exposure of cells to the composition.For example, the composition can be administered to mammal more than once (for example, 2, 3, 4, 5, 6, 6, 8, 9, or 10 times or more) during the treatment period.

[0070] Accordingly, the present disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a gene therapy vector comprising a nucleic acid sequence encoding APOE2 and a sequence that inhibits APOE4 expression.

[0071] subject The subject can be any animal, including human and non-human animals.Non-human animals include all vertebrates, for example, mammals and non-mammals, for example, 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 considered as subjects.The subject can also be livestock, for example, cows, pigs, sheep, poultry, and horses, or pets, for example, dogs and cats.

[0072] In one embodiment, the subject includes a human subject suffering from or at risk for the medical diseases and disorders described herein. The subject will generally be diagnosed with the condition by one of ordinary skill in the art, e.g., a physician.

[0073] The methods described herein can be used for subjects of any species, sex, age, ethnic group, or genotype. Thus, the term subject includes males and females, and includes elderly people, adult subjects in the transition age between elderly and adulthood, subjects in the transition age between adulthood and pre-adulthood, and pre-adults, including adolescents, children, and infants.

[0074] Examples of human ethnic groups include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semitic peoples, and Pacific Islanders. The method may be more suitable for some ethnic groups, such as Caucasians, particularly Northern European populations, and Asian populations.

[0075] The term subject also includes subjects of any genotype or phenotype, as described above, as long as they require treatment.In addition, subjects may have any genotype or phenotype for any hair color, eye color, skin color, or any combination thereof.The term subject includes subjects of any height, weight, or size or shape of any organ or body part.

[0076] Exemplary Nanoparticle Formulations Biodegradable nanoparticles (e.g., containing gene therapy vectors or isolated nucleic acids or vectors for RNAi expression) have been developed using a variety of nanoparticles, including 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), polypropylene fumarate, poly(orthoesters), polyol / diketene acetal addition polymers, poly-alkyl-cyano-acrylates (PAC), poly(methyl-2-methyl-2-propanol ... The polymeric material may comprise or be formed from biodegradable polymer molecules, including, but not limited to, poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxyhexone) (PCPP), poly[bis(p-carboxyphenoxy)methane] (PCPM), copolymers of PSA, PCPP, and PCPM, poly(amino acids), poly(pseudoamino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] and poly[(organo)phosphazenes], polyhydroxybutyric acid, or S-caproic acid, elastin, or gelatin (see, e.g., 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, 5,981,707, 19, 5,871,747, 5,723,269, 5,603,960, and 5,578,709, as well as U.S. Application Publication No. 2007 / 0081972, and International Application Publication Nos. WO2012 / 115806 and WO2012 / 054425, the contents of which are incorporated herein by reference in their entireties.

[0077] 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, Suppl. 3, 2012, pages 16-23; Cismaru et al. Rev., Roum. Chim., 2010, 55(8), 433-442; and International Application Publication Nos. WO2012 / 115806 and WO2012 / 054425, the contents of which are incorporated herein by reference in their entirety.) Suitable methods for preparing nanoparticles can include methods utilizing preformed polymer dispersions, including, but not limited to, solvent evaporation, nanoprecipitation, emulsification / solvent diffusion, salting out, dialysis, and supercritical fluid techniques. In some embodiments, nanoparticles can be prepared by forming a double emulsion (e.g., water-in-oil-in-water) followed by solvent evaporation. The nanoparticles obtained by the disclosed methods can be subjected to further processing steps, such as washing and lyophilization, if desired. Optionally, the nanoparticles can be combined with a preservative (e.g., trehalose).

[0078] Typically, the nanoparticles have an average effective diameter of less than 1 micron, e.g., the nanoparticles have an average effective diameter of about 25 nm to about 500 nm, e.g., about 50 nm to about 250 nm, about 100 nm to about 150 nm, or about 450 nm to 650 nm. Particle size (e.g., average effective diameter) can be assessed by methods known in the art, including, but not limited to, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), photon correlation spectroscopy (PCS), nanoparticle surface area monitor (NSAM), condensation particle counter (CPC), differential mobility analyzer (DMA), scanning mobility particle sizer (SMPS), nanoparticle tracking analysis (NTA), X-ray diffraction (XRD), aerosol time-of-flight mass spectrometry (ATFMS), and aerosol particle mass spectrometry (APM).

[0079] Biodegradable nanoparticles can have a zeta potential that facilitates uptake by target cells. Typically, nanoparticles have a zeta potential greater than 0. In some embodiments, nanoparticles have a zeta potential of about 5 mV to about 45 mV, about 15 mV to about 35 mV, or about 20 mV to about 40 mV. Zeta potential can be determined by characteristics including electrophoretic mobility or dynamic electrophoretic mobility. Electrokinetic and electroacoustic phenomena can be used to calculate zeta potential.

[0080] In one embodiment, the non-viral delivery vehicle comprises poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), linear and / or branched PEI with different molecular weights (e.g., 2, 22, and 25 kDa), dendrimers such as polyamidoamine (PAMAM) and polymethacrylate, cationic liposomes, cationic emulsions, lipids including, but not limited to, DOTAP, DOTMA, DMRIE, DOSPA, distearoylphosphatidylcholine (DSPC), DOPE, or DC-cholesterol, peptide-based vectors including, but not limited to, poly-L-lysine or protamine, or polymers including, but not limited to, poly(β-amino ester), chitosan, PEI-polyethylene glycol, PEI-mannose-dextrose, DOTAP-cholesterol, or RNAiMAX.

[0081] In one embodiment, the delivery vehicle is poly(glycamidoamine) (PGAA), a glycopolymer-based delivery vehicle capable of complexing with various polynucleotide types and forming nanoparticles. These materials are prepared by polymerizing methyl ester or lactone derivatives of various carbohydrates (D-glucarate (D), meso-galactarate (G), D-mannarate (M), and L-tartrate (T)) with a series of oligoethyleneamine monomers (containing one to four ethyleneamines) (Liu and Reineke, 2006). A subset consisting of these carbohydrates and four ethyleneamines in the polymer repeat unit resulted in excellent delivery efficiency.

[0082] In one embodiment, the delivery vehicle comprises polyethyleneimine (PEI), polyamidoamine (PAMAM), PEI-PEG, PEI-PEG-mannose, dextran-PEI, OVA conjugate, PLGA microparticles, or PLGA microparticles coated with PAMAM, or any combination thereof. The disclosed cationic polymers may include, but are not limited to, polyamidoamine (PAMAM) dendrimers. Suitable polyamidoamine dendrimers for preparing the nanoparticles of the present disclosure may include third, fourth, fifth, or at least sixth generation dendrimers.

[0083] In one embodiment, the delivery vehicle comprises a lipid, such as N-[1-(2,3-dioleoyloxy)propan]-N,N,N-trimethylammonium (DOTMA), 2,3-dioleyloxy-N-[2-sperminecarboxamido]ethyl-N,N-dimethyl-1-propaneammonium trifluoracetate (DOSPA, Lipofectamine), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dimyristoloxy)propyl], N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 3-β-[N-(N,N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), dioctadecylamidoglyceryl spermine (DOGS, Transfectamine), or dimethyldioctadecylammonium bromide (DDAB). The positively charged hydrophilic head group of cationic lipids typically consists of monoamines such as tertiary and quaternary amines, polyamines, amidinium, or guanidinium groups. A series of pyridinium lipids have been developed (Zhu et al., 2008; van der Woude et al., 1997; Ilies et al., 2004). In addition to pyridinium cationic lipids, other types of heterocyclic head groups include imidazole, piperidine, and amino acids. The main function of the cationic head group is to condense negatively charged nucleic acids into slightly positively charged nanoparticles through electrostatic interactions, resulting in enhanced cellular uptake and endosomal escape.

[0084] Lipids with two linear fatty acid chains, such as DOTMA, DOTAP, and SAINT-2, or DODAC, can be used as delivery vehicles, as can dimers of the tetraalkyl lipid chain surfactant N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Regardless of their hydrophobic chain length (C 16:1 , C 18:1 , and C 20:1 ), all trans-oriented lipids appear to enhance transfection efficiency compared to their cis-oriented counterparts.

[0085] The structures of cationic polymers useful as delivery vehicles include, but are not limited to, linear polymers such as chitosan and linear poly(ethyleneimine), branched polymers such as branched poly(ethyleneimine) (PEI), ring-like polymers such as cyclodextrin, network (cross-linked) polymers such as cross-linked poly(amino acids) (PAA), and dendrimers. Dendrimers consist of a central core molecule from which several highly branched arms "grow" to form a tree-like structure in a symmetric or asymmetric manner. Examples of dendrimers include polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers.

[0086] DOPE and cholesterol are neutral co-lipids commonly used to prepare cationic liposomes. Branched PEI-cholesterol water-soluble lipopolymer conjugates self-assemble into cationic micelles. Nonionic polymers such as Pluronic (poloxamer) and SP1017, a combination of Pluronic L61 and F127, can also be used.

[0087] In one embodiment, PLGA particles are used to increase encapsulation frequency, although complexation with PLL may also increase encapsulation efficiency. Other cationic materials, such as PEI, DOTMA, DC-Chol, or CTAB, can be used to create nanospheres.

[0088] In one embodiment, the complex is embedded in or applied to a material including, but not limited to, a hydrogel of poloxamer, polyacrylamide, poly(2-hydroxyethyl methacrylate), carboxyvinyl polymer (e.g., Carbopol 934, Goodrich Chemical Co.), cellulose derivatives such as methylcellulose, cellulose acetate, and hydroxypropylcellulose, polyvinylpyrrolidone or polyvinyl alcohol, or combinations thereof.

[0089] In some embodiments, the biocompatible polymeric material is derived from a biodegradable polymer such as collagen, e.g., hydroxylated collagen, fibrin, polylactic acid-polyglycolic acid, or a 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-glycolide) block copolymer, polyglycolide, polylactide (PLLA or PDLA), poly(caprolactone) (PCL), or poly(dioxanone) (PPS).

[0090] In another embodiment, the biocompatible material includes polyethylene terephthalate, polytetrafluoroethylene, copolymers of polyethylene oxide and polypropylene oxide, combinations of polyglycolic acid and polyhydroxyalkanoate, gelatin, alginate, poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, and polyhydroxyoctanoate, and polyacrylonitrile polyvinyl chloride.

[0091] In one embodiment, the following polymers include natural polymers such as starch, chitin, glycosaminoglycans, e.g., hyaluronic acid, dermatan sulfate, and chlordrin sulfate, and microbial polyesters, e.g., hydroxyalkanoates such as hydroxyvalerate and hydroxybutyrate copolymers, and synthetic polymers such as poly(orthoesters) and polyanhydrides, and homo- and copolymers of 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-coglycolide), poly(collidine lactate), and polycaprolactone may be used).

[0092] In one embodiment, the biocompatible material is derived from isolated extracellular matrix (ECM). ECM can be isolated from any organ or tissue source, including the endothelial layer of various cell populations, tissues, and / or organs, such as the skin, liver, digestive tract, respiratory tract, intestinal tract, urinary tract, or genital tract of warm-blooded vertebrates. The ECM used in the present invention can be from a combination of sources. The isolated ECM can be prepared as a sheet, in particulate form, gel form, etc.

[0093] Biocompatible scaffold polymers may include silk, elastin, chitin, chitosan, poly(d-hydroxy acid), poly(anhydride), or poly(orthoester). More specifically, biocompatible polymers include 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 / diketene acetal addition polymer, poly(sebacic anhydride) (PSA), poly(carboxybis(carboxyphenoxyphenoxy)-2-hydroxybenzoyl)benzoate, poly( ... The polymeric polymer may be formed of poly(amino acid), poly(pseudoamino acid), polyphosphazene, derivatives of poly[(dichloro)phosphazene] or poly[(organo)phosphazene], polyhydroxybutyric acid, or S-caproic acid, polylactide-co-glycolide, polylactic acid, polyethylene glycol, cellulose, oxidized cellulose, alginate, gelatin, or derivatives thereof.

[0094] Thus, the polymer may be formed from any of a wide range of materials, including polymers comprising naturally occurring polymers, synthetic polymers, or combinations thereof. In one embodiment, the scaffold comprises a biodegradable polymer. In one embodiment, a naturally occurring biodegradable polymer may be modified to provide a synthetic biodegradable polymer derived from a naturally occurring polymer. In one embodiment, the polymer is poly(lactic acid) (“PLA”) or poly(lactic-co-glycolic acid) (“PLGA”). In one embodiment, scaffold polymers include, but are not limited to, alginate, chitosan, poly(2-hydroxyethyl methacrylate), xyloglucan, copolymers of 2-methacryloyloxyethyl phosphorylcholine, poly(vinyl alcohol), silicone, hydrophobic and hydrophilic polyesters, poly(lactide-co-glycolide), N-isopropylacrylamide copolymers, poly(ethylene oxide) / poly(propylene oxide), polylactic acid, poly(orthoesters), polyanhydrides, polyurethanes, copolymers of 2-hydroxyethyl methacrylate and sodium methacrylate, phosphorylcholine, cyclodextrin, polysulfone and polyvinylpyrrolidine, starch, poly-D,L-lactic acid-para-dioxanone-polyethylene glycol block copolymer, polypropylene, poly(ethylene terephthalate), poly(tetrafluoroethylene), poly-epsilon-caprolactone, or cross-linked chitosan hydrogels.

[0095] Alternatively, the nucleic acid or vector can be administered at a dosage 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 dosages may provide beneficial results.

[0096] Alternatively, the nucleic acid or vector can be administered at a dosage 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 dosages may provide beneficial results.

[0097] Illustrative Embodiments In one embodiment, a gene therapy vector is provided, comprising: a promoter operably linked to a nucleic acid sequence comprising 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 inhibiting APOE4 mRNA. In one embodiment, the vector comprises the nucleotide sequence. In one embodiment, the nucleotide sequence is 5' or 3' relative to the open reading frame. In one embodiment, the nucleotide sequence is 5' and 3' relative to the open reading frame. In one embodiment, the nucleotide sequences are on different vectors. 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 AAVrhlO. 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 Pol III promoter. In one embodiment, the RNAi comprises an miRNA comprising multiple miRNA sequences. In one embodiment, the RNAi comprises an siRNA comprising 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.

[0098] Also provided is a host cell or mammal comprising the vector. 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.

[0099] Further provided is a method for preventing, inhibiting, or treating Alzheimer's disease in a mammal, comprising administering to the mammal an effective amount of a composition comprising a gene therapy vector.

[0100] A method for preventing, inhibiting, or treating a disease associated with APOE4 expression in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising a gene therapy vector.

[0101] In one embodiment, the composition comprises a liposome comprising the vector. In one embodiment, the composition comprises a nanoparticle 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, e.g., identical miRNA sequences.

[0102] The invention is illustrated by the following non-limiting examples. [Example]

[0103] Example 1 Alzheimer's disease (AD) affects 5 million Americans and is rapidly increasing in prevalence. Existing drugs have little effect on the underlying disease process, and no preventive therapies are currently available. Inheritance of the APOE4 allele confers a high risk for disease development, whereas inheritance of the APOE2 allele is protective, reducing the risk of developing AD by ≥50% and delaying the age of onset. Adeno-associated virus (AAV) delivery of the human APOE2 gene into a mouse model of AD expressing human APOE4 (homozygous expression) showed reduced amyloid-β peptide and amyloid burden. The odds ratio for developing AD is reduced in E2 / E4 heterozygotes compared with E4 / E4 homozygotes (2.6 vs. 14.9). Suppression of APOE4 (e.g., via AAV vector delivery) while simultaneously expressing human APOE2 may further reduce the risk for AD. In one embodiment, gene therapy, such as AAV therapy, is designed to deliver both human APOE2 gene coding sequence and artificial RNA, for example, the microRNA (miRNA) that targets endogenous APOE4.The knockdown of harmful endogenous APOE4 expression combined with the expression of beneficial APOE2 allele can provide the homozygous individual of APOE4 allele with enhanced protection from developing AD.

[0104] In one embodiment, siRNA interacts with mRNA and silences translation.In order to express siRNA from DNA sequence such as gene therapy expression vector, targeting sequence must be embedded in short hairpin RNA (shRNA) or miRNA scaffold.The artificial miRNA expressed by vector is similar to endogenous RNAi and undergoes two processing steps.Because miRNA is expressed at a lower level, it is less likely to induce liver and CNS toxicity when delivered by gene therapy vector.

[0105] In one embodiment, the knockdown of all endogenous APOE isoforms can be achieved by using multiple miRNAs that target different sections of APOE mRNA, thereby enhancing silencing. In one embodiment, the human APOE2 derived vector can contain silent mutations in the coding sequence to prevent silencing.

[0106] As shown in Figure 3, miRNAs with RNAi sequences for inhibiting APOE4 expression can be inserted into 5' non-coding sequences, such as introns, and / or 3' non-coding sequences.For example, to enhance the silencing of APOE4, multiple miRNAs can be placed in tandem.It was found that the expression levels of hAPOE2-HA and miRNA are similar.There is a lower level of miRNA expression (compared to the U6 promoter), which in turn leads to fewer off-target effects and less potential toxicity.

[0107] In one embodiment (see Figure 4), a constitutive promoter such as CAG drives hAPOE2-HA, and a U6 promoter (an exemplary Pol III promoter) drives the miRNA. In one embodiment, multiple miRNAs are arranged in tandem to enhance APOE4 silencing (e.g., two, three, four, or more miRNAs). In one embodiment, a Pol III promoter is used for transcription of rRNA, tRNA, and / or miRNA. In one embodiment, the vector may have a defined terminator; for example, polyA is not required, because Pol III transcription is terminated by an oligo(dT) stretch in the non-template strand (dA in the template strand). In one embodiment, a two-vector system may be used, in which the second vector contains a stuffer sequence (e.g., for a reporter gene) to maintain length and track expression.

[0108] Therefore, the present disclosure provides a vector, such as a viral vector such as an AAV vector, that delivers both the human APOE2 gene and the artificial miRNA that targets human APOE4. These gene therapy vectors can be used to reduce the risk of AD development in APOE4 homozygous individuals (as well as E2 / E4 heterozygotes) by shifting the balance towards the expression of beneficial APOE2 alleles.

[0109] In one embodiment, the vector is useful in disorders or diseases that may benefit from increasing APOE2 and / or decreasing APOE4. In one embodiment, the vector is delivered to a mammal, such as a human, at risk for AD. AD currently affects 5 million people in the United States, and the global prevalence is expected to rise to 65 million by 2030. The overall prevalence of the APOE4 allele is 15%, with approximately 50% of AD patients carrying at least one APOE4 allele. The harmful APOE4 gene is targeted for reduced expression, while providing protective APOE2 expression, further reducing the risks associated with APOE4 compared to gene therapy that only delivers APOE2.

[0110] Example 2 Figure 5 shows a system in which miRNA knocks down all APOE isoform expression, and vector-derived APOE2 is resistant to miRNA.For example, by using CAG promoter, the expression levels of hApoE2-HA and miRNA are similar, and lower levels of miRNA expression (compared to U6 promoter) may mean that silencing is lower, but there are also fewer off-targets and toxicity.In one embodiment, miRNA can be inserted into the CAG intron or 3' untranslated region.

[0111] Figure 6 shows the test of APOE knockdown efficiency by siRNA in U87 cells. Based on the comparison of multiple siRNA design algorithms, four different siRNAs targeting the coding sequence of APOE were generated. The siRNAs were transfected into U87 cells (astroglioma cell line), and APOE mRNA copies were quantified by RT-qPCR. The identified sequences were as follows: TIFF2026041903000007.tif24128 non-targeting siRNA TIFF2026041903000008.tif4128. Other sequences for siRNA include: TIFF2026041903000009.tif18128

[0112] The sequence from one siRNA (#2 above) was converted into a miRNA. A scaffold based on a modified version of mir155 (Fowler et al. 2015 Nucl. Acids Res., 44:e48, the disclosure of which is incorporated herein by reference) was used. However, any miRNA backbone could be used (e.g., mir21, mir30, or mir33).

[0113] For example, for the miR from siRNA#2, the following may be used: TIFF2026041903000010.tif77128

[0114] In one embodiment, the miRNA has a U or A at guide position 1 relative to the 5' microprocessor cleavage site, a U or A at positions 2-7, 10-14, and 17, and a G or C at positions 19-21, and / or a G / C content of 36.4% to 45.5%, and / or a guide strand that is two nucleotides longer than the passenger strand, and / or one or more of the following mismatches: (1) a loop in which 3-5 adjacent nucleotides of the guide strand do not base-pair with the target strand, (2) mismatches spaced 3 bp apart, in which two single guide strand nucleotide mismatches are separated by three guide / passenger base pairs, and / or (3) mismatches spaced 4 bp apart, in which two single guide strand nucleotide mismatches are separated by four guide / passenger base pairs. Mismatches in the passenger strand were selected for optimal GC content and location. Mfold was used to predict the miRNA hairpin secondary structure. Two tandem copies of the miRNA were cloned into either the CAG intron or the 3' untranslated region of the pAAV expression cassette. Up to four copies of the miRNA (of that length) can be inserted within the AAV size limit.

[0115] The vector-derived APOE2 was modified to be resistant to silencing by the above-mentioned targeting miRNA (see underlined sequence below). Silent changes were made in the nucleotide sequence of the miRNA targeting region (red / bold).

[0116] APOE2 from vector: TIFF2026041903000011.tif137128Modified APOE2: TIFF2026041903000012.tif137128

[0117] The above sequence silently changes all possible nucleotides while taking into account the codon usage within the composite recognition sites for the three miRNAs derived from siRNA#2. However, other examples are provided below. Modified APOE: TIFF2026041903000013.tif137128Modified APOE: TIFF2026041903000014.tif137128Modified APOE: TIFF2026041903000015.tif137128Modified APOE: TIFF2026041903000016.tif137128Modified APOE: TIFF2026041903000017.tif137128Modified APOE: TIFF2026041903000018.tif137128 or, Modified APOE: TIFF2026041903000019.tif137128

[0118] The vector can be tested in non-human animals such as mice. In one embodiment, the vector comprises sequences from an AAV9-CAG-APOE2 vector (AAV9-APOE2), an adeno-associated virus vector serotype 9 that expresses APOE2 behind the chicken β-actin promoter, or from an AAVrh.10-CAG-APOE2 vector (AAVrh.10-APOE2), a rhesus monkey adeno-associated virus vector serotype 10 that expresses an APOE2 transgene behind the chicken β-actin promoter.

[0119] AAVrh.10 and AAV9 vectors can be produced and purified as previously described (Sondhi et al., 2007, 2012; Zolotukhin et al., 2002). Briefly, vectors are produced by cotransfection of HEK293T cells with the expression cassette plasmid and adenovirus helper plasmid. The packaging cell line, HEK293T, is maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin at 37°C and 5% CO2. Cells are plated in CellSTACKS (Corning, Tewksbury, MA) at 30%-40% confluence for 24 hours (or when 70%-80% confluence) and subsequently transfected with the plasmids using the PEIpro protocol. After incubation at 37°C for 3 days, the cells are harvested and lysed by five freeze / thaw cycles. The resulting cell lysate is treated with 50 U / mL benzonase for 30 minutes at 37°C. For AAVrh.10 vectors, the cell lysate is purified by iodixanol density gradient followed by Q-HP ion exchange chromatography. For AAV9 vectors, the cell lysate is precipitated overnight in PEG (final PEG concentration: 8%). After centrifugation, the supernatant is discarded, and the pellet is resuspended in 15 mL of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.5). The sample is purified by centrifugation through 1.37 g / mL CsCl in a 38.5 mL polyallomer tube using an SW28 rotor at 24,000 rpm (182,000 g) at 20°C for 24 hours. Insert a 21-gauge needle (Hamilton, Reno, NV) through the bottom of the centrifuge tube and collect 1 mL fractions. Vector-containing fractions are those containing the sequence of the vector construct. 32The titer is determined by dot blot analysis using a P-labeled probe. The positive fractions are then pooled and diluted with 1.37 g / mL CsCl. The sample is loaded into a 13.5 mL Quick-Seal tube and centrifuged at 67,000 rpm (384,000 g) at 20°C for 16–20 hours in an ultracentrifuge (Beckman LE-80K; Beckman Coulter, Fullerton, CA) 90Ti rotor. Fractions (0.5 mL) are collected, and the positive fractions are pooled. The purified AAVrh.10 or AAV9 vector is concentrated in phosphate-buffered saline (PBS). The vector genome titer is determined by Taq-Man quantitative polymerase chain reaction. The purified vector is sterile filtered and tested for growth on media supporting aerobic, anaerobic, or fungal growth for 14 days, tested for endotoxin, and demonstrated to be mycoplasma-free.

[0120] AAV preparation (2 mL, 1.0 x 10 10 vg or another dose) can be injected at a rate of 0.2 mL / min using, for example, a 33-gauge needle (Hamilton) and a syringe pump (KD Scientific, Holliston, Mass.).

[0121] References Baek et al.,PLoS One,5:e13468 (2010). Bales et al., J. Neurosci., 29:6771 (2009). Cearley and Wolfe, J. Neurosci., 27:9928 (2007). Bales et al., Nat. Genet., 17:263e264 (1997). Boyles et al., J. Clin. Invest., 76:1501e1513 (1985). Carrasquillo et al., Neurobiol. Aging, 36:60e67 (2015). Castellano et al.,Sci.Transl.Med.,3:89ra57 (2011). Corder et al.,Nat.Genet.,7:180e184 (1994). Corder et al.,Science,261:921e923 (1993). Deane et al.,J.Clin.Invest.,118:4002e4013 (2008). DeMattos et al.,Neurochem.Int.,39:415e425 (2001). DiBattista et al.,Curr.Alzheimer Res.,13:1200 (2016). Dodart et al.,Proc.Natl.Acad.Sci.U.S.A.,102:1211e1216 (2005). Fagan et al.,Neurobiol.Dis.,9:305e318 (2002). Fan et al.,Biofactors,35:239e248 (2009). Farrer et al.,JAMA,278:1349e1356 (1997). Franklin and Paxinos,The mouse brain in Stereotaxic Coordinates,3rd edition.Elsevier Inc,New York (2007). Games et al.,Nature,373:523e527 (1995). Haass and Selkoe,Nat.Rev.Mol.Cell Biol.,8:101e112 (2007). Hardy and Selkoe,Science,297:353e356 (2002). Hashimoto et al.,J.Neurosci.,32:15181e15192 (2012). Hatters et al.,Trends Biochem.Sci.,31:445e454 (2006). Heffernan Aet al.,J.Mol.Neurosci.,60:316 (2016). Holtzman et al.,Proc.Natl.Acad.Sci.U.S.A.,97:2892e2897 (2000). Holtzman et al.,Cold Spring Harb.Perspect.Med.,2:a006312 (2012). Hudry et al.,Sci.Transl.Med.,5:212ra161 (2013). Johnson-Wood et al.,Proc.Natl.Acad.Sci.U.S.A.,94:1550e1555 (1997). Kells et al.,Proc.Natl.Acad.Sci.U.S.A.,106:2407e2411 (2009). Kim et al.,Neuron,63:287e303 (2009). Kim et al.,J.Neurosci.,31:18007e18012 (2011). Lambert et al.,Nat.Genet.,45:1452e1458 (2013). Lemere and Masliah,Nat.Rev.Neurol.,6:108e119 (2010). Li et al.,J.Biol.Chem.,287:44593e44601 (2012). Liu et al.,Nat.Rev.Neurol.,9:106e118 (2013). Manelli et al.,J.Mol.Neurosci.,23:235e246 (2004). Morris et al.,Ann.Neurol.,67:122e131 (2010). Ramanan et al.,Mol.Psychiatry,19:351e357 (2013). Rebeck et al.,Neuron,11:575e580 (1993). Reiman et al.,Proc.Natl.Acad.Sci.U.S.A.,106:6820e6825 (2009). Rosenberg et al.,Hum.Gene Ther.Clin.Dev.,29:24 (2018). Safieh et al.,BMC Medicine,17:64 (2019). Saunders et al.,Neurology,43:1467e1472 (1993). Schmechel et al.,Proc.Natl.Acad.Sci.U.S.A.,90:9649e9653 (1993). Schmued and Hopkins,Brain Res.,874:123 (2000). Sondhi et al.,Mol.Ther.,15:481e491 (2007). Sondhi et al.,Hum.Gene Ther.Method,23:324e335 (2012). Strittmatter et al.,Proc.Natl.Acad.Sci.U.S.A.,90:1977e1981 (1993). Sullivan et al.,Neurobiol.Aging,32:791e801 (2011). Sullivan et al.,J.Biol.Chem.,272:17972e17980 (1997). Suri et al.,Neurosci.Biobehav.Rev.,37:2878e2886 (2013). Talbot et al.,Lancet,343:1432e1433 (1994). Tsirka et al.,Proc.Natl.Acad.Sci.,94:9779 (1997). Walker et al., Acta Neuropathol., 100:36e42 (2000). Youmans et al., J. Biol. Chem., 287:41774e41786 (2012). Yu et al.,Annu.Rev.Neurosci.,37:79e100 (2014). Zhao et al.,J.Neurosci.,29:3603e3612 (2009). Zhao et al., Neurobiol Aging, 44:159 (2016). Zolotukhin et al., Methods, 28:158e167 (2002).

[0122] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described in connection with specific embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that some of the details described herein may be varied considerably without departing from the basic principles of the invention.

[0123] Sequence information SEQUENCE LISTING <110> Cornell University <120> GENE THERAPY FOR ALZHEIMER'S DISEASE <150> US 62 / 915,988 <151> 2019-10-16 <160> 32 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 1 gguggagcaa gcgguggagu u 21 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 2 ggaguugaag gccuacaaau u 21 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 3 ggaagacaug cagcgccagu u 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 4 gcgcgcggau ggaggagauu u 21 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 5 gtagcgacta aacacatcau u 21 <210> 6 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 6 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 ctgatggacg agaccatgaa ggagttgaag gcctacaaat cggaactgga ggaacaactg 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 7 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 7 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cgggctggcg cggagatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggac aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gccacaatca c 951 <210> 8 <211> 317 <212> PRT <213> Homo sapiens <400> 8 Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys 1 5 10 15 Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30 Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45 Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55 60 Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala 65 70 75 80 Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95 Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110 Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125 Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140 Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg 145 150 155 160 Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170 175 Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu 180 185 190 Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val 195 200 205 Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220 Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly 225 230 235 240 Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255 Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270 Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285 Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295 300 Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His 305 310 315 <210> 9 <211> 299 <212> PRT <213> Artificial Sequence <220> <223> A synthetic polypeptide <400> 9 Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln 1 5 10 15 Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg 20 25 30 Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln 35 40 45 Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met 50 55 60 Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu 65 70 75 80 Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu 85 90 95 Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Cys 100 105 110 Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln 115 120 125 Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu 130 135 140 Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Cys Leu Ala 145 150 155 160 Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala 165 170 175 Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala 180 185 190 Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln 195 200 205 Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg 210 215 220 Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg 225 230 235 240 Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala 245 250 255 Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu Asp Met 260 265 270 Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala Val Gly 275 280 285 Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His 290 295 <210> 10 <211> 299 <212> PRT <213> Artificial Sequence <220> <223> A synthetic polypeptide <400> 10 Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu Arg Gln 1 5 10 15 Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu Gly Arg 20 25 30 Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln Val Gln 35 40 45 Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala Leu Met 50 55 60 Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu Glu Glu 65 70 75 80 Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser Lys Glu 85 90 95 Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp Val Arg 100 105 110 Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu Gly Gln 115 120 125 Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu 130 135 140 Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala 145 150 155 160 Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu Ser Ala 165 170 175 Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val Arg Ala 180 185 190 Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg Ala Gln 195 200 205 Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly Ser Arg 210 215 220 Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu Val Arg 225 230 235 240 Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala Glu Ala 245 250 255 Phe Gln Ala Arg Leu Lys Ser on Phe Glu Pro Leu Val Glu Asp Met 260 265 270 Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala Val Gly 275 280 285 Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His 290,295 <210> 11 <211> 1098 <212> DNA <213> Homo sapiens <400> 11 ggaacttgat gctcagagag zgaagtcat ttgcccagg tcacacagct ggcaactggc 60 agagccagga ttcacgccct ggcaatttga ctccagaatc ctaccttaa cccagaagca 120 cggcttcaag cccctggaaa ccacaatacc tgtggcagcc agggggt gctggaatct 180 cattcacat gtggggagggg ggctcccctg tgtcaggt cacaaccaaa gaggaagctg 240 tgattaaaac ccaggtccca tttgcaaagc ctcgactttt agcaggtgca tcatactgtt 300 cccacccctc ccatcccact tctgtccagc cgcctagccc cactttcttt tttttctttt 360 tttgagacag tctccctctt gctgaggctg gagtgcagtg gcgagatctc ggctcactgt 420 aacctccgcc tcccgggttc aagcgattct cctgcctcag cctcccaagt agctaggatt 480 acaggcgccc gccaccacgc ctggctaact tttgtatttt tagtagagat ggggtttcac 540 catgttggcc aggctggtct caaactcctg accttaagtg attcgcccac tgtggcctcc 600 caaagtgctg ggattacagg cgtgagctac cgcccccagc ccctcccatc ccacttctgt 660 ccagccccct agccctactt tctttctggg atccaggagt ccagatcccc agccccctct 720 ccagattaca ttcatccagg cacaggaaag gacagggtca ggaaaggagg actctgggcg 780 gcagcctcca cattcccctt ccacgcttgg cccccagaat ggaggagggt gtctggatta 840 ctgggcgagg tgtcctccct tcctggggac tgtggggggt ggtcaaaaga cctctatgcc 900 ccacctcctt cctccctctg ccctgctgtg cctggggcag ggggagaaca gcccacctcg 960 tgactggggg ctggcccagc ccgccctatc cctgggggag ggggcgggac agggggagcc 1020 ctataattgg acaagtctgg gatccttgag tcctactcag ccccagcgga ggtgaaggac 1080 gtccttcccc aggagccg 1098 <210> 12 <211> 1157 <212> DNA <213> Homo sapiens <400> 12 ccccagcgga ggtgaaggac gtccttcccc aggagccgac tggccaatca caggcaggaa 60 gatgaaggtt ctgtgggctg cgttgctggt cacattcctg gcaggatgcc aggccaaggt 120 ggagcaagcg gtggagacag agccggagcc cgagctgcgc cagcagaccg agtggcagag 180 cggccagcgc tgggaactgg cactgggtcg cttttgggat tacctgcgct gggtgcagac 240 actgtctgag caggtgcagg aggagctgct cagctcccaa gtcacccaag aactgagggc 300 gctgatggac gagaccatga aggagttgaa ggcctacaaa tcggaactgg aggaacaact 360 gaccccggta gcggaggaga cgcgggcacg gctgtccaag gagctgcaga cggcgcaggc 420 ccggctgggc gcggacatgg aggacgtgtg cggccgcctg gtgcagtacc gcggcgaggt 480 gcaggccatg ctcggccaga gcaccgagga gctgcgggtg cgcctcgcct cccacctgcg 540 caagctgcgt aagcggctcc tccgcgatcc cgatgacctg cagaagcgcc tggcagtgta 600 ccaggccggg gcccgcgagg gcgccgagcg cggcctcagc gccatccgcg agcgcctggg 660 gcccctggtg gaacagggcc gcgtgcgggc cgccactgtg ggctccctgg ccggccagcc 720 gctacaggag cgggcccagg cctggggcga gcggctgcgc gcgcggatgg aggagatggg 780 cagtcggacc cgcgaccgcc tggacgaggt gaaggagcag gtggcggagg tgcgcgccaa 840 gctggaggag caggcccagc agatacgcct gcaggccgag gccttccagg cccgcctcaa 900 gagctggttc gagcccctgg tggaagacat gcagcgccag tgggccgggc tggtggagaa 960 ggtgcaggct gccgtgggca ccagcgccgc ccctgtgccc agcgacaatc actgaacgcc 1020 gaagcctgca gccatgcgac cccacgccac cccgtgcctc ctgcctccgc gcagcctgca 1080 gcgggagacc ctgtccccgc cccagccgtc ctcctggggt ggaccctagt ttaataaaga 1140 ttcaccaagt ttcacgc 1157 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 13 gaaagaactc aaagcttata agagcgagct ggagg 35 <210> 14 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <220> <221> misc_feature <222> (21)...(22) <223> n = A,U,C or G <400> 14 gcacaagcug gaguacaauu nn 22 <210> 15 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 15 aauuguacuc cagcuugugc cc 22 <210> 16 <211> 61 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 16 gcacaagcug gaguacaauu cuguaaagcc acagauggga auuguacucc agcuugugcu 60 u 61 <210> 17 <211> 86 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 17 cucgagugag cgaggcacaa gcuggaguac aauucuguaa agccacagau gggaauugua 60 cuccagcuug ugccccgccu acuagu 86 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <220> <221> misc_feature <222> (1)...(5) <223> n = A,T,C or G <220> <221> misc_feature <222> (28)...(32) <223> n = A,T,C or G <400> 18 nnnnngggca caagctggag tacaactnnn nn 32 <210> 19 <400> 19 000 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 20 gccgatgacc tgcagaagcu u 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 21 gcgcgcggat ggaggagatu u 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 22 gtaagcggct cctccgcgau u 21 <210> 23 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 23 ctggaggctt gctgaaggct gtatgctgat ttgtaggcct tcaactcctg ttttggccac 60 tgactgacag gagtgaggcc tacaaatcag gacacaggc ctgttactag cactcacatg 120 gaacaaatgg cc 132 <210> 24 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 24 ctggaggctt gctttgggct gtatgctgat ttgtaggcct tcaactcctg ttttggccac 60 tgactgacag gagttgaagt cacaaatcag gacacaagc cctttatcag cactcacatg 120 gaacaaatgg ccaccgtggg aggatgacaa 150 <210> 25 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 25 ctggaggctt gctttgggct gtatgctgtt ccgatttgta ggccttcaag ttttggccac 60 tgactgactt gaagtcacaa atcggaacag gacacaaggc cctttatcag cactcacatg 120 gaacaaatgg ccaccgtggg aggatgacaa 150 <210> 26 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 26 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 ctgatggacg agaccatgaa agaacttaaa gcatataaga gtgagctgga ggaacaactg 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 27 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 27 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cgggctggcg cggagatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggac aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 28 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 28 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 ctgatggacg agaccatgaa agaacttaaa gcttataaga gtgagctgga ggaacaactg 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 29 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 29 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cgggctggcg cggagatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggac aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 30 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 30 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 ctgatggacg agaccatgaa agaactcaaa gcttataaga gtgagctgga ggaacaactg 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 31 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 31 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cgggctggcg cggagatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggac aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 32 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide <400> 32 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120 ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180 ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240 ctgatggacg agaccatgaa agaacttaaa gcttataaga gcgagctgga ggaacaactg 300 accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360 cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420 caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480 aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagtgcct ggcagtgtac 540 caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600 cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660 ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720 agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780 ctggaggac aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951

Claims

1. a first promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding APOE2 and a 3' untranslated region; An isolated nucleotide sequence comprising one or more RNAi nucleic acid sequences for the inhibition of APOE4 mRNA; A gene therapy vector comprising:

2. The vector of claim 1 comprising the nucleotide sequence.

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

4. The vector of claim 2 , wherein the nucleotide sequences are inserted 5′ and 3′ to the open reading frame.

5. The vector of claim 1 , wherein the nucleotide sequences are on different vectors.

6. The vector of any one of claims 1 to 5, wherein the isolated nucleotide sequence comprises a second promoter operably 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 of claim 5 , wherein the different vector is a viral vector.

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

10. The vector of claim 9, wherein the AAV is AAV5, AAV9, or AAVrhlO.

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

12. The vector 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 a Pol I promoter.

14. The vector of claim 6 , wherein the second promoter is a Pol III promoter.

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

16. The vector of any one of claims 1 to 14, wherein the RNAi comprises an siRNA comprising multiple siRNA sequences.

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

6.

18. 18. The vector of claim 17, wherein the multiple silent nucleotide substitutions in the open reading frame of the APOE2 are absent in the RNAi nucleic acid sequence in the isolated nucleotide sequence.

19. 19. The vector of 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. 19. The vector of 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 of any one of claims 1 to 20, wherein the APOE4 to be inhibited has a sequence having at least 80%, 85%, 90%, 95% or more amino acid sequence identity to a polypeptide comprising SEQ ID NO:

10.

22. 22. The vector of 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 to the polypeptide encoded by SEQ ID NO:

11.

23. 23. The vector of 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 to one of SEQ ID NOs: 1-4 or complements thereof.

24. a first promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding human APOE2; An isolated nucleotide sequence having one or more RNAi nucleic acid sequences for the inhibition of human APOE4 mRNA. The vector of claim 1 , comprising:

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

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

27. 25. The vector of claim 24, wherein the nucleotide sequences are inserted 5' and 3' to the open reading frame.

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

29. A gene therapy vector according to any one of claims 1 to 28; Optionally, a pharmaceutically acceptable carrier A composition comprising:

30. 1. A method for preventing, inhibiting, or treating Alzheimer's disease in a mammal, comprising: administering to said mammal an effective amount of a composition comprising a gene therapy vector according to any one of claims 1 to 28 or a composition according to claim 29. A method comprising:

31. 1. A method for preventing, inhibiting, or treating a disease associated with APOE4 expression in a mammal, comprising: administering to said mammal an effective amount of a composition comprising a gene therapy vector according to any one of claims 1 to 28 or a composition according to claim 29. A method comprising:

32. 32. The method of claim 30 or 31, wherein the composition comprises a liposome containing the gene therapy vector or the different vector, or both.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

51. 46. ​​The method of any one of claims 30 to 45, wherein the vector comprises a Pol III promoter operably linked to the RNAi sequence.

52. 46. ​​The method of any one of claims 30 to 45, wherein the second vector comprises a Pol III promoter operably linked to the RNAi sequence.

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