Gene therapies for neurodegenerative disease

Abstract

To provide, in some aspects, compositions and methods for treatment of neurodegenerative disease such as Alzheimer's disease.SOLUTION: In some embodiments, the disclosure provides expression constructs comprising a transgene encoding an APOE protein isoform or a portion thereof, an inhibitory nucleic acid targeting an APOE gene or a portion thereof, or any combination of them. In some embodiments, the disclosure provides methods of treating Alzheimer's disease by administering an expression construct to a subject in need thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application No. 62 / 772,230, entitled "Gene Therapy for Neurodegenerative Diseases," filed November 28, 2018, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Alzheimer's disease (AD) is the most common form of dementia, affecting more than 5 million people in the United States alone. Alzheimer's disease is an irreversible, progressive brain disorder characterized by the presence of abnormal protein deposits throughout the brain that inhibit neuronal function, destroy connections between neurons, and ultimately lead to cell death. These deposits include plaques of amyloid-β and tangles formed by phosphorylated tau protein. Patients with mild AD experience memory loss, leading to wandering, difficulty handling money, repetitive questioning, and personality and behavioral changes. Patients with moderate AD exhibit increased memory loss, become confused and have difficulty recognizing friends and family, are unable to learn new things, and lead to hallucinations, delusions, and paranoia. Patients with severe AD are unable to communicate and are completely dependent on others for their care. Eventually, protein plaques and tangles spread throughout the brain, leading to significant tissue atrophy. Summary of the Invention

[0003] overview Most Alzheimer's disease (AD) patients have late-onset AD, with symptoms appearing in subjects in their mid-60s. The Apolipoprotein E (APOE) gene is involved in the development of late-onset AD. APOE has several isoforms, including APOE2, which is protective against AD, and APOE4, which is associated with an increased risk of developing late-onset AD. Homozygous patients who carry two copies of APOE4 (e.g., APOE4 + / + Subjects who are heterozygous (subjects who have one copy of APOE and one copy of either APOE2 or APOE3) are at greater risk of developing late-onset AD compared to heterozygous patients who carry one copy of APOE and one copy of either APOE2 or APOE3.

[0004] Aspects of the present disclosure relate to compositions and methods for treating subjects having or suspected of having AD. The present disclosure is based, in part, on expression constructs encoding inhibitory RNAs (e.g., shRNAs, miRNAs, amiRNAs, etc.) that target AD-associated genes (e.g., APOE, such as APOE4).

[0005] In some aspects, the present disclosure is based on an expression construct (e.g., a vector) encoding APOE2 (or a portion thereof), and optionally one or more additional gene products from AD-related genes (e.g., an inhibitory nucleic acid targeting APOE4). Without wishing to be bound by any particular theory, the combination of gene products described herein, when expressed in a subject, act together (e.g., synergistically) to reduce one or more signs and symptoms of AD. Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding an APOE2 protein, wherein the isolated nucleic acid comprises a sequence represented by SEQ ID NO:4.

[0006] In some embodiments, the present disclosure provides an isolated nucleic acid comprising an expression construct that encodes a transgene that expresses APOE2 and an inhibitory nucleic acid that inhibits the expression or activity of APOE4. In some embodiments, the expression construct is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, the ITRs are AAV2 ITRs.

[0007] In some embodiments, the inhibitory nucleic acid is complementary to at least 6 contiguous nucleotides of the sequence represented by SEQ ID NO: 1. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA comprising (or encoded by) a nucleic acid sequence represented by any one of SEQ ID NOs: 5-8, 12-15, and 17-20. In some embodiments, the inhibitory nucleic acid comprises (or is encoded by) a sequence represented by any one of SEQ ID NOs: 7, 8, 14, 15, 19, and 20.

[0008] In some embodiments, the transgene expressing APOE2 encodes a protein having an amino acid sequence represented by SEQ ID NO: 3. In some embodiments, the transgene expressing APOE2 comprises a codon-optimized nucleic acid sequence. In some embodiments, the codon-optimized nucleic acid sequence encoding APOE2 is represented by SEQ ID NO: 4. In some embodiments, the disclosure provides an isolated nucleic acid comprising a sequence represented by any one of SEQ ID NOs:11, 16, and 21.

[0009] In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding an APOE2 protein, wherein the isolated nucleic acid comprises a sequence represented by SEQ ID NO: 4. In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of APOE4. In some embodiments, the expression construct is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), where optionally the ITRs are AAV2 ITRs.

[0010] In some embodiments, the isolated nucleic acid further comprises one or more promoters. In some embodiments, the promoter is a chicken beta actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter. In some embodiments, the present disclosure provides a vector comprising the isolated nucleic acid described by the present disclosure.In some embodiments, the vector is a plasmid.In some embodiments, the vector is a viral vector.In some embodiments, the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector.

[0011] In some aspects, the disclosure provides compositions comprising the isolated nucleic acids or vectors described herein. In some embodiments, the disclosure provides host cells comprising the isolated nucleic acids or vectors described herein.

[0012] In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid protein; and an isolated nucleic acid or vector described herein. In some embodiments, the capsid protein can cross the blood-brain barrier. In some embodiments, the capsid protein is AAV9 capsid protein or AAVrh.10 capsid protein. In some embodiments, the rAAV transduces neural and / or non-neuronal cells of the central nervous system (CNS).

[0013] In some aspects, the disclosure provides a method of treating a subject having or suspected of having Alzheimer's disease, the method comprising administering to the subject an isolated nucleic acid, vector, composition, or rAAV described in this disclosure.

[0014] In some embodiments, administering comprises direct injection into the CNS of the subject. In some embodiments, direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, or any combination thereof. In some embodiments, direct injection into the CNS of the subject comprises convection-enhanced delivery (CED). In some embodiments, administering comprises peripheral injection. In some embodiments, peripheral injection is intravenous injection. In some embodiments, the subject is homozygous for APOE4 allele (e.g., APOE4+ / +). [Brief description of the drawings]

[0015] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 1 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector comprising an expression construct encoding an inhibitory RNA targeting APOE (e.g., APOE transcript variant 4 ApoE4). The inhibitory RNA is operably linked to the H1 promoter. [Diagram 2] 2 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector containing an expression construct encoding an inhibitory RNA targeting APOE (e.g., APOE transcript variant 4 ApoE4). The inhibitory RNA is located within an intron and operably linked to a promoter sequence. [Diagram 3] 3 is a schematic diagram showing one embodiment of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting APOE (for example, APOE transcript variant 4 ApoE4). The inhibitory RNA is located within an intron between the promoter sequence and the APOE2 protein coding sequence. [Figure 4] 4 is a schematic diagram showing one embodiment of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting APOE (for example, APOE transcript variant 4 ApoE4). The inhibitory RNA is located within an intron between the promoter sequence and the APOE2 protein coding sequence. [Diagram 5]5 is a schematic diagram showing one embodiment of a plasmid containing an rAAV vector containing an expression construct encoding an inhibitory RNA targeting APOE (for example, APOE transcript variant 4 ApoE4). The inhibitory RNA is located within an intron between the promoter sequence and the APOE2 protein coding sequence. [Figure 6] FIG. 6 is a schematic diagram showing an rAAV vector (top) that includes a "D" region located "outside" the ITRs (e.g., proximal to the end of the ITR relative to a transgene insert or expression construct), and a wild-type rAAV vector that has an ITR "inside" the vector (e.g., proximal to the transgene insert of the vector). [Figure 7A] Figures 7A and 7B show in vitro validation of rAAV vectors carrying different shRNAs against APOE4 and codon-optimized APOE2 coding sequences by qRT-PCR. Figure 7A shows that several candidate vectors successfully reduced endogenous APOE expression. [Figure 7B] FIG. 7B shows that the shRNAs expressed by these vectors do not affect the expression of codon-optimized APOE2. [Figure 8] FIG. 8 is a schematic diagram showing the experimental design for in vivo selection of different shRNAs against APOE4 and rAAV vectors carrying a codon-optimized APOE2 coding sequence using APOE4 knock-in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description The present disclosure is based in part on compositions and methods for expressing combinations of AD-related gene products in subjects.Gene products can be proteins, protein fragments (e.g., parts), interference nucleic acids that inhibit AD-related genes, etc. In some embodiments, gene products are proteins or protein fragments that are encoded by AD-related genes.In some embodiments, gene products are interference nucleic acids that inhibit AD-related genes (e.g., shRNA, siRNA, miRNA, amiRNA, etc.).

[0017] AD-related gene refers to a gene that encodes a gene product that is genetically, biochemically, or functionally related to Alzheimer's disease (AD).For example, individuals with at least one copy of APOE4 are at increased risk of developing late-onset AD.In another example, APOE2 shows neuroprotective effects in mouse models of AD.As used herein, the term "neuroprotection" refers to the preservation of the neurostructure and / or function of cells or subjects, compared to the preservation of the neurostructure and / or function of cells or subjects in the absence of neuroprotection (for example, the absence of neuroprotective agents or proteins).

[0018] Isolated Nucleic Acids and Vectors The isolated nucleic acid can be DNA or RNA. In some aspects, the present disclosure provides an isolated nucleic acid comprising an expression construct that encodes a transgene 4 encoding an APOE2 protein or a portion thereof and / or an inhibitory nucleic acid that targets APOE. Generally, the isolated nucleic acid described herein may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more inhibitory nucleic acids (e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, etc.). In some embodiments, the isolated nucleic acid encodes more than 10 inhibitory nucleic acids. In some embodiments, each of the one or more inhibitory nucleic acids targets a different gene or part of a gene (e.g., a first miRNA targets a first target sequence of a gene, and a second miRNA targets a second target sequence of a gene that is different from the first target sequence). In some embodiments, each of the one or more inhibitory nucleic acids targets the same target sequence of the same gene (e.g., the isolated nucleic acid encodes multiple copies of the same miRNA).

[0019] An aspect of the present disclosure relates to an isolated nucleic acid, including an expression construct encoding one or more interfering nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.) that target APOE4 protein (e.g., isoform E4 of the APOE gene). APOE protein refers to apolipoprotein E, which is a fat-binding protein that plays a role in the catabolism of triglyceride-rich lipoproteins. There are three major isoforms of APOE, designated APOE2, APOE3, and APOE4. Each isoform differs from the others at two positions, amino acid 130 and amino acid 176 (also designated positions 112 and 158, respectively, when the signal peptide of the protein is removed). APOE2 contains Cys130 / Cys176 and has been observed to be associated with type III hyperlipidemia and other diseases, but also plays a neuroprotective role. APOE3 contains Cys130 / Arg176 and is the most common APOE allele.

[0020] APOE4 contains Arg130 / Arg176 and has been observed to be associated with late-onset Alzheimer's disease, atherosclerosis, unfavorable outcomes in traumatic brain injury (TBI) and other diseases.In humans, the APOE gene is located on chromosome 19.In some embodiments, APOE4 is encoded by the nucleic acid sequence represented by SEQ ID NO:1 (for example, NCBI reference sequence NM_001302690.1).In some embodiments, APOE2 is encoded by the nucleic acid sequence represented by SEQ ID NO:2 (for example, NCBI reference sequence NM_000041.3).

[0021] An inhibitory nucleic acid targeting an APOE gene (e.g., APOE4) may include a region of complementarity (e.g., a region of the inhibitory nucleic acid that hybridizes to a target gene, e.g., a gene encoding APOE4) that is 6-50 nucleotides in length. In some embodiments, the inhibitory nucleic acid includes a region of complementarity to APOE that is about 6-30, about 8-20, or about 10-19 nucleotides in length. In some embodiments, the inhibitory nucleic acid is complementary to at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the APOE sequence. In some embodiments, the inhibitory nucleic acid targeting an APOE gene is non-allele specific (e.g., the inhibitory nucleic acid silences all isoforms of the APOE gene). In some embodiments, the inhibitory nucleic acid targets one or more specific alleles of APOE, such as one or more of APOE2, APOE3, and / or APOE4.

[0022] In some embodiments, the gene product (e.g., a transgene encoding APOE2) is encoded by a coding portion (e.g., a cDNA) of a naturally occurring gene. In some embodiments, the gene product is a protein (or a fragment thereof) encoded by the APOE2 isoform of the APOE gene. In some embodiments, the APOE2 gene comprises a nucleic acid sequence represented by SEQ ID NO:3. In some embodiments, the gene product is an inhibitory nucleic acid that targets (e.g., includes a region that hybridizes to or has complementarity with) an AD-related gene (e.g., the APOE4 isoform of the APOE gene). Those skilled in the art will recognize that the order of expression of a first gene product (e.g., APOE2) and a second gene product (e.g., an inhibitory RNA that targets the APOE4 isoform of the APOE gene) can generally be reversed (e.g., the inhibitory RNA is the first gene product and APOE2 is the second gene product). In some embodiments, the gene product is a fragment (e.g., a portion) of the APOE gene. The protein fragment may comprise about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the protein encoded by the APOE gene. In some embodiments, the protein fragment comprises 50% to 99.9% (e.g., any value between 50% and 99.9%) of the protein having the amino acid sequence represented by SEQ ID NO:3. In some embodiments, the gene product (e.g., inhibitory RNA) hybridizes to a portion of the target gene (e.g., is complementary to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more consecutive nucleotides of the target gene, e.g., the APOE4 isoform of APOE, such as the sequence represented by SEQ ID NO:1).

[0023] In some embodiments, the expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein that includes a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (e.g., the expression construct encodes two different gene products, e.g., two different proteins or protein fragments).

[0024] A polycistronic expression vector can include one or more (e.g., 1, 2, 3, 4, 5, or more) promoters. Any suitable promoter can be used, such as a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS-specific promoter), etc. In some embodiments, the promoter is a chicken beta-actin promoter (CBA promoter), a CAG promoter (e.g., as described in Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi: 10.1186 / 1471-2121-9-2), a CD68 promoter, or a JeT promoter (e.g., as described in Tornoee et al. (2002) Gene 297(1-2):21-32). In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a first gene product, a second gene product, or a first and a second gene product. In some embodiments, the expression cassette contains one or more additional regulatory sequences, including, but not limited to, transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences, repressor binding sites, or any combination thereof.

[0025] In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). Examples of IRES sites are described, for example, in Mokrejs et al. (2006) Nucleic Acids Res. 34(Database issue):D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, and those described in Liu et al. (2017) Sci Rep. 7: 2193. In some embodiments, the self-cleaving peptide is a T2A peptide.

[0026] In some embodiments, disorders such as AD are associated with the expression of at least one copy of APOE4.Thus, in some embodiments, the isolated nucleic acid described herein comprises an inhibitory nucleic acid that reduces or prevents the expression of APOE4 (e.g., APOE).The sequence encoding the inhibitory nucleic acid can be placed in the untranslated region (e.g., intron, 5'UTR, 3'UTR, etc.) of the expression vector.

[0027] In some embodiments, the inhibitory nucleic acid is placed in an intron of the expression construct, e.g., an intron upstream of a sequence encoding a first gene product. The inhibitory nucleic acid can be a double-stranded RNA (dsRNA), shRNA, siRNA, microRNA (miRNA), artificial miRNA (amiRNA), or RNA aptamer. In general, the inhibitory nucleic acid binds (e.g., hybridizes) to about 6 to about 30 (e.g., any integer between 6 and 30) consecutive nucleotides of a target RNA (e.g., mRNA). In some embodiments, the inhibitory nucleic acid molecule is an miRNA or amiRNA, e.g., an miRNA that targets the APOE4 isoform of APOE (the gene encoding the APOE4 protein). In some embodiments, the miRNA does not contain any mismatches with the region of the APOE mRNA to which it hybridizes (e.g., the miRNA is "complete"). In some embodiments, the inhibitory nucleic acid is an shRNA (e.g., an shRNA that targets APOE), e.g., one represented by any one of SEQ ID NOs: 7, 14, and 19. In some embodiments, the miRNA contains at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mismatch with the region of the APOE mRNA to which it hybridizes.

[0028] In some embodiments, the inhibitory nucleic acid is an artificial microRNA (amiRNA). MicroRNA (miRNA) typically refers to small non-coding RNA found in plants and animals, which functions in transcriptional and post-translational regulation of gene expression. miRNA is transcribed by RNA polymerase to form a hairpin loop structure called pri-miRNA; this is then processed by enzymes (Drosha, Pasha, spliceosome, etc.) to form a pre-miRNA hairpin structure, which is then processed by Dicer to form the miRNA / miRNA * Double strand ( *indicates the passenger strand of the miRNA duplex, one strand of which is then incorporated into the RNA-induced silencing complex (RISC). In some embodiments, the inhibitory RNA described herein is a miRNA that targets the APOE4 isoform of APOE (the gene encoding the APOE4 protein).

[0029] In some embodiments, the inhibitory nucleic acid targeting APOE (e.g., the APOE4 isoform of APOE) is an miRNA / miRNA * In some embodiments, the miRNA / miRNA * The miRNA strand of the duplex comprises or consists of a sequence or a portion thereof set forth in any one of SEQ ID NOs: 5, 6, 12, 13, 17, and 18. In some embodiments, the miRNA / miRNA * Double-stranded miRNA * The strand comprises or consists of a sequence represented by any one of SEQ ID NOs: 5, 6, 12, 13, 17, and 18.

[0030] Artificial microRNAs (amiRNAs) are derived by modifying natural miRNAs to replace the natural targeting region of the pre-mRNA with the targeting region of interest. For example, a naturally expressed miRNA can be used as a scaffold or backbone (e.g., a pri-miRNA scaffold), with the stem sequence replaced by that of the miRNA targeting the gene of interest. Artificial precursor microRNAs (pre-amiRNAs) are usually processed to preferentially generate a single stable small RNA. In some embodiments, the rAAV vectors and rAAVs described herein contain a nucleic acid encoding an amiRNA. In some embodiments, the pri-miRNA scaffold of the amiRNA is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451. In some embodiments, the amiRNA comprises a nucleic acid sequence targeting APOE (e.g., the APOE4 isoform of APOE) and an eSIBR amiRNA scaffold, e.g., as described in Fowler et al. Nucleic Acids Res. 2016 Mar 18; 44(5): e48.

[0031] In some embodiments, the amiRNA targeting APOE (eg, the APOE4 isoform of APOE) comprises or consists of a sequence represented by any one of SEQ ID NOs: 8, 15, and 20. The isolated nucleic acid described herein may exist by itself or as part of a vector. Generally, the vector is a plasmid, a cosmid, a phagemid, a bacterial artificial chromosome (BAC), or a viral vector (e.g., an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a baculovirus vector, etc.). In some embodiments, the vector is a plasmid (e.g., a plasmid containing an isolated nucleic acid described herein). In some embodiments, the vector is a recombinant AAV (rAAV) vector. The rAAV may comprise either the "plus strand" or the "minus strand" of the rAAV vector. In some embodiments, the rAAV vector is single-stranded (e.g., single-stranded DNA). In some embodiments, the vector is a baculovirus vector (e.g., an Autographa californica nuclear polyhedrosis (AcNPV) vector).

[0032] Typically, a rAAV vector comprises a transgene (e.g., an expression construct comprising one or more of each of the following: promoter, intron, enhancer sequence, protein coding sequence, inhibitory RNA coding sequence, polyA tail sequence, etc.) flanked by two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the transgene of the rAAV vector comprises an isolated nucleic acid described in this disclosure. In some embodiments, each of the two ITR sequences of the rAAV vector is a full-length ITR (e.g., about 145 bp in length and including a functional Rep binding site (RBS) and terminal resolution site (trs)). In some embodiments, one of the ITRs of the rAAV vector is truncated (e.g., shortened or not full-length). In some embodiments, the truncated ITR lacks a functional terminal resolution site (trs) and is used for the generation of self-complementary AAV vectors (scAAV vectors). In some embodiments, the truncated ITR is a ΔITR, e.g., as described in McCarty et al. (2003) Gene Ther. 10(26):2112-8.

[0033] Aspects of the present disclosure relate to isolated nucleic acids (e.g., rAAV vectors) that include an ITR that has one or more modifications (e.g., additions, deletions, substitutions, etc. of nucleic acids) compared to a wild-type AAV ITR, e.g., compared to a wild-type AAV2 ITR (e.g., SEQ ID NO: 25). The structure of the wild-type AAV2 ITR is shown in FIG. 6. Generally, wild-type ITRs include a 125 nucleotide region that self-anneals to form a palindromic double-stranded T-shaped hairpin structure consisting of two cross arms (formed by sequences designated B / B' and C / C', respectively), a longer stem region (formed by sequences A / A'), and a single-stranded terminal region designated the "D" region (FIG. 6). Generally, the "D" region of the ITR is located between the stem region formed by the A / A' sequences and the transgene-containing insert of the rAAV vector (e.g., located "inside" the ITR relative to the end of the ITR or proximal to the transgene insert or expression construct of the rAAV vector). In some embodiments, the "D" region comprises the sequence set forth in SEQ ID NO: 23. The "D" region has been observed to play an important role in encapsidation of rAAV vectors by capsid proteins, e.g., as disclosed in Ling et al. (2015) J Mol Genet Med 9(3).

[0034] This disclosure is based, in part, on the surprising discovery that rAAV vectors containing a "D" region located "outside" the ITRs (e.g., closer to the end of the ITR relative to a transgene insert or expression construct) are more efficiently encapsidated by AAV capsid proteins than rAAV vectors with unmodified (e.g., wild-type) ITRs. In some embodiments, rAAV vectors with modified "D" regions have reduced toxicity compared to rAAV vectors with wild-type ITR sequences.

[0035] In some embodiments, the modified "D" sequence contains at least one nucleotide substitution compared to the wild-type "D" sequence (e.g., SEQ ID NO:23). The modified "D" sequence can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide substitutions compared to the wild-type "D" sequence (e.g., SEQ ID NO:23). In some embodiments, the modified "D" sequence contains at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotide substitutions compared to the wild-type "D" sequence (e.g., SEQ ID NO:23). In some embodiments, the modified "D" sequence is about 10% to about 99% (e.g., 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) identical to the wild-type "D" sequence (e.g., SEQ ID NO: 23). In some embodiments, the modified "D" sequence comprises the sequence set forth in SEQ ID NO: 22, also referred to as the "S" sequence, as described in Wang et al. (1995) J Mol Biol 250(5):573-80.

[0036] An isolated nucleic acid or rAAV vector described in this disclosure can further comprise a "TRY" sequence, e.g., as set forth in SEQ ID NO:24, or as described in Francois, et al. 2005. J Virol The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element. In some embodiments, the TRY sequence is located between the ITR (e.g., 5'ITR) of the isolated nucleic acid or rAAV vector and the expression construct (e.g., an insert encoding a transgene).

[0037] In some aspects, the present disclosure relates to a baculovirus vector, comprising the isolated nucleic acid or rAAV vector described in the present disclosure.In some embodiments, the baculovirus vector is an Autographa californica nuclear polyhedrosis (AcNPV) vector, for example, as described in Urabe et al. (2002) Hum Gene Ther 13(16):1935-43 and Smith et al. (2009) Mol Ther 17(11):1888-1896.

[0038] In some aspects, the present disclosure provides a host cell comprising the isolated nucleic acid or vector described herein.The host cell can be a prokaryotic cell or a eukaryotic cell.For example, the host cell can be a mammalian cell, a bacterial cell, a yeast cell, an insect cell, etc.In some embodiments, the host cell is a mammalian cell, for example, a HEK293T cell.In some embodiments, the host cell is a bacterial cell, for example, an E. coli cell.

[0039] rAAV In some aspects, the present disclosure relates to recombinant AAV (rAAV) comprising a transgene encoding a nucleic acid described herein (e.g., an rAAV vector described herein). The term "rAAV" generally refers to a viral particle comprising a rAAV vector encapsidated by one or more AAV capsid proteins. The rAAV described in this disclosure may comprise a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10. In some embodiments, the rAAV comprises a capsid protein from a non-human host, e.g., a rhesus AAV capsid protein such as AAVrh.10, AAVrh.39. In some embodiments, the rAAV described herein comprises a capsid protein that is a variant of a wild-type capsid protein, e.g., a capsid protein variant that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) relative to the wild-type AAV capsid protein from which it is derived.

[0040] In some embodiments, the rAAV described in this disclosure spreads easily through the CNS, especially when introduced into the CSF space or directly into the brain parenchyma.Thus, in some embodiments, the rAAV described in this disclosure comprises a capsid protein that can cross the blood-brain barrier (BBB).For example, in some embodiments, the rAAV comprises a capsid protein with AAV9 or AAVrh.10 serotype.The generation of rAAV is described, for example, in Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7): 698-706.

[0041] In some embodiments, the rAAV described in this disclosure (e.g., comprising a recombinant rAAV genome that is encapsidated by AAV capsid proteins to form rAAV capsid particles) is produced in a baculovirus vector expression system (BEVS). The production of rAAV using BEVS is described, for example, in Urabe et al. (2002) Hum Gene Ther 13(16):1935-43, Smith et al. (2009) Mol Ther 17(11):1888-1896, U.S. Patent No. 8,945,918, U.S. Patent No. 9,879,282, and International PCT Publication WO 2017 / 184879. However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).

[0042] Pharmaceutical Compositions In some aspects, the present disclosure provides a pharmaceutical composition comprising the isolated nucleic acid or rAAV described herein and a pharma- ceutically acceptable carrier.As used herein, the term "pharma-ceutically acceptable" refers to a material, such as a carrier or diluent, that does not destroy the biological activity or properties of a compound and is relatively non-toxic, e.g., the material may be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained.

[0043] As used herein, the term "pharmaceutical acceptable carrier" refers to a pharmaceutical acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which is associated with carrying or transporting a compound useful within the present invention into or to a patient so that it can perform its intended function. Additional components that can be included in the pharmaceutical composition used in the practice of the present invention are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA); which is incorporated herein by reference.

[0044] The compositions (e.g., pharmaceutical compositions) provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual; intratracheal instillation, bronchial instillation, and / or inhalation; and / or as oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), topical administration via the blood and / or lymphatic supply, and / or direct administration to the affected site. Generally, the most appropriate route of administration will depend on various factors, such as the properties of the agent (e.g., its stability in the environment of the gastrointestinal tract) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of a subject.

[0045] method The present disclosure is based in part on a composition for expressing a combination of AD-related gene products in a subject, which act together (e.g., synergistically) to treat Alzheimer's disease.As used herein, "treat" or "treating" refers to (a) preventing or delaying the onset of Alzheimer's disease; (b) reducing the severity of Alzheimer's disease; (c) reducing or preventing the onset of symptoms characteristic of Alzheimer's disease; (d) and / or preventing the deterioration of symptoms characteristic of Alzheimer's disease.Alzheimer's disease symptoms include, for example, cognitive dysfunction (e.g., dementia, hallucinations, memory loss, etc.), motor dysfunction (e.g., difficulty performing daily tasks, etc.), and emotional and behavioral dysfunction.

[0046] Thus, in some aspects, the disclosure provides a method of treating a subject having or suspected of having Alzheimer's disease, the method comprising administering to the subject a composition described in the disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV).

[0047] The subject is typically a mammal, such as a human, dog, cat, pig, hamster, rat, mouse, etc. In some embodiments, the subject is a human. In some embodiments, the subject is characterized by an APOE4 allele. The subject is homozygous for APOE4 (e.g., APOE4 + / + ) or heterozygous. In some embodiments, the subject is heterozygous for APOE4 and the subject's second APOE allele is selected from APOE2 and APOE3.

[0048] In some embodiments, the composition is administered directly to the CNS of a subject, for example, by direct injection into the brain and / or spinal cord of a subject.Examples of CNS direct administration methods include, but are not limited to, intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the above.In some embodiments, direct injection into the CNS of a subject results in transgene expression (e.g., expression of the first gene product, the second gene product, and if applicable, the third gene product) in the midbrain, striatum, and / or cerebral cortex of the subject.In some embodiments, direct injection into the CNS results in transgene expression (e.g., expression of the first gene product, the second gene product, and if applicable, the third gene product) in the spinal cord and / or CSF of a subject.

[0049] In some embodiments, direct injection into the CNS of the subject comprises convection-enhanced delivery (CED).Convection-enhanced delivery is a therapeutic strategy that comprises surgical exposure of the brain and placing a small-diameter catheter directly into the target area of ​​the brain, followed by injecting a therapeutic agent (for example, the composition described herein or rAAV) directly into the brain of the subject.CED is described, for example, in Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27. In some embodiments, the composition is administered to the subject's periphery, for example by peripheral injection.Examples of peripheral injection include subcutaneous injection, intravenous injection, intraarterial injection, intraperitoneal injection, or any combination thereof.In some embodiments, the peripheral injection is intraarterial injection, for example injection into the carotid artery of the subject.

[0050] In some embodiments, the composition described in this disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) is administered both peripherally and directly to the CNS of a subject. For example, in some embodiments, the subject is administered the composition by intra-arterial injection (e.g., injection into the carotid artery) and intraparenchymal injection (e.g., intraparenchymal injection by CED). In some embodiments, the direct injection into the CNS and the peripheral injection are simultaneous (e.g., occur simultaneously). In some embodiments, the direct injection is performed before the peripheral injection (e.g., between 1 minute and 1 week, or more). In some embodiments, the direct injection is performed after the peripheral injection (e.g., between 1 minute and 1 week, or more).

[0051] The amount of a composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) administered to a subject will vary depending on the method of administration. For example, in some embodiments, the rAAV described herein is administered to a subject at a dose of about 10 9 genome copies (GC) / kg and approximately 10 14 Between GC / kg (e.g., about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11 GC / kg, approx. 10 12 GC / kg, approx. 10 12 GC / kg, or approximately 10 14 In some embodiments, the subject is administered a high titer (e.g., >10 12 The rAAV (genome copies GC / kg) is administered by injection into the CSF space or by intraparenchymal injection.

[0052] A composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) can be administered to a subject one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more). In some embodiments, the composition is administered to a subject continuously (e.g., chronically), e.g., via an infusion pump. EXAMPLES

[0053] example Example 1: Nucleic Acids and rAAV Vectors This example describes an isolated nucleic acid (e.g., a vector, such as an rAAV vector, and an rAAV containing the isolated nucleic acid) that includes a transgene encoding an APOE2 protein or a portion thereof and / or an inhibitory nucleic acid that targets APOE4. In some embodiments, the constructs described in this example are useful for treating a subject having or suspected of having Alzheimer's disease (AD) that has at least one copy of the APOE4 isoform. In some embodiments, the subject is homozygous for an APOE4 isoform (e.g., APO44 + / + ).

[0054] The isolated nucleic acid encoding shRNA is specifically utilized to knock down the expression of APOE4 isoform both in vitro and in vivo.In some embodiments, shRNA is non-allele specific, for example, they can also knock down the expression of other APOE isoforms (for example, E2, E3, or E4). An isolated nucleic acid encoding a transgene encoding APOE2 is utilized to overexpress APOE2. The APOE2 transgene is codon-optimized to be sufficiently different from the endogenous APOE2 sequence in the cell so that it is not recognized by shRNAs targeting wild-type APOE, regardless of isoform.

[0055] The shRNA and the transgene may be operably linked to the same promoter or to another promoter. The shRNA is expressed under another promoter, typically a Pol III promoter (e.g., H1 promoter), or a Pol II promoter (e.g., CBA, T7, etc.). In general, the shRNA is operably linked to a Pol II promoter located in an intron sequence upstream of the open reading frame containing the codon-optimized APOE2 transgene. Examples of expression constructs described by the present disclosure are shown in Figures 1-5 and Table 1 below.

[0056] Recombinant adeno-associated viruses (rAAV) containing the isolated nucleic acid are produced using cells such as HEK293 cells for triple plasmid transfection. ITR sequences typically flank an expression construct that includes one or more of the following: at least one promoter / enhancer element, a 3' polyA signal, and a post-translational signal such as a WPRE element. Multiple gene products, such as the APOE2 isoform of APOE and one or more inhibitory nucleic acids (e.g., inhibitory nucleic acids targeting the APOE4 isoform of APOE), are expressed simultaneously. The presence of a short intron sequence that is efficiently spliced ​​upstream of the expressed gene can improve expression levels. shRNAs and other regulatory RNAs can potentially be included within these sequences.

[0057] Table 1 [Table 1]

[0058] Example 2: APOE4 + / + Cell-based assays for viral transduction of cells The cells are obtained, for example fibroblasts, monocytes, or hES cells from AD patients, or patient-derived induced pluripotent stem cells (iPSCs). These cells accumulate proteinaceous plaques containing the amyloid-β protein and tangles containing twisted strands of the protein tau.

[0059] Using such cell models, neurodegenerative features associated with AD are quantified in terms of accumulation of protein aggregates such as plaques and tangles, for example, utilizing α-amyloid-β or α-phosphorylated tau antibodies, followed by imaging using fluorescence microscopy. Imaging of neurodegenerative features associated with AD by ICC of protein markers such as amyloid-β, phosphorylated tau, or APOE4 is also performed. Western blotting, ELISA, and / or qPCR are used to quantify APOE4 expression levels in these cells.

[0060] Therapeutic endpoints (e.g., reduction in pathology associated with AD) will be measured in the context of rAAV transduction expression, activity and function will be confirmed and quantified. Amyloid-β and phosphorylated tau levels will also be quantified using Western blotting, ELISA, and / or qPCR.

[0061] Example 3: In vivo assay using mice expressing human APOE4 This example describes the in vivo assay of rAAV using mutant mice. The in vivo study of the above rAAV in mutant mice is performed using the assays described by, for example, Liao et al. (2018) J. Clin. Invest 128(5): 2144-2155; Rosenberg, et al. (2018) Hum Gene Ther Clin Dev 29(1): 24-47; Zhao et al. (2016) Neurobiol Aging 44: 159-172. These mutant mice carry the human APOE4 isoform at the mouse APOE locus. Vehicle control and rAAV (e.g., 2 × 10 11 Intrathecal or intraventricular delivery of 100 mg / mouse (dose of 100 mg / vg / mouse) is performed using concentrated rAAV stocks, e.g., in an injection volume of 5-10 µL. Intraparenchymal delivery via convection-enhanced delivery is performed. Treatment is initiated either prior to or following the onset of symptoms. The endpoints measured are the levels of APOE4 and APOE2 expression in the CNS and CSF.

[0062] Example 4: In vivo assay of mouse models of AD This example describes an in vivo assay of rAAV using mutant mice. In vivo studies of the above rAAV in mutant mice are performed using assays described, for example, by Liao et al. (2018) J. Clin. Invest 128(5): 2144-2155; Rosenberg, et al. (2018) Hum Gene Ther Clin Dev 29(1): 24-47; Zhao et al. (2016) Neurobiol Aging 44: 159-172. These mutant mice carry the human APOE4 isoform at the mouse APOE locus. In some cases, to model the development of amyloid-β plaques in human AD, these mice also express mutant human amyloid precursor protein (APP), mutant human presenilin 1 (PS1) protein, and / or mutant human presenilin 2 (PS2) protein.

[0063] Vehicle control and rAAV (e.g., 2 × 10 11 Intrathecal or intraventricular delivery of 100 mg / mouse (dose of 100 mg / vg / mouse) is performed using concentrated rAAV stocks, e.g., in an injection volume of 5-10 µL. Intraparenchymal delivery by convection-enhanced delivery is performed. Peripheral delivery is achieved by tail vein injection. Treatment is initiated either prior to or following the onset of symptoms. Endpoints measured are levels of APOE4 and APOE2 expression in the CNS and CSF, Aβ expression, and β-blockers. 42 These include accumulation of longer amyloid-β (Aβ) species such as sarcoidosis, an increase in all Aβ species, motor and cognitive endpoints, and accumulation of amyloid-β plaques and tau tangles.

[0064] Example 5: Clinical trial of AD patients This example describes a clinical trial to assess the safety and efficacy of the rAAV described by this disclosure in patients with AD. Clinical trials of the disclosed rAAV for the treatment of AD will be conducted using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39. The rAAV will be delivered into the CSF, intraparenchyma, to the hippocampus, or to another brain region, or peripherally. Endpoints measured are amyloid-β plaque levels, tau tangles, motor and cognitive endpoints, and APOE4 and APOE2 protein levels.

[0065] Example 6: Clinical trial in AD patients in combination with amyloid-β antibodies This example describes a clinical trial to assess the safety and efficacy of the rAAV described by this disclosure utilized in combination with an amyloid-β antibody (e.g., bapineuzumab and solanezumab) in patients with AD. Clinical trials of the disclosed rAAV in combination with anti-amyloid-β antibodies for the treatment of AD will be conducted using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39. The rAAV will be delivered to the CSF, intraparenchyma, to the hippocampus, or to another brain region, or peripherally.

[0066] In some embodiments, the rAAV of the present disclosure synergizes with anti-amyloid-β antibody to reduce the likelihood that AD patients will develop amyloid-associated imaging abnormalities (ARIA), which are highly correlated with APOE genotype.ARIA is a series of abnormalities observed in AD patients, specifically related to amyloid-modifying therapy with human monoclonal antibodies.There are two types of ARIA: ARIA-E, which refers to cerebral edema, and ARIA-H, which refers to cerebral microbleeds.

[0067] The endpoints to be evaluated are pre- and post-treatment brain imaging to determine whether ARIA occurs, and whether the rAAV of the present disclosure reduces the likelihood of ARIA, levels of amyloid-β plaques, tau tangles, motor and cognitive endpoints, and levels of APOE4 and APOE2 proteins.

[0068] Example 7: APOE4 + / + , APOE4 + / - , and APOE4 - / - Clinical trials in patients with AD This example shows APOE4 + / - or APOE4 - / - APOE4 compared with patients who + / + Clinical trials are described to assess the efficacy of the rAAV described by this disclosure in improving patients with AD, who are at increased risk for other pathologies, including stroke, coronary artery disease, atherosclerosis, poor recovery from head trauma, and cognitive recovery from surgery with bypass devices.

[0069] AD Treatment and APOE4 + / + Clinical trials of the rAAV of the present disclosure for amelioration of the increased risk of other conditions associated with patients with RA will be conducted using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39. The rAAV will be delivered to the CSF, intraparenchyma, to the hippocampus, or to another brain region, or peripherally. Endpoints assessed before and after treatment with the rAAV of the present disclosure include blood pressure, blood cholesterol and blood glucose levels, motor and cognitive endpoints, MRI, PET, and ultrasound imaging of the coronary arteries, recovery from cognitive trauma, and recovery from surgery with bypass devices.

[0070] Example 8: Prevention of AD or treatment of AD in patient carriers of the APOE4 isoform This example describes a clinical trial to assess the efficacy of the rAAV described by the present disclosure in reducing the risk of developing AD in subjects with at least one APOE4 isoform, and in treating AD in patients with at least one APOE4 isoform. + / + or APOE4 + / - It can be either of the following. Clinical trials of the disclosed rAAV for the prevention or treatment of AD in carriers of the APOE4 allele will be conducted using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39. The rAAV will be delivered to the CSF, intraparenchyma, to the hippocampus, or to another brain region, or peripherally. Endpoints assessed before and after treatment with the rAAV of the present disclosure are APOE4 and APOE2 levels in the CSF and blood, as well as cognitive and motor endpoints.

[0071] Example 9: Testing ITR "D" sequence configuration and cell transduction The effect of ITR "D" sequence placement on rAAV cell transduction is investigated. HEK293 cells are transduced with rAAV encoding ApoE2 with 1) wild-type ITR (e.g., the "D" sequence proximal to the transgene insert and distal to the end of the ITR) or 2) an ITR containing a "D" located "outside" the vector (e.g., the "D" sequence proximal to the end of the ITR and distal to the transgene insert), as shown in FIG.

[0072] Example 9: In vitro validation of shRNAs for endogenous APOE silencing and APOE2 overexpression Multiple plasmids containing unique shRNAs against APOE and codon-optimized coding sequences for APOE2 were evaluated in an in vitro transfection screen to assess the degree of APOE (e.g., APOE4) knockdown and heterologous expression of APOE2. Plasmids were specifically designed to selectively knockdown endogenous APOE genes without affecting expression of the vector-encoded APOE2 protein. Multiple plasmids demonstrate reduction of endogenous APOE (Figure 7A) and expression of codon-optimized APOE2 (Figure 7B) via qRT-PCR. shRNA candidates demonstrated significant reduction of endogenous APOE without affecting codon-optimized APOE2.

[0073] Example 10: In vivo validation of shRNAs for endogenous APOE silencing and APOE2 overexpression shRNA candidates that demonstrate a significant reduction of endogenous APOE without affecting codon-optimized APOE2 are selected for further in vivo studies. APOE4 knock-in (KI) mouse model is used to evaluate the in vivo efficacy of candidate shRNAs against APOE4. In APOE4 KI mice, both mouse Apoe alleles are replaced with human APOE-ε4. Mice (n=5) receive vectors carrying candidate shRNAs against APOE4 via intracerebroventricular injection (ICV), and biodistribution of human APOE4 mRNA is analyzed 60 days after injection (Figure 8).

[0074] Equivalent Having thus described several aspects of at least one embodiment of this invention, it should be understood that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

[0075] Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0076] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The term "and / or" as used in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Unless expressly indicated to the contrary, optionally other elements may also be present, whether related to the specifically identified elements or not, other than the elements specifically identified in the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B" when used in conjunction with open-ended language such as "comprising" can, in one embodiment, refer to A without B (optionally including elements other than B); in another embodiment, refer to B without A (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so forth.

[0077] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of, and also including a plurality of, a number or list of elements, and optionally including additional items not listed. When terms to the contrary are clearly indicated, such as "only one" or "exactly one," or when used in the claims, "consisting of" refers to including exactly one of, a number or list of elements. In general, the term "or" as used herein, when preceded by terms of exclusivity, such as "either," "one," or "only one," or "exactly one," should be interpreted as referring to exclusive alternatives (i.e., one or the other, but not both). When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0078] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements; but not necessarily including at least one of every respective element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") means, in one embodiment, at least one, optionally more than one, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one, B, and no A (optionally including elements other than A); in yet another embodiment, at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements); and so forth.

[0079] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, are to be understood as open-ended, i.e., to mean inclusive without limitation. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure. The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, indicate a priority, precedence, or order of one claim element over another, or the temporal order in which acts of a method are performed, but is used solely as a label to distinguish a given claim element having a particular name from another element of the same name (but differing only in ordinal terms) to distinguish between the claim elements. It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes one or more steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are presented.

[0080] array In some embodiments, an expression cassette encoding one or more gene products (e.g., a first, second and / or third gene product) comprises or consists of a sequence represented by any one of SEQ ID NOs: 1-21. In some embodiments, a gene product is encoded by a portion (e.g., a fragment) of a sequence represented by any one of SEQ ID NOs: 1-21. One skilled in the art will appreciate that a nucleic acid sequence encoding an inhibitory nucleic acid may describe a sequence in which all "T"s are replaced by "U".

[0081] > Human APOE4 nucleic acid sequence (NM_001302690.1) (SEQ ID NO: 1) [Table 2-1] [Table 2-2]

[0082] >Human APOE2 nucleic acid sequence (NM_000041.3) (SEQ ID NO:2) [Table 3]

[0083] > Human ApoE2 amino acid sequence (SEQ ID NO:3) [Table 4-1] [Table 4-2]

[0084] >ApoE2 nucleic acid sequence (codon optimized) (SEQ ID NO: 4) [Table 5]

[0085] >ApoE4 shRNA1 nucleic acid sequence (SEQ ID NO:5) [Table 6] >ApoE4 shRNA1 nucleic acid sequence (SEQ ID NO:6) [Table 7] >ApoE4 shRNA1 with loop (SEQ ID NO: 7) [Table 8] >ApoE4 amiRNA1 (SEQ ID NO:8) [Table 9] >ApoE4 amiRNA rAAV vector H1 (SEQ ID NO: 9) [Table 10-1] [Table 10-2] >ApoE4 amiRNA rAAV vector 1 (SEQ ID NO: 10) [Table 11] >ApoE4 amiRNA-ApoE2opt rAAV vector 1 (SEQ ID NO: 11) [Table 12-1] [Table 12-2] >ApoE4 shRNA2 nucleic acid sequence (SEQ ID NO: 12) [Table 13] >ApoE4 shRNA2 nucleic acid sequence (SEQ ID NO: 13) [Table 14] >ApoE4 shRNA2 with loop (SEQ ID NO: 14) [Table 15] >ApoE4 amiRNA2 (SEQ ID NO: 15) [Table 16] >ApoE4 amiRNA-ApoE2opt rAAV vector 2 (SEQ ID NO: 16) [Table 17-1] [Table 17-2] >ApoE4 shRNA3 nucleic acid sequence (SEQ ID NO: 17) [Table 18] >ApoE4 shRNA3 nucleic acid sequence (SEQ ID NO: 18) [Table 19] >ApoE4 shRNA3 with loop (SEQ ID NO: 19) [Table 20] >ApoE4 amiRNA3 (SEQ ID NO: 20) [Table 21] >ApoE4 amiRNA-ApoE2opt rAAV vector 3 (SEQ ID NO: 21) [Table 22-1] [Table 22-2] >AAV2 ITR D region "S" sequence (SEQ ID NO:22) [Table 23] >AAV2 ITR D region "D" sequence (SEQ ID NO:23) [Table 24] >TRY motif sequence (SEQ ID NO: 24) [Table 25] >Wild type AAV2 ITR nucleic acid sequence (SEQ ID NO: 25) [Table 26]

Claims

1. An isolated nucleic acid comprising an expression construct encoding a transgene that expresses APOE2 and an inhibitory nucleic acid that inhibits the expression or activity of APOE4.

2. 2. The isolated nucleic acid of claim 1, wherein the inhibitory nucleic acid is encoded by a sequence represented by any one of SEQ ID NOs: 5-8, 12-15, and 17-20.

3. 3. The isolated nucleic acid of claim 1 or 2, wherein the inhibitory nucleic acid is encoded by a sequence represented by any one of SEQ ID NOs: 7, 8, 14, 15, 19, and 20.

4. The isolated nucleic acid according to any one of claims 1 to 3, wherein the transgene expressing APOE2 encodes a protein having the amino acid sequence represented by SEQ ID NO:

3.

5. 5. The isolated nucleic acid of any one of claims 1 to 4, wherein the transgene expressing APOE2 comprises a codon-optimized nucleic acid sequence, optionally wherein the nucleic acid sequence is represented by SEQ ID NO:

4.

6. 6. The isolated nucleic acid of any one of claims 1 to 5, wherein the expression construct is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).

7. The isolated nucleic acid of claim 6 , wherein the ITR is an AAV2 ITR.

8. The isolated nucleic acid according to any one of claims 1 to 7, wherein the isolated nucleic acid comprises a sequence represented by any one of SEQ ID NOs: 11, 16, and 21.

9. An isolated nucleic acid comprising an expression construct encoding an APOE2 protein, wherein the isolated nucleic acid comprises the sequence represented by SEQ ID NO:

4.

10. An isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of APOE4.

11. 11. The isolated nucleic acid of claim 9 or 10, wherein the expression construct is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), optionally wherein the ITRs are AAV2 ITRs.

12. 12. The isolated nucleic acid of any one of claims 1 to 11, further comprising one or more promoters, optionally each of the one or more promoters independently is a chicken beta actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter.

13. A vector comprising the isolated nucleic acid of any one of claims 1 to 12.

14. The vector of claim 13 , wherein the vector is a plasmid.

15. 14. The vector of claim 13, wherein the vector is a viral vector, optionally wherein the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector.

16. A composition comprising an isolated nucleic acid according to any one of claims 1 to 12, or a vector according to any one of claims 13 to 15.

17. A host cell comprising an isolated nucleic acid according to any one of claims 1 to 12, or a vector according to any one of claims 13 to 15.

18. (i) a capsid protein; and (ii) An isolated nucleic acid according to any one of claims 1 to 12, or a vector according to claim 15. A recombinant adeno-associated virus (rAAV).

19. 19. The rAAV of claim 18, wherein the capsid protein is capable of crossing the blood-brain barrier, and optionally the capsid protein is an AAV9 capsid protein or an AAVrh.10 capsid protein.

20. 20. The rAAV of claim 18 or claim 19, wherein the rAAV transduces neuronal and non-neuronal cells of the central nervous system (CNS).

21. A method for treating a subject having or suspected of having Alzheimer's disease comprising administering to the subject an isolated nucleic acid according to any one of claims 1 to 12, a vector according to any one of claims 13 to 15, a composition according to claim 16, or an rAAV according to any one of claims 18 to 20.

22. 22. The method of claim 21, wherein the administration comprises direct injection into the CNS of the subject, optionally wherein the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, or any combination thereof.

23. 23. The method of claim 22, wherein direct injection into the CNS of the subject comprises convection-enhanced delivery (CED).

24. 24. The method of any one of claims 21 to 23, wherein the administration comprises a peripheral injection, optionally wherein the peripheral injection is an intravenous injection.

25. The method of any one of claims 21 to 24, wherein the subject is homozygous for the APOE4 allele.

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