APOE gene therapy

Abstract

To provide a gene therapy vector comprising an expression cassette coding for a mammalian apolipoprotein E (APOE); a pharmaceutical composition; and a method for preventing, inhibiting or treating Alzheimer's disease in mammals.SOLUTION: The invention provides a gene therapy vector comprising an expression cassette coding for a mammalian apolipoprotein E that has a residue other than arginine at at least one of positions 112, 136 or 158, but is not a mammalian apolipoprotein E that has R112, R136 and R158 or a mammalian apolipoprotein E that has C112, R136 and C158, or coding for an antibody that binds to APOE4 or disrupts the binding of APOE to heparan sulfate proteoglycans.SELECTED DRAWING: None

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 / 939999, filed November 25, 2019, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] background Alzheimer's disease (AD) directly affects 5 million Americans and is rapidly increasing in both prevalence and economic impact. Existing medications have little effect on the underlying disease course, and no preventive therapies are currently available. While inheritance of the APOE4 variant confers a high risk for developing AD, 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. Previous studies have shown that adeno-associated virus (AAV) gene delivery of the human APOE2 coding sequence into the CNS of a mouse model expressing human APOE4 reduces the amount of amyloid-β peptide and amyloid burden (Zhao et al. 2016). In humans, the odds ratio for developing AD for the E4 / E4 homozygous genotype is 14.9, which is reduced to 2.6 for E2 / E4 heterozygotes. Recent reports suggest that APOE4, in addition to its role in promoting amyloid production, is associated with abnormal brain function. Summary of the Invention

[0003] overview The present disclosure provides a gene therapy vector comprising an expression cassette encoding a mammalian apolipoprotein E (APOE) that is protective or includes a substitution, e.g., relative to APOE4, in a region that binds to a lipoprotein receptor, e.g., LDLR, or that binds to heparan sulfate proteoglycan (HSPG), e.g., in the region between positions 135 and 151. In one embodiment, the mammalian APOE in the gene therapy vector has a residue other than arginine, histidine, or lysine at at least one of positions 112, 136, or 158. In one embodiment, the mammalian APOE in the gene therapy vector has residues other than arginine at positions 112 and 136. In one embodiment, the mammalian APOE in the gene therapy vector has residues other than arginine at positions 136 and 158. In one embodiment, the mammalian APOE in the gene therapy vector has residues other than arginine at positions 112, 136, and 158. In one embodiment, the apolipoprotein E is human apolipoprotein E. In one embodiment, the residue other than arginine is serine, leucine, valine, glycine, isoleucine, alanine, threonine, asparagine, cysteine, or methionine. In one embodiment, the residue other than arginine is serine, threonine, asparagine, cysteine, or glutamine. In one embodiment, position 112 has cysteine, methionine, valine, threonine, alanine, serine, arginine, glycine, or isoleucine. In one embodiment, position 112 has cysteine. In one embodiment, position 136 has threonine, cysteine, methionine, arginine, or serine. In one embodiment, position 136 has serine. In one embodiment, position 158 has cysteine, methionine, valine, threonine, alanine, serine, arginine, glycine, or isoleucine. In one embodiment, position 158 has an arginine or a cysteine. In one embodiment, two of positions 112, 136, or 158 have arginines. In one embodiment, position 112 does not have an arginine. In one embodiment, position 136 does not have an arginine. In one embodiment, position 158 does not have an arginine.In one embodiment, the mammalian APOE in the gene therapy vector has a Cys at position 112, a Ser at position 136, and a Cys at position 158. In one embodiment, the mammalian APOE in the gene therapy vector has a Cys at position 112, a Ser at position 136, and an Arg at position 158. In one embodiment, the gene therapy vector is a viral gene therapy vector. In one embodiment, the gene therapy vector is an adenovirus, adeno-associated virus (AAV), retrovirus, or lentivirus vector. In one embodiment, the gene therapy vector is a rAAV vector. In one embodiment, the AAV vector is pseudotyped with, for example, AAVrh.10, AAV8, AAV9, AAV5, AAVhu.37, AAVhu.20, AAVhu.43, AAVhu.8, AAVhu.2, or AAV7 capsid. In one embodiment, the AAV vector is pseudotyped with AAVrh.10, AAV8, or AAV5. In one embodiment, the AAV is AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh.10. In one embodiment, the gene therapy vector further comprises a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibiting APOE4 mRNA. In one embodiment, a second gene therapy vector comprises a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibiting APOE4 mRNA. 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 vector is a plasmid. In one embodiment, the mammalian apolipoprotein gene in the vector has C112, S136, and R158, or C112, S136, and C158. In one embodiment, the second gene therapy vector comprises a nucleotide sequence having a nucleic acid sequence encoding an anti-APOE4 or anti-heparan antibody.

[0004] Also provided is a pharmaceutical composition comprising a gene therapy vector. In one embodiment, the vector is a viral vector. In one embodiment, the vector is an rAAV vector. In one embodiment, the amount of vector in the composition is about 1 x 10 11 ~Approx. 1×10 16 In one embodiment, the amount of vector in the composition is about 1 x 10 genome copies. 12 ~Approx. 1×10 15 In one embodiment, the amount of vector in the composition is about 1 x 10 genome copies. 11 ~Approx. 1×10 13 In one embodiment, the amount of vector in the composition is about 1 x 10 genome copies. 13 ~Approx. 1×10 15 In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0005] Further provided are methods for preventing, inhibiting, or treating Alzheimer's disease, comprising preventing or treating one or more symptoms thereof, e.g., dementia, or other tauopathy or cognitive impairment, in a mammal. In one embodiment, prior to administration, the mammal has or is at risk for increased amyloid plaques and / or increased tau in the brain, e.g., compared to an age-matched mammal or a mammal at low risk for Alzheimer's disease or other tauopathy or cognitive impairment. In one embodiment, the mammal has a cognitive impairment. The method includes 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 vector. In one embodiment, the composition comprises nanoparticles comprising a nucleic acid. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is an E3 / E4 heterozygote. In one embodiment, the mammal is an E2 / E3 heterozygote. In one embodiment, the mammal is a human. In one embodiment, the human is an adult, e.g., 20 years of age or older, e.g., at least 40, 50, or 60 years of age. In one embodiment, the human is a young person, e.g., 10-20 years of age. In one embodiment, the human is a child, e.g., under 10 years of age. In one embodiment, the composition is administered to the human at birth or within 1, 2, 3, 4, or 5 years after birth. 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 administered by injection. 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, administration of the gene therapy vector is effective to prevent or inhibit amyloid plaque accumulation. In one embodiment, administration of the gene therapy vector is effective to prevent or inhibit tau pathology. In one embodiment, administration of the gene therapy vector is effective to prevent or inhibit the accumulation of amyloid plaques and to prevent or inhibit tau pathology.

[0006] Additionally, methods are provided for preventing, inhibiting, or treating diseases associated with APOE4 expression in a mammal. The methods include 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 vector. In one embodiment, the composition comprises a nanoparticle comprising a nucleic acid. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is an E3 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is a human. 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.

[0007] A method of reducing binding to heparin sulfate proteoglycans in a mammal is provided, 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 vector. In one embodiment, the composition comprises a nanoparticle comprising a nucleic acid. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is an E3 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is a human. 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.

[0008] In one embodiment, the composition is administered via a catheter. In one embodiment, the composition is administered intraventricularly. In one embodiment, the composition is administered intracranially. In one embodiment, the composition is administered in the lumbar region. In one embodiment, the composition is administered into the cisterna magna. In one embodiment, the composition is administered to the brain via a burr hole. In one embodiment, the composition is administered below C1-C2.

[0009] In one embodiment, the composition is administered so that the expression of the encoded gene product, e.g., modified APOE or an antibody that binds heparan, is similar to the expression level of APOE4 in the mammal. In one embodiment, the ratio of the gene product encoded by the secreted vector to the secreted APOE4 in the mammal is 1:1. In one embodiment, the ratio of the gene product encoded by the secreted vector to the secreted APOE4 in the mammal is 2:1. In one embodiment, the ratio of the gene product encoded by the secreted vector to the secreted APOE4 in the mammal is 0.5:1. In one embodiment, the ratio of the gene product encoded by the secreted vector to the secreted APOE4 in the mammal is 0.1:1. In one embodiment, the ratio of the gene product encoded by the secreted vector to the secreted APOE4 in the mammal is 3:1. In one embodiment, the level of expression of the encoded gene product in the mammal prevents or inhibits cognitive decline (worsening). In one embodiment, the level of expression of the encoded gene product in the mammal reduces tau tangles and amyloid accumulation.

[0010]

[0010] Methods for preventing, inhibiting, or treating a lipid disorder in a mammal are provided, 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 vector. In one embodiment, the composition comprises a nanoparticle comprising a nucleic acid. In one embodiment, the mammal is an E2 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is an E3 / E4 heterozygote. In one embodiment, the mammal is an E4 / E4 homozygote. In one embodiment, the mammal is a human. 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.

[0011] In one embodiment, the gene therapy vector comprises an AAV expression vector encoding a modified human APOE gene as described herein and an artificial miRNA targeting endogenous APOE4 in either cis or trans. This vector system silences the harmful expression of endogenous APOE4 in combination with supplementation of a beneficial APOE gene from a gene therapy vector, e.g., an AAV vector. Exemplary microRNA sequences are incorporated into the CAG promoter intron or polyA tail of a vector transgene plasmid encoding a human APOE coding sequence. Alternatively, the microRNA may be inserted between a Pol III promoter, e.g., a U6 promoter, and the terminator following the polyA site of the APOE expression cassette. The human APOE DNA sequence derived from the vector contains silent nucleotide changes to ensure that it is not suppressed by the microRNA and 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 into an AAV capsid of a serotype (e.g., AAV9, but could be other vectors) that targets astrocytes and glial cells, the predominant sites of endogenous APOE expression in the CNS.

[0012] The vectors described herein may be used to prevent, inhibit, or treat cognitive impairment, dementia, Alzheimer's disease (e.g., autosomal dominant, late-onset, or early-onset). In one embodiment, a mammal has a reduced fibrillar amyloid beta plaque burden, reduced levels of paired helical filament tau, or decreased neurodegeneration after administration of the vector. In one embodiment, the vectors described herein may be used to prevent, inhibit, or treat, for example, cognitive impairment associated with traumatic brain injury, stroke, transient ischemic attack, dementia, Creutzfeldt-Jakob disease, multiple sclerosis, prion disease, Pick's disease, corticobasal degeneration, Parkinson's disease, dementia with Lewy bodies, progressive supranuclear palsy; dementia pugilistica (chronic traumatic encephalopathy); frontotemporal dementia, and chromosome 17-linked parkinsonism; Ritiko-Bodig disease; neurofibrillary tangle-predominant dementia; gangliogliomas and gangliocytomas; meningioangiomatosis; subacute sclerosing panencephalitis; lead encephalopathy, tuberculous sclerosis, Hallervorden-Spatz syndrome, and lipofuscinosis; argyrophilic grain disease; or frontotemporal lobar degeneration.

[0013] The vectors described herein may also be used to prevent, inhibit, or treat one or more symptoms associated with hyperlipoproteinemia, e.g., Type I, Type 2, Type 3, Type 4, or Type 5. In one embodiment, the administered amount of the vector reduces circulating cholesterol, e.g., reduces VLDL and / or LDL, reduces intermediate density lipoproteins, reduces plasma levels of cholesterol and / or triglycerides, reduces triglyceride containing VLDL, reduces lipid accumulation in glomerular capillaries, reduces intraglomerular lipoproteins, or reduces thrombus formation. [The present invention 1001] An expression cassette encoding a mammalian apolipoprotein E having a residue other than arginine at at least one of positions 112, 136, or 158, but which is not a mammalian apolipoprotein E having R112, R136, and R158, or a mammalian apolipoprotein E having C112, R136, and C158, or encoding an antibody that binds to APOE4 or disrupts the binding of APOE to heparan sulfate proteoglycans. A gene therapy vector comprising: [The present invention 1002] 1001. The gene therapy vector of the present invention, wherein said apolipoprotein E is human apolipoprotein E. [The present invention 1003] The gene therapy vector of the present invention 1001 or 1002, wherein the residue other than arginine in the apolipoprotein E is serine, threonine, asparagine, cysteine, or glutamine. [The present invention 1004] 1004. The gene therapy vector of any one of claims 1001 to 1003, wherein position 112 of the apolipoprotein E has cysteine. [The present invention 1005] The gene therapy vector of any one of claims 1001 to 1004, wherein position 136 of the apolipoprotein E has arginine or serine. [The present invention 1006] 1006. The gene therapy vector of any one of claims 1001 to 1005, wherein position 158 of the apolipoprotein E has arginine or cysteine. [The present invention 1007] A gene therapy vector according to any one of claims 1001 to 1003, wherein two of positions 112, 136 and 158 of said apolipoprotein E have arginine. [The present invention 1008] A gene therapy vector according to any one of claims 1001 to 1003, wherein position 112 of said apolipoprotein E does not have arginine. [The present invention 1009] A gene therapy vector according to any one of claims 1001 to 1003, wherein said apolipoprotein E does not have arginine at position 136. [The present invention 1010] A gene therapy vector according to any one of claims 1001 to 1003, wherein said apolipoprotein E does not have arginine at position 158. [The present invention 1011] The gene therapy vector of any one of 1001 to 1010, which is a viral gene therapy vector. [The present invention 1012] The gene therapy vector of the present invention 1011, which is an adenoviral, adeno-associated viral (AAV), retroviral, or lentiviral vector. [The present invention 1013] The gene therapy vector of the present invention, wherein the viral gene therapy vector is an rAAV vector. [The present invention 1014] The gene therapy vector of the present invention 1013, wherein the AAV vector is pseudotyped. [The present invention 1015] 1014. The gene therapy vector of the present invention, wherein the AAV vector is pseudotyped with an AAVrh.10, AAV8, AAV9, AAV5, AAVhu.37, AAVhu.20, AAVhu.43, AAVhu.8, AAVhu.2, or AAV7 capsid. [The present invention 1016] 1015. The gene therapy vector of the present invention, wherein said AAV vector is pseudotyped with AAVrh.10, AAV8, or AAV5. [The present invention 1017] The gene therapy vector of any of claims 1013 to 1016, wherein the AAV vector is AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh.10. [The present invention 1018] A gene therapy vector according to any one of claims 1001 to 1017, further comprising a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibiting APOE4 mRNA. [The present invention 1019] The gene therapy vector of the present invention, wherein the nucleotide sequence is linked to a second promoter. [The present invention 1020] 1019. The gene therapy vector of the present invention, wherein the second promoter is a PolIII promoter. [The present invention 1021] The gene therapy vector of any one of claims 1018 to 1020, wherein the RNAi comprises a miRNA comprising multiple miRNA sequences. [The present invention 1022] 1022. The gene therapy vector of any one of claims 1018 to 1021, wherein the RNAi comprises an siRNA comprising multiple siRNA sequences. [The present invention 1023] A pharmaceutical composition comprising any one of the gene therapy vectors of the present inventions 1001 to 1022. [The present invention 1024] The pharmaceutical composition of the present invention 1023, wherein the vector is a viral vector. [The present invention 1025] The pharmaceutical composition of the present invention 1024, wherein the vector is an rAAV vector. [The present invention 1026] The amount of the vector is about 1×10 11 ~Approx. 1×10 16 The pharmaceutical composition of the present invention 1024 or 1025, which is a genome copy. [The present invention 1027] 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 present inventions 1001 to 1022. [The present invention 1028] A method for preventing or inhibiting cognitive decline in a mammal, comprising administering to the mammal an effective amount of a composition comprising any one of the gene therapy vectors of present inventions 1001 to 1022. [The present invention 1029] 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 present inventions 1001 to 1022. [The present invention 1030] A method for preventing, inhibiting, or treating a lipid disorder in a mammal, comprising administering to the mammal an effective amount of a composition comprising any one of the gene therapy vectors of present inventions 1001 to 1022. [The present invention 1031] 1031. The method of claim 1027, 1028, 1029, or 1030, wherein the mammal is an E2 / E4 heterozygote, an E4 / E4 homozygote, or an E3 / E4 heterozygote. [The present invention 1032] The method of any one of claims 1027 to 1031, wherein the mammal is a human. [The present invention 1033] 1032. The method of any one of claims 1027 to 1031, wherein the composition is administered systemically. [The present invention 1034] 1033. The method of any one of claims 1027 to 1032, wherein the composition is injected. [This invention 1035] The method of any one of claims 1027 to 1032 or 1034, wherein the composition is administered to the central nervous system. [The present invention 1036] The method of any one of claims 1027 to 1032 or 1034, wherein the composition is administered to the brain. [This invention 1037] The method of any one of claims 1027 to 1036, wherein a catheter is used to administer the composition. [The present invention 1038] The method of any one of claims 1027 to 1037, wherein the composition is a sustained-release composition. [This invention 1039] The vector is Apolipoprotein E having C112, S136, and R158 or having C112, S136, and C158 The method of any one of claims 1027 to 1038, wherein the [The present invention 1040] 1039. The method of any of claims 1027 to 1038, wherein the vector comprises a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibiting APOE4 mRNA. [This invention 1041] 1039. The method of any of claims 1027 to 1038, wherein said mammal is further administered a second composition comprising a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibition of APOE4 mRNA. [The present invention 1042] 1042. The method of claim 1041, wherein said second composition comprises a liposome. [This invention 1043] 1042. The method of claim 1041, wherein said second composition comprises nanoparticles. [This invention 1044] Any of the methods of claims 1027 to 1038, wherein the mammal is further administered an anti-heparan antibody, or an antibody that binds to APOE4 or disrupts the binding of APOE to heparan sulfate proteoglycans, or a second composition comprising a nucleotide sequence encoding said antibody. [This invention 1045] 104. The method of claim 1044, wherein a viral vector comprises said nucleotide sequence encoding said antibody. [The present invention 1046] The method of any one of claims 1041 to 1045, wherein said second composition is administered systemically. [This invention 1047] The method of any one of claims 1041 to 1045, wherein said second composition is administered topically. [This invention 1048] The vector of any one of claims 1027 to 1047, wherein the vector is an AAV vector. [Brief explanation of the drawings]

[0014] [Figure 1] APOE alleles, prevalence, and associated risk of developing Alzheimer's disease. [Figure 2] Exemplary viral gene therapy. [Figure 3] 1 is an exemplary administration route for gene therapy. [Figure 4] Selected amino acid residues for different APOE alleles, prevalence, and associated risk of developing Alzheimer's disease. [Figure 5] Nucleotide residues at two positions in different APOE alleles. [Figure 6] APOE3ch mutation. [Figure 7] This is the effect of the APOE3ch mutation. [Figure 8] Affinity of APOE3ch for heparin. [Figure 9] Possible mechanisms for the binding of APOE or tau to HSPGs. [Figure 10] 1 is an exemplary gene therapy approach for preventing, inhibiting, or treating diseases associated with the expression of certain APOE alleles. [Figure 11] Association of APOE alleles with overt type III hyperlipoproteinemia and lipoprotein glomerulopathy. [Figure 12] AAV-mediated gene therapy for APOE AD reduces risk variants. Based on the finding that APOE4 homozygotes have a 15- to 20-fold increased risk for AD ( Reiman et al., 2020 , Corder et al., 1993 , Hefferman et al., 2016 ), we used AAV vectors to deliver coding sequences for APOE2 and APOE3 variants, along with the Christchurch variant, to reduce risk for AD. [Figure 13]APOE variants, risk for AD, and proposed gene therapy to protect against APOE4. APOE2, 3, and 4 are common variants (Hefferman et al., 2016; ALXFORUM, 2010). APOE3ChC is a variant superimposed on PSEN1-E290A in a case report of a Christchurch mutation on an APOE3 background that protects against the dominant PSEN1-E290A variant, which causes early-onset AD19. APOE3ChC and APOE2ChC variants may be therapeutics to reduce the high risk of AD in APOE4 homozygotes. The APOE2ChC variant, although not observed naturally, may be more effective than APOE3ChC. *Low risk in a PSEN1-E290A background. [Figure 14] This study supports the relative effects of APOE3ChC in females with the PSEN1-E280 mutation. Quantitative data are shown for mean cortical amyloid plaque burden (Pittsburgh Compound 8, PET), inferior temporal cortex PHF tau burden (Florataucipir PET), hippocampal volume (MRI), and precuneus glucose metabolism (Fludeoxyglucose PET). Black dots represent PSEN1 carriers with typical early-onset MCI, gray dots represent PSEN1 carriers who have not yet developed MCI, and PSEN1 carriers with APOE3ChC variants (red dots with arrows). Figure modified from Arboleda-Velasquez et al. (2019). [Figure 15] The Christchurch mutation impairs heparin binding of APOE. ELISA was used to quantify differences in the NaCl elution patterns of different APOE isoforms from a heparin column. Figure adapted from Arboleda-Velasquez et al. (2019). Arrows indicate subjects with APOEChC variants. [Figure 16]The AAVrh.10hAPOE2 vector expresses the human APOE2 transgene behind a constitutive CAG promoter consisting of a cytomegalovirus (CMV) enhancer, chicken β-actin promoter, splice donor and intron, and rabbit β-globin splice acceptor, followed by a rabbit β-globin polyadenylation signal (An). The hAPOE2 expression cassette is flanked by AAV2 inverted terminal repeats (ITRs) and packaged into a rhesus adeno-associated virus vector serotype 10 (AAVrh.10) capsid. All APOE vectors used in this proposal are identical except for the APOE coding sequence. The AAVrh.10Null vector is identical, except that the expression cassette AAVrh.10hAPOE2 is replaced by a non-translatable sequence. [Figure 17] Using AAVrh.10hAPOE2 as an example, an SDS polyacrylamide gel electrophoresis is shown, which shows three bands associated with the three AAV vector capsid proteins that match the expected molecular weights. [Figure 18] Assessment of APOE2 protein distribution in the non-human primate brain after intracisternal administration of AAVrh.10APOE2. NHPs administered AAVrh.10hAPOE2 (5 x 1013 gc, APOE2 transgene) were evaluated 8 weeks after treatment. The right hemisphere was sectioned into 1 cm3 cubes and analyzed for APOE2 protein levels by ELISA. A. Human APOE2 protein in brain sections. B. CSF (10 μl / time point) was sampled from three NHPs at three time points: pre-treatment (day 0), 28 days, and 56 days post-treatment, and analyzed by SOS-PAGE followed by antibody detection of APOE2 by Western blotting. See Rosenberg et al. (2018). [Figure 19]Figures 19A and 19B show the resolution of amyloid pathology in a PDAPP mouse model. AAVrh.10hAPOE2 or control AAVrh.10mCherry (1010gc) was administered bilaterally to the hippocampus of 9-month-old PDAPP mice. Eight weeks after administration, the mice were sacrificed, and the left hemisphere hippocampus was dissected, homogenized, and sequentially extracted with RIPA (representing soluble Aβ) and 5.5 M guanidine (representing insoluble Aβ). Aβ1-42 and Aβ1-40 levels were determined by ELISA. (A) Tissue levels of Aβ1-42 and (B) tissue levels of Aβ1-40 in the hippocampus of PDAPP mice injected with AAVrh.10hAPOE2 or AAVrh.10mCherry (n = 10-12 animals per group). Data are presented as mean ± SD. *p<0.05, ***p<0.001. See Zhao et al. (2016). [Figure 20] Figures 20A and 20B show the dose-dependent effect of intrahippocampal delivery of APOE2 on AP levels in the hippocampus of 2.5-month-old APP.PS1 / TRE4 mice. AAVrh.10hAPOE2 (0.25 × 10 10 , 0.5 × 10 10 , or 1 × 10 10 gc) or AAVrh.10mCherry (1 × 10 10 gc) was injected bilaterally into the hippocampus of 2.5-month-old male APP.PS1 / TRE4 mice. Eight weeks after administration, the mice were sacrificed. The left hemisphere hippocampus was dissected, homogenized, and sequentially extracted with RIPA (representing soluble Aβ) and 5.5 M guanidine (representing insoluble Aβ). Aβ1-42 levels in each extract were determined by ELISA. (A) Insoluble Aβ1-42 levels (guanidine-extractable) in the hippocampus. (B) Soluble Aβ1-42 levels in the hippocampus (RIPA extractable). n = 3-5 animals per group. Data are presented as mean ± SD. *p<0.05, ***p<0.001. Zhao et al. (2016). [Figure 21]Figures 21A and 21B show intrathalamic delivery of AAV9-hAPOE2 on APOE and Aβ levels in the brain of APP.PS1 / TRE4 mice. Ten-week-old mice were administered AAV9-hAPOE2 or AAV9-GFP by bilateral intrathalamic injection (1010gc) and sacrificed 8 weeks later. The thalamus, hippocampus, and cerebral cortex of the left hemisphere were dissected and homogenized. (A) APOE and (B) Aβ1-42 levels were quantified by ELISA. Adapted from Zhao et al. [Figure 22] In vitro APOE knockdown. U87 human astrocytoma cells were transfected with APOE-targeting siRNA or non-targeting (NT) control (5 pmol). Cells were harvested 72 hours later, and endogenous APOE mRNA levels were quantified by RT-qPCR. Based on a comparison of multiple siRNA design algorithms, four different siRNAs targeting the APOE coding sequence were generated. The identified sequences were as follows: GGUGGAGCAAGCGGUGGAGuu (SEQ ID NO: 20), GGAGUUGAAGGCCUACAAAuu (SEQ ID NO: 21), GGAAGACAUGCAGCGCCAGuu (SEQ ID NO: 22), and GCGCGCGGAUGGAGGAGAUuu (SEQ ID NO: 23). [Figure 23]Figures 23A and 23B show the hAPOE2-mirAPOE4 expression cassette. (A) Schematic diagram of mirAPOE4. An siRNA targeting sequence (guide strand) and an antisense sequence (passenger strand) with mismatches selected to facilitate processing are incorporated into an enhanced mir155 backbone. (B) Schematic diagram of the AAV9-hAPOE2-mirAPOE4 expression construct. The construct contains the human APOE2 coding sequence fused to a hemagglutinin tag (HA) behind a constitutive CAG promoter (cytomegalovirus (CMV) enhancer, chicken β-actin promoter, splice donor and intron, and rabbit β-globin splice acceptor), followed by a rabbit β-globin polyadenylation (polyA) signal. The mirAPOE4 tandem repeat is inserted either within the CAG intron and / or between the transgene stop codon and the polyA sequence. The hAPOE2 expression cassette is flanked by AAV2 inverted terminal repeats (ITRs) and packaged into AAV9 capsids. A miR from siRNA#2, for example, CTGGAGGCTTGCTGAAGGCTGTATGCTGATTTGTAGGCCTTCAACTCCT GTTTTGGCCACTGACTGACAGGAGTGAGGCCTACAAATCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 24), CTGGAGGCTTGCTTTGGGCTGTATGCTGATTTGTAGGCCTTCAACTCCTGTTTTGGCCACTGACTGACAGGAGTTGAAGTCACAAATCAGGACACAAGGCCCTTTATCAGCACTCACATGGAACAAATGGCCACCGTGGGAGGATGACAA (SEQ ID NO: 25), or CTGGAGGCTTGCTTTGGGCTGTATGCTGTTCCGATTTGTAGGCCTTCAAGTTTTGGCCACTGACTGACTTGAAGTCACAAATCGGAACAGGACACAAGGCCCTTTATCAGCACTCACATGGAACAAATGGCCACCGTGGGAGGATGACAA (SEQ ID NO: 26) may also be used. [Figure 24]The knockdown efficiency of intron- and polyA-located miRs is tested. [Figure 25] Luciferase provides knockdown efficiency. [Figure 26] APOE target site only controls are tested. [Figure 27] Identify miRNA target sites in vector-derived APOE2 with silent nucleotide changes. [Figure 28] Generate pAAV-miRNA-APOE-HA. [Figure 29] This is an in vivo test. [Figure 30] Luciferase expression in the control. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description In the following description, reference is made to the accompanying drawings, which form a part hereof, and which are shown for the purpose of illustrating specific embodiments that may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the invention, it being understood that other embodiments may be utilized and that logical changes may be made without departing from the scope of the present invention. Therefore, the following description of exemplary embodiments is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

[0016] 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.

[0017] As used herein, "transduction," "transfection," "transformation," or "transducing" refers to the process of introducing an exogenous polynucleotide into a host cell, resulting in the 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.

[0018] "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.

[0019] "Gene transfer" refers to the introduction of an exogenous polynucleotide into a cell, which can include targeting, binding, uptake, transport, localization, and replicon integration, but is distinct from and does not imply subsequent expression of a gene.

[0020] "Gene expression" or "expression" refers to the processes of transcription, translation, and post-translational modification of a gene.

[0021] 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.

[0022] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can contain modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure can be added before or after the polymer is assembled. 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 that are known or expected to form the double-stranded form.

[0023] 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 where the substance or similar substance naturally occurs or where it is 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 setting 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.

[0024] "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.

[0025] "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.

[0026] "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.

[0027] "Terminator" refers to a polynucleotide sequence that tends to attenuate or prevent read-through transcription (i.e., attenuate or prevent 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.

[0028] "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.

[0029] "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.

[0030] 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.

[0031] 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 to which they are operably linked 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.

[0032] 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., disulfide bond formation, glycosylation, acetylation, phosphonylation, lipid attachment, or conjugation with a labeling component).

[0033] 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).

[0034] "Transformed" or "transgenic" are used herein to include any host cell or cell line that has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells of 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.

[0035] 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.

[0036] 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.

[0037] 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."

[0038] The term "sequence identity" means that two polynucleotide sequences are identical (i.e., nucleotide-by-nucleotide) over a comparison window. The term "sequence identity percentage" means that two polynucleotide sequences are identical (i.e., nucleotide-by-nucleotide) over a comparison window. 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., the window size), and multiplying the result by 100 to obtain the sequence identity percentage. 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.

[0039] "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.

[0040] The present disclosure also contemplates polypeptides with non-conservative substitutions, which entail exchanging a member of one of the above classes for another.

[0041] Rationale Existing drugs have little effect on the underlying disease process of AD, and no preventive therapies are currently available. Because there are no effective therapies for AD, an effective therapy for AD would be a major advance (Conrado et al., 2020). Globally, there are approximately 35 million cases of AD, affecting 6% of individuals over the age of 65 (Fann et al., 2018), and it is responsible for 1.9 million deaths annually (Collaborators GBDD, 2019; Hebert et al., 2013). In the United States, more than 122,000 people die annually. The estimated economic burden of caring for AD patients in the United States is $305 billion annually. The APOE4 allele affects 13.7% of the global population and is much more frequent in patients with AD (Safieh et al., 2019). Inheritance of APOE4 is associated with earlier cognitive decline, early-onset AD, and more severe disease with premature death (Safieh et al., 2019; Williams 2020; Raber et al., 2004). There have been numerous attempts at AD therapies targeting amyloid, tau, and other pathologies. Inheritance of the APOE4 allele is the strongest genetic risk factor for the development of late-onset AD, while the APOE2 allele is protective. Gene therapy to durably deliver APOE2 while reducing endogenous APOE4 expression could effectively convert APOE4 homozygous individuals to APOE2 / 4 heterozygotes with reduced APOE4 expression, significantly reducing the risk of developing AD. Furthermore, APOE4 homozygous individuals could receive AAV gene therapy according to their genotype, years before the onset of symptoms. This strategy represents an expansion of the finding that the APOE3ChC variant protected individuals in PSEN1-E290A pedigrees from developing early-onset AD (Arboleda-Velasquez et al., 2019). AAV-mediated gene transfer of APOE3ChC and APOE2ChC may be effective in treating amyloid and / or tau pathology, and may be more effective than AAV delivery of APOE2 alone.

[0042] Based on the finding that APOE4 increases the risk of amyloid- and tau-based AD pathology, it has been demonstrated that CNS gene therapy with APOE2 can prevent APOE4-based AD pathology in mouse models (Zhao et al., 2016, Shi et al., 2017, Dodart et al., 2005, Hudry et al., 2013). The main innovation of this proposal is to translate unique clinical observations (Arboleda-Velasquez et al., 2019) into a second-generation, more effective gene therapy. Extensive epidemiological data indicate that APOE genotype plays a key role in AD pathogenesis, with APOE2 protecting against the development of AD-related pathology and APOE4 increasing its risk (Corder et al., 1994, Raber 2004, Genin et al., 2011, Sando et al., 2008). As a result of homozygous co-inheritance of homozygous APOE3ChC, we observed that APOE3ChC gene therapy-based genetic modification of the CNS of APOE4 homozygotes prevented the high risk of APOE4-driven AD-related pathology, thereby preventing the onset of early-onset AD in individuals of Colombian PSEN1-E290A descent. Furthermore, based on previous studies demonstrating that APOE2 gene therapy suppresses E4-driven amyloid-related pathology, APOE2ChC gene therapy may prevent both APOE4-driven amyloid and tau pathology, and do so more effectively than APOE2 or APOE3ChC.

[0043] Exemplary APOE Nucleic Acid and Amino Acid Sequences Apolipoprotein E (APOE) is a 299-amino acid protein involved in the metabolism of fat in the body. It is a family of proteins that binds to fat and interacts with the low-density lipoprotein receptor (LDLR), which is important for the normal processing of triglyceride-rich lipoproteins. In peripheral tissues, APOE is produced by the liver and macrophages and mediates cholesterol metabolism. In the central nervous system, APOE is produced by astrocytes and transports cholesterol to neurons via the APO receptor, a member of the LDLR family. There are three major APOE alleles: APOE2 (Cys112, Cys158), APOE3 (Cys112, Arg158), and APOE4 (Arg112, Arg158).

[0044] Exemplary human APOE sequences include: TIFF2025163078000001.tif34128, and sequences having at least 80%, 85%, 90%, 95%, or more, e.g., 99% or 100%, sequence identity thereto, where in one embodiment, APOE4 may have 31K, 46P, 79T, 130R, 163C, 292H, and / or 314R, and APOE2 may have 43C, 152Q, 154C / S, 163C / P, 164Q, 172A, 176C, 242Q, 246C, 254E.

[0045] Exemplary human APOE sequences, such as those encoding APOE2, include the following sequences for silent nucleotide substitutions: TIFF2025163078000002.tif113128 or TIFF2025163078000003.tif119128 or TIFF2025163078000004.tif28164, and sequences encoding APOE that have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more, e.g., 99% or 100% sequence identity thereto.

[0046] Other exemplary APOE sequences include: Contains TIFF2025163078000005.tif71159.

[0047] At positions 112 and 158, E2 contains two cysteine ​​residues, E3 contains a cysteine ​​and an arginine, and E4 contains an arginine residue at both positions. In one embodiment, AAV vectors are prepared to express Christchurch variants in the ApoE2 or ApoE3 background. The vectors can be introduced into wild-type and disease model mice to determine biological measures of expression and efficacy.

[0048] Gene Delivery Vectors Gene delivery vectors within the scope of the present invention 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 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. Gene therapy vectors may encode APOE, RNAi, and / or antibodies or fragments thereof disclosed herein. Exemplary viral gene delivery vectors are described below. The gene delivery vector 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 scaffolding such as an extracellular matrix or hydrogel, e.g., a hydrogel patch. In one embodiment, no penetration enhancer is used to facilitate indirect delivery to the CNS.

[0049] 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.

[0050] 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.For example, lentivirus vectors based on human immunodeficiency virus genome can efficiently transduce cardiomyocytes in vivo.Although lentiviruses have specific tropism, pseudotyping of viral envelope with vesicular stomatitis virus results in a virus with a wider range (Schnepp et al.Meth.Mol.Med.,69:427(2002)).

[0051] Adenovirus vectors Adenoviral vectors can be made replication-incompetent 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, and in particular, these vectors have been shown to efficiently infect cardiomyocytes in vivo, for example, after directional injection or perfusion. Adenoviral vectors have been shown to produce transient expression of therapeutic genes in vivo, peaking at 7 days and persisting for approximately 4 weeks. The duration of transgene expression can be improved in systems utilizing neural-specific promoters. In addition, adenoviral vectors can be produced at very high titers, allowing efficient gene transfer with small amounts of virus.

[0052] Adeno-associated virus vector Recombinant adeno-associated viruses (rAAVs) are derived from nonpathogenic parvoviruses, induce 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 have the ability to infect replicating and non-replicating cells and are considered nonpathogenic to humans. Furthermore, they appear promising for sustained cardiac gene transfer (Hoshijima et al., Nat. Med., 8:864 (2002); Lynch et al., Circ. Res., 80:197 (1997)).

[0053] 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).

[0054] Plasmid DNA vectors Plasmid DNA is often referred to as "naked DNA" to indicate the absence of more elaborate packaging systems. Direct injection of plasmid DNA into cardiac myocytes 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. For example, expression of secreted angiogenic factors after intramuscular injection of plasmid DNA has shown significant biological effects in animal models and appears clinically promising, despite relatively low levels of localized transgene expression (Isner, Nature, 415:234 (2002)). Furthermore, plasmid DNA is rapidly degraded in the bloodstream, thus negligible transgene expression potential in distant organ systems. Plasmid DNA may also be delivered to cells as part of macromolecular complexes, such as liposomes or DNA-protein complexes (see, e.g., below), and delivery can be enhanced using techniques including electroporation.

[0055] Exemplary Non-Viral Formulations For example, biodegradable particles containing isolated nucleic acids for protective APOE expression, vectors for antibody expression, or vectors for RNAi expression may be prepared using a variety of polymers, 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-cyanoacrylates (P The polymeric materials may comprise or be formed from biodegradable polymer molecules, which may include, but are 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, the entire contents of which are incorporated herein by reference). 75(2010)1-18, as well as 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, 6,0 22,564, 5,981,719, 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).

[0056] 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, pp. 16-23; Cismaru et al., Rev. Roum. Chim., 2010, 55(8), 433-442; and International Application Publication Nos. WO2012 / 115806 and WO2012 / 054425, the entire contents of which are incorporated herein by reference.) 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 optionally subjected to further processing steps, such as washing and lyophilization. Optionally, the nanoparticles can be combined with a preservative (e.g., trehalose).

[0057] 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).

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 trifluoroacetate (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, Transfectam), 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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).

[0069] 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.

[0070] In one embodiment, the following polymers may be used: natural polymers such as starch, chitin, glycosaminoglycans, e.g., hyaluronic acid, dermatan sulfate, and chlorotin sulfate; and microbial polyesters, e.g., hydroxyalkanoates, such as hydroxyvalerate and hydroxybutyrate copolymers; and synthetic polymers, including 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).

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] Pharmaceutical Composition The present invention provides compositions comprising, consisting essentially of, or consisting of the above-described gene transfer vector and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. When a composition consists essentially of a gene transfer 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 transfer vector of the present invention and a pharmaceutically acceptable carrier, the composition does not contain any additional components. Any suitable carrier can be used within the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by 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 transfer 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).

[0077] 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 transfer vector of the present invention is administered in a composition formulated to protect the gene transfer vector from damage prior to administration. For example, the composition may be formulated to reduce loss of the gene transfer vector on devices used to prepare, store, or administer the gene transfer vector, such as glassware, syringes, or needles. The composition may be formulated to reduce the light and / or temperature sensitivity of the gene transfer vector. To this end, the composition may contain, 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 extends the shelf life of the gene transfer vector, facilitates administration, and increases the efficiency of the methods of the present invention. Formulations for gene transfer 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).

[0078] The composition can also be formulated to enhance transduction efficiency. In addition, those skilled in the art will understand that the gene transfer vector of the present invention can be present in the composition together with other therapeutic or bioactive agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the gene transfer vector. Immune system stimulants or adjuvants, such as interleukins, lipopolysaccharides, and double-stranded RNA, can be present. Antibiotics, i.e., bactericides and fungicides, can be present to treat existing infections and / or reduce the risk of future infections, such as those associated with gene transfer procedures.

[0079] 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.

[0080] 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.

[0081] 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 transfer 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).

[0082] The dose of the gene transfer 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 of the present invention involves administering a "therapeutically effective amount" of a composition comprising a gene transfer vector described herein. A "therapeutically effective amount" refers to an amount effective, for a period and dosage as necessary, to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the extent of the disease or disorder, the age, sex, and weight of the individual, and the ability of the gene transfer vector to elicit the desired response in the individual. The dose of the gene transfer vector in the composition 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 of body weight (gc / kg). One of skill in the art can easily determine the appropriate gene transfer 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 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 14A therapeutically effective dose may be 1×10 genome copies. 7 Genome copies ~1 x 10 10 A therapeutically effective dose may be 1×10 genome copies. 14 Genome copies ~1 x 10 17 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 × 10 11 gc / kg~1.4×10 14 It can be gc / kg.

[0083] In one embodiment, the composition is administered to mammals once.It is believed that a single administration of the composition can result in sustained expression in mammals 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 mammals two or more times (for example, 2, 3, 4, 5, 6, 6, 8, 9, or 10 times or more) during the treatment period.

[0084] The present disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a gene transfer vector comprising the above-described nucleic acid sequence.

[0085] Route of Administration, Dosage, and Dosage Form For example, the administration of the gene delivery vector according to the present invention can be continuous or intermittent, depending on, for example, the physiological state of the recipient and other factors known to skilled practitioners.The administration of the gene delivery vector can be essentially continuous over a preselected period of time, or can be carried out in a series of spaced doses.Both local administration, for example, intracranial, intranasal, or intrathecal, and systemic administration, for example, using a virus that crosses the blood-brain barrier, are contemplated.Any administration route can be used, for example, intravenous, intranasal, or intrabronchial, direct administration to the lung, and intrapleural.In one embodiment, the composition can be delivered to the pleura.

[0086] One or more suitable unit dosage forms containing gene delivery vectors, which may optionally be formulated for sustained release, can be administered by various routes, including intracranial, intrathecal, or intranasal, or by other means for CNS or oral or parenteral delivery, including rectal, oral, vaginal, and sublingual, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, or intrapulmonary routes.The formulations may conveniently be presented in individual unit dosage forms, if appropriate, and may be prepared by any method known in the pharmaceutical industry.Such methods may include associating the vector with a liquid carrier, a solid matrix, a semi-solid carrier, a finely divided solid carrier, or a combination thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0087] The amount of gene delivery vector administered to achieve a particular outcome will vary depending on a variety of factors, including, but not limited to, the gene and promoter selected, the condition, patient-specific parameters such as height, weight, and age, and whether prevention or treatment is being achieved.

[0088] The vector of the present invention can be conveniently provided in the form of a formulation suitable for administration to, for example, the brain.The suitable mode of administration can best be determined individually for each patient by a physician according to standard procedures.Suitable pharmaceutically acceptable carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences.The term "pharmaceutically acceptable" refers to carriers, diluents, excipients, and / or salts that are compatible with other components of the formulation and are not harmful to the recipient.

[0089] The vectors of the present invention can be formulated in solutions at neutral pH, e.g., about pH 6.5 to about pH 8.5, or about pH 7 to 8, using excipients to render the solution approximately isotonic, e.g., 4.5% mannitol or 0.9% sodium chloride, buffering the pH with buffers known in the art, such as sodium phosphate, generally considered safe, along with an acceptable preservative, such as 0.1% to 0.75% meta-cresol or 0.15% to 0.4% meta-cresol. Achieving the desired isotonicity can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (e.g., mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is useful in buffers containing sodium ions. If desired, solutions of the above compositions can also be prepared to improve shelf life and stability. The therapeutically useful compositions of the present invention can be prepared by mixing the ingredients according to generally accepted procedures. For example, selected components can be mixed to produce a concentrated mixture, which may then be adjusted to a final concentration and viscosity by the addition of water and / or buffer to control pH, or additional solutes to control tonicity.

[0090] The vector can be provided in a dosage form containing an effective amount of the vector in one or more doses. For viral vectors, an effective dose is at least about 10 7 Viral particles, e.g., about 109 virus particles, or approximately 10 11 The number of virus particles added can range from 10 to 10. 14 For example, when a viral expression vector is used, the 8 ~about 10 60 The viral vector of gc can be administered as a nucleic acid or as packaged virions. In some embodiments, for example, about 10 per 0.5-10 mL. 9 ~about 10 15 Copies of the viral vector can be administered as nucleic acids or as packaged virions. 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. The amount administered will vary depending on a variety of factors, including, but not limited to, the nucleic acid or vector selected for administration, the disease, and the mammal's weight, physical condition, health, and / or age. Such factors can be readily determined by the clinician using animal models or other test systems available in the art. As noted above, the exact dose administered is determined by the attending physician, but may be in 1 mL of phosphate-buffered saline. For delivery of plasmid DNA alone or complexed with other macromolecules, the amount of DNA administered will be that amount that provides a beneficial effect in the recipient. For example, 0.0001 to 1 mg or more, eg, up to 1 g, eg, 0.001 to 0.5 mg, or 0.01 to 0.1 mg of DNA can be administered in individual or divided doses.

[0091] For example, when a viral expression vector is used, about 10 8 ~about 10 60 The viral vector of gc can be administered as a nucleic acid or as packaged virions. In some embodiments, for example, about 10 per 0.5-10 mL.9 ~about 10 15 Copies of the viral vector can be administered as nucleic acids or as packaged virions. 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 of body weight, although other dosages may provide beneficial results.

[0092] In one embodiment, administration may be performed by intracranial, intraventricular, intravesical, lumbar, intrahepatic, intratracheal, or intrabronchial injection or infusion using an appropriate catheter or needle. As known in the art, various catheters can be used to achieve delivery. For example, various general-purpose catheters, as well as modified catheters suitable for use in the present invention, are available from commercial suppliers. Also, when delivery is achieved by direct injection into a specific region of the brain or lung, the catheter can be introduced into that region using a number of approaches, as known in the art.

[0093] For illustrative purposes, liposomes and other lipid-containing gene delivery complexes can be used to deliver one or more transgenes. The principles of preparing and using such complexes for gene delivery have been described in the art (see, for example, Ledley, (1995); Miller et al., (1995); Chonn et al., (1995); Schofield et al., (1995); Brigham et al., (1993)).

[0094] Pharmaceutical formulations containing gene delivery vectors can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the agents can be formulated with common excipients, diluents, or carriers and formed into tablets, capsules, suspensions, powders, etc. The vectors of the present invention can also be formulated as elixirs or solutions suitable for parenteral administration, for example, by intramuscular, subcutaneous, or intravenous routes.

[0095] The pharmaceutical formulation of the vector may take the form of an aqueous or anhydrous solution, for example a lyophilized formulation, or dispersion, or alternatively the form of an emulsion or suspension.

[0096] In one embodiment, the vector may be formulated for administration by injection, e.g., bolus injection or continuous infusion via a catheter, and may be presented in unit-dose form in ampoules, prefilled syringes, small-volume infusion containers, or multi-dose containers with added preservatives. The active ingredient may take such forms as a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0097] These formulations may contain pharmaceutically acceptable vehicles and adjuvants that are well known in the art. For example, solutions may be prepared using one or more physiologically acceptable organic solvents.

[0098] For administration to the upper (nasal) or lower respiratory tract by inhalation, the vector is conveniently delivered from an insufflator, nebulizer, pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0099] Alternatively, for administration by inhalation or insufflation, the compositions may take the form of a dry powder, for example a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, for example, gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered dose inhaler.

[0100] For intranasal administration, the vector may be administered via nose drops, a liquid spray, e.g., via a plastic bottle atomizer or a metered dose inhaler, typical of which are the Mistometer (Wintrop) and Medihaler (Riker).

[0101] Local delivery of vectors can also be achieved by various techniques, for example, using a catheter or needle to administer the vector at or near the site of disease. Examples of site-specific or targeted local delivery techniques are not intended to be limiting, but rather to illustrate available techniques. Examples include local delivery catheters, such as injection or indwelling catheters, e.g., needle injection catheters, shunts and stents, or other implantable devices, site-specific carriers, direct injection, or direct application.

[0102] The formulations and compositions described herein may also contain other ingredients, such as antimicrobial agents or preservatives.

[0103] subject The subject can be any animal, including humans, humans, 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 can also be subjects.The subject can also be livestock, for example, cows, pigs, sheep, poultry, and horses, or pets, for example, dogs and cats.

[0104] In one embodiment, the subject includes a human subject. The subject will generally be diagnosed with the condition by a person skilled in the art, e.g., a physician.

[0105] The methods of the invention described herein can be used with subjects of any species, sex, age, ethnic group, or genotype. Thus, the term subject includes males and females, and includes elderly, elderly-to-adult transition age subjects, adults, adult-to-pre-adult transition age subjects, and pre-adults, including adolescents, children, and infants.

[0106] Examples of human ethnic groups include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semitic peoples, and Pacific Islanders. The methods of the present invention may be more suitable for some ethnic groups, such as Caucasians, particularly Northern European populations, and Asian populations.

[0107] The term subject also includes subjects of any genotype or phenotype, as described above, so long as they are in need of treatment. In addition, subjects may have a genotype or phenotype for any hair color, eye color, skin color, or any combination thereof.

[0108] The term subject includes subjects of any height, weight, or size or shape of any organ or body part.

[0109] Illustrative Embodiments In one embodiment, a gene therapy vector is provided that includes an expression cassette encoding a mammalian apolipoprotein E having a residue other than arginine at at least one of positions 112, 136, or 158, or encoding an antibody or antigen-binding fragment thereof that specifically binds to APOE4, e.g., antibodies having binding specificity for or derived from 9D11, 4E4 (binding to residues 100-150), 5B5, E29, 1343A (Arboleda-Velasquez et al., 2019), or those disclosed in U.S. Patent No. 8,741,298, the disclosure of which is incorporated herein by reference, or that binds to heparan sulfate, e.g., heparan sulfate proteoglycan (HPSG). In one embodiment, the mammalian apolipoprotein E is not APOE2, APOE3, or APOE4, but rather a modified APOE, which may be protective, e.g., reduce or delay cognitive impairment or deterioration, or have reduced binding to HSPG, when exogenously expressed in a mammal. In one embodiment, the apolipoprotein E in the vector is human apolipoprotein E, e.g., modified as described herein. In one embodiment, the residue in the APOE in the vector other than arginine is serine, threonine, asparagine, cysteine, or glutamine. In one embodiment, position 112 of the APOE in the vector is cysteine. In one embodiment, position 136 of the APOE in the vector is serine. In one embodiment, position 158 of the APOE in the vector is arginine or cysteine. In one embodiment, two of positions 112, 136, or 158 of the APOE in the vector have arginine. In one embodiment, the 112th position of APOE in the vector does not have arginine.In one embodiment, the 158th position of APOE in the vector does not have arginine.In one embodiment, gene therapy is a viral gene therapy vector, for example, adenovirus, adeno-associated virus (AAV), retrovirus or lentivirus vector.In one embodiment, gene therapy vector is rAAV vector.In one embodiment, the AAV vector has an AAVrh.10, AAV8, AAV9, AAV5, AAVhu.37, AAVhu.20, AAVhu.43, AAVhu.8, AAVhu.2, or AAV7 capsid. In one embodiment, the AAV is AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh.10. The vector may be present in a pharmaceutical composition. In one embodiment, the amount of viral vector in the composition is about 1 x 10. 11 ~Approx. 1×10 16 It is a genome copy.

[0110] The vector may be introduced into a cell in vitro or in vivo. In one embodiment, a method for preventing, inhibiting, or treating Alzheimer's disease in a mammal comprises administering to the mammal an effective amount of a gene therapy vector. In one embodiment, a method for preventing, inhibiting, or treating a disease associated with APOE4 expression in a mammal comprises administering to the mammal an effective amount of a gene therapy vector. In one embodiment, a method for preventing, inhibiting, or treating a lipid disorder in a mammal comprises administering to the mammal an effective amount of a gene therapy 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 mammal is an E2 / E2 homozygote. In one embodiment, the mammal is an E3 / E3 homozygote. In one embodiment, the mammal is an E3 / E4 heterozygote. In one embodiment, the mammal is an E2 / E3 heterozygote. In one embodiment, the mammal is a human. In one embodiment, the vector is administered systemically. In one embodiment, the vector is injected. In one embodiment, the vector is administered to the central nervous system. In one embodiment, the vector is administered to the brain. In one embodiment, the vector encodes apolipoprotein E having C112, S136, and R158 or having C112, S136, and C158.

[0111] In one embodiment, a gene therapy vector comprises a promoter operably linked to a nucleic acid sequence comprising an open reading frame encoding a modified, protective APOE, but not APOE2, APOE3, or APOE4, 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 nucleotide sequence is linked to a second promoter. In one embodiment, the second promoter is a PolIII 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, the vector has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of codons with silent nucleotide substitutions. In one embodiment, the open reading frame further comprises a peptide tag, such as an HA, histidine tag, AviTag, maltose-binding tag, Strep tag, FLAG tag, V5 tag, Myc tag, Spot tag, T7 tag, or NE tag. In one embodiment, the vector is within a host cell, for example, a host organism such as a mammal, or a non-human primate or human.The host mammal may be administered an amount of the vector effective to, for example, prevent, inhibit, or treat Alzheimer's disease in the mammal, or to prevent, inhibit, or treat a disease associated with APOE4 expression in the mammal. In one embodiment, the vector and / or nucleotide sequence is administered systemically. In one embodiment, the vector and / or nucleotide sequence is administered orally. In one embodiment, the vector and / or nucleotide sequence is administered intravenously.

[0112] The present invention is further illustrated by the following non-limiting examples. [Example]

[0113] Example 1 overview The pathogenesis of Alzheimer's disease (AD) is complex and characterized by the accumulation of amyloid beta (Aβ) in the central nervous system (CNS) and the formation of amyloid plaques, abnormal tau phosphorylation, tau tangles, inflammation, and progressive loss of neurons, leading to progressive cognitive decline. Genetics plays a major role in the risk of these pathogenic processes (DeTure & Dickson, 2019, Holtzman et al., 2012, Safieh et al., 2019, Fernandez et al., 2019). Early-onset autosomal dominant AD is caused by mutations in amyloid protein precursor (APP) and presenilin (PSEN) 1 and 2 (Campion et al., 1999; Carmona et al., 2018). These genes affect APP processing, altering the production of Aβ peptides, leading to aggregation and plaque formation (Carmona et al., 2018; Dai et al., 2018). The primary genetic factor for sporadic late-onset AD is variants in the lipid transport protein apolipoprotein E (APOE) (Tzioras et al., 2019; Wolters et al., 2019). APOE has three common isoforms: the common ε3 isoform is associated with an average risk of AD, ε4 is associated with a 15-20-fold increased risk, and ε2 is associated with a 66-99% reduced risk compared to ε3 and ε4, respectively (Wolters et al., 2019, Corder et al., 1994, Naj et al., 2011, Reiman et al., 2020). At a biological level, APOE genotype (ε4>ε3>ε2) predicts the timing and abundance of cerebral amyloid, p-tau, and tau tangles in humans and AD mouse models (Tzioras et al., 2019, Shi et al., 2017, Abner et al., 2018). The difference between E2 and E4 is determined by amino acids 112 and 158, with APOE2 (C112 C158) representing low risk and APOE4 (R112 R158) representing high risk (Corder et al., 1994, Reiman et al., 2020, Corder et al., 1993).Within this region 112–158, the “Alzheimer's risk region” encodes the LDL receptor and binds to heparan proteoglycans ( Mahley et al., 1999 ).

[0114] Because APOE4 represents high risk and APOE2 represents low risk, we prepared a serotype rh.10 adeno-associated virus (AAV) gene transfer vector encoding human APOE2 to develop a gene therapy for preventing / treating the development of AD-related pathology in APOE4 homozygotes using AAVrh.10hAPOE2. CNS administration of AAVrh.10hAPOE2 prevented APOE4-associated Aβ and amyloid burden in a mouse model (Zhao et al., 2016), supporting the idea that APOE2-associated risk of AD could be mitigated by using APOE2 gene therapy to convert APOE4 homozygous brains to an APOE2-APOE4 heterozygous state (Zhao et al., 2016).

[0115] The importance of the APOE112-158 region in mediating AD risk was highlighted by the report of a female carrier of the PSEN1 E280 mutation, as well as a homozygote for the Christchurch R136S APOE3 variant, APOE3ChC (Arboleda-Velasquez et al., 2019). The dominant PSEN1-E290A gene defines a Colombian family of over 1,200 individuals, all of whom have early-onset CNS Aβ and amyloid accumulation, with heterozygotes developing dementia in their mid-40s (Lopera et al., 1997). Strikingly, a woman with co-inheritance of APOE3ChC and PSEN1-E290A was cognitively healthy at age 70, well beyond the age at which her relatives typically develop cognitive decline (Arboleda-Velasquez et al., 2019). Clinical evaluation revealed the accumulation of amyloid plaques but low levels of tau pathology, leading to the notion that the APOE3ChC variant blocked tau pathology progression. This was confirmed by in vitro experiments demonstrating that APOE3ChC behaved like the APOE2 allele and both bind poorly to heparin compared with the tightly binding APOE4 (APOE3ChC>APOE2) (Mahley et al., 1999; Zhao et al., 2016; Rosenberg et al., 2018; Arboleda-Velasquez et al., 2019). The observation that inheritance of APOE3ChC prevents the progression of PSEN1-E280A-driven tau pathology but not amyloid pathology (Arboleda-Velasquez et al., 2019), coupled with gene therapy studies showing that APOE4-driven amyloid pathology can be suppressed by AAV-mediated delivery of APOE2 to the CNS, led us to test whether AAVrh.10-mediated delivery of APOE3ChC (APOE3 with the Christchurch variant) to the CNS suppresses tau-related pathology, and whether delivery of APOE2ChC (APOE2 with the Christchurch variant) suppresses both amyloid and tau pathology in mouse models of AD amyloid and tau pathology.Two mouse, human E4-based models are used: APP.PS1 / TRE4, which exhibits APP and APOE4-associated amyloid pathology (Zhao et al., 2016; Rosenberg et al., 2018; Arboleda-Velasquez et al., 2019; Lopera et al., 1997; Kim et al., 2011), and P301S / E4, which exhibits tau and APOE4-associated tau pathology (Liu et al., 2016; Allen et al., 2002).

[0116] strategy Genetics plays a major role in Alzheimer's disease risk (DeTure & Dickson, 2019; Holtzman et al., 2012; Safieh et al., 2019; Fernandez et al., 2019). Early-onset autosomal dominant AD is caused by mutations in amyloid protein precursor (APP) and presenilin 1 (PSEN1) and 2 (PSEN2) (Campion et al., 1999; Carmona et al., 2018). These genes affect APP processing, altering the production of Aβ peptides and leading to increased aggregation and plaque formation (Carmona et al., 2018; Dai et al., 2018). The primary genetic factor for sporadic late-onset AD is variants in the lipid transport protein apolipoprotein E (APOE) (Tzioras et al., 2019; Wolters et al., 2019). APOE has three common isoforms, ε2, ε3, and ε4; the common ε3 isoform is associated with an average risk of AD, whereas ε4 increases the risk and decreases the age of onset, whereas ε2 decreases the risk and delays the age of onset (Wolters et al., 2019, Corder et al., 1994, Naj et al., 2011). At the biological level, the APOE genotype ε4>ε3>ε2 predicts the timing and abundance of cerebral amyloid, p-tau, and tau tangles in humans and mouse models of AD (Tzioras et al., 2019, Shi et al., 2017, Abner et al., 2018). Based on the finding that inheritance of different variants of the APOE gene can determine AD risk, age at onset, and severity ( DeTure & Dickson, 2019 ; Shinohara et al., 2016 ), it was postulated that a therapy to reduce CNS-related AD pathology in APOE4 homozygotes could be achieved by delivering “APOE AD risk-reducing” variants to the CNS.Previous studies demonstrated that APOE2 delivery to the CNS mediated by AAV serotype rh.10 (AAVrh.10) reduced Aβ and amyloid burden in mouse models of AD (Zhao et al., 2016). The ability of APOE2 to reduce brain Aβ burden in these mouse models was dependent on gene dosage and the amount of pre-existing Aβ1-42 deposits, suggesting that gene delivery of APOE2 using AAV vectors is a viable therapeutic approach for treating or preventing AD if sufficient brain APOE2 levels are achieved early in the disease process (Zhao et al., 2016). This data, along with studies demonstrating the ability to deliver and safely achieve high levels of APOE2 expression in the CNS of non-human primates using the intracisternal route (Rosenberg et al., 2018), has led to an ongoing first-in-human clinical trial to treat APOE4 homozygotes with intracisternal administration of AAVrh.10hAPOE2.

[0117] An "APOE AD risk reduction" gene therapy strategy is envisioned that would be more effective than APOE2 gene therapy in mitigating the progression of APOE4-driven AD-related pathology. This approach is based on recent reports of the effects of the APOE3-Christchurch (APOE3ChC) mutation superimposed on the Colombian PSEN1-E290A mutation, a familial PSEN1 variant affecting over 1,200 individuals (Lopera et al., 1997; Acosta-Baena et al., 2011). The PSEN1-E280A mutation is associated with increased Aβ production, leading to amyloid deposition. Heterozygotes develop dementia in their mid-40s (Lopera et al., 1997). A woman from this family, who carries the autosomal dominant PSEN1 E280A mutation but is also homozygous for the APOE3ChC variant, remains cognitively healthy and independent at age 70, well beyond the age of 40 when individuals with the same PSEN1 mutation typically show cognitive decline (Arboleda-Velsquez et al., 2019). Clinical evaluation revealed significant amyloid plaque accumulation but low tau / p-tau pathology, suggesting that the APOE3ChC mutation may have blocked the progression of tau pathology (Arboleda-Velsquez et al., 2019). Interestingly, the APOE3ChC variant (R136S) is located within the same region of the APOE protein (112–158) as the common ε2, ε3, and ε4 alleles, consistent with the notion that this region of APOE is associated with AD risk (Mahley et al., 1999).

[0118] The ability of the APOE3ChC variant to suppress tau pathology was confirmed by in vitro experiments demonstrating that APOE3ChC behaves like the APOE2 allele (both have poor binding to heparin (APOE3ChC < APOE2)) (Mahley et al., 1999; Arboleda-Velsquez et al., 2019). These findings suggest that the AD-related chain of pathogenic events is likely disrupted by APOE3ChC after amyloid formation, and that, independent of mechanism, the Christchurch mutation is protective and could be used to treat APOE4 homozygotes, and is likely more effective than gene therapy with the APOE2 allele (Figs. 12–13). Using the AAVrh.10 capsid as a delivery system in relevant AD mouse models, we evaluate that AAVrh.10-mediated delivery of either APOE3 or APOE2 each carrying the Christchurch mutation to the CNS is superior to AAVrh.10hAPOE2 as a treatment for Alzheimer's disease in APOE4 homozygotes. Based on previous demonstration that AAVrh.10hAPOE2 suppresses amyloid accumulation in mouse models (Zhao et al., 2016), and clinical case reports on the Christchurch variant, AAVrh.10hAPOE3ChC is likely to suppress tau rather than amyloid pathology, and AAVrh.10hAPOE2ChC is likely more effective as it suppresses both amyloid and tau pathology.

[0119] PSEN1-APOE3ChC. While the report of an individual inheriting the APOE3ChC variant in conjunction with the PSEN1-E280 mutation of Colombian descent represents only a single case, this report included additional studies supporting the conclusion that APOE3ChC is an "APOE AD risk-reducing" variant. Amyloid PET scans revealed significant accumulation of CNS amyloid plaques, but little tau pathology was observed, mostly confined to the medial temporal lobe. In addition, cerebral glucose metabolism was nearly normal (Figure 14). The idea that the APOE3ChC mutation blocks tau pathology progression is similar to that in a study by Reiman et al. (2020), which observed that APOE2 was associated with reduced tau pathology in 24 autopsy samples from APOE2 homozygotes. The ability of the APOE3ChC variant to suppress tau pathology was supported by in vitro experiments demonstrating that APOE3ChC behaves like the APOE2 allele and that both bind poorly to heparin compared with E4, E2, and E3ChC (binding: E4>E3>E2>>E3ChC; Figure 15). HSPGs have been implicated in promoting amyloid plaque accumulation and the microglial response to amyloid (Hefferman et al., 2016; ALXFORUM, 2010; Snow et al., 1988; Liu et al., 2016; O'Callaghan et al., 2018). HSPGs have also been implicated in tau pathology, suggesting that they facilitate the attachment of tau fibrils to neurons and the uptake and propagation of toxic forms of tau (Rauch et al., 2018; Zhao et al., 2019). In vitro studies also showed that APOE3 bound with an antibody to residues 130–143 (including the region of the Christchurch mutation) functionally converted APOE3 to APOE3ChC, as measured by heparin binding.Together, these findings suggest that the Alzheimer-associated chain of pathogenic events is likely disrupted by APOE3ChC after amyloid formation and, regardless of the mechanism, the Christchurch mutation is protective and could therefore be used to treat APOE4 homozygotes.

[0120] APOE4-Associated Risk of AD. APOE, a lipid-binding protein, is the primary carrier of cholesterol in the CNS (Puglielli et al., 2003; Williams et al., 2020). In the brain, it is primarily produced by astrocytes and microglia (Flowers & Rebeck, 2020; Pitas et al., 1987; Holtzman et al., 2012). APOE particles form in the extracellular environment (Mahley et al., 1999; Flowers & Rebeck, 2020). APOE particles transport cholesterol to neurons via the APOE receptor, a member of the low-density lipoprotein receptor gene family (Liu et al., 2013; Mondadoori et al., 2007; Zhang et al., 2013). The APOE protein (299 amino acids) contains an N-terminal (1–167) domain and a C-terminal (206–299) domain connected by a hinge region (Safieh et al., 2019; Flowers & Rebeck, 2020). The C-terminal region contains the low-density lipoprotein receptor binding site 41 (Figure 2). In addition to its role in transporting cholesterol within the CNS, APOE has numerous other functions (Holtzman et al., 2012; Castellano et al., 2011; Deane et al., 2008; Hashimoto et al., 2012; Hatters et al., 2006a; Hatters et al., 2006b; Li et al., 2012; Manelli et al., 2005; Walker et al., 2000; Yu et al., 2014; Zhao et al., 2009). Human APOE is generally expressed in three isoforms (APOE2, 3, and 4) that differ by only two residues (Mahley et al., 1996; Zannis et al., 1981) (Figure 13). Inheritance of APOE4 is the most important genetic risk factor for AD.APOE3 is neutral, the E4 allele increases risk and delays age of onset, and E2 decreases risk and delays age of onset ( Reiman et al., 2020 , Mahley, 2016 , Conejero-Goldberg et al., 2014 , Nagy et al., 1995 , Raber et al., 2004 ). Epidemiological data suggest that APOE4 and APOE2 are codominant, i.e., instead of a fourfold increased risk of AD for E3 / E4 heterozygotes, E2 / E4 heterozygotes are closer to the normal risk of E3 / E3 homozygotes (Corder et al., 1994, Liu et al., 2013, Genin et al., 2011), i.e., the nearly equal expression of E2 counteracts the deleterious effect of the E4 allele (Corder et al., 1994, Farrer et al., 1997, Coon et al., 2007). Key conclusions from epidemiological studies of the protective effects of E2 include that the E2 genotype is significantly underrepresented in AD (Corder et al., 1994; Reiman et al., 2020), that the E2 allele is associated with a delayed age of onset of AD (Reiman et al., 2020; Benjamin et al., 1994), that E2 carriers have reduced AD-related pathology (Reiman et al., 2020; Nagy et al., 1995), that E2 is associated with slower cognitive decline (Small et al., 2004), that E2 carriers possess more robust white matter integrity that may be associated with reduced vulnerability to AD progression (Chiang et al., 2012), and that in normal elderly individuals, E2 carriers have lower levels of cerebrospinal fluid p-tau and slightly lower tau (Chiang et al., 2010). Experimental studies indicate that E2 promotes Aβ metabolism, neuronal repair and neurite outgrowth, and functions as an antioxidant and anti-inflammatory agent (Rebeck et al., 2002).Furthermore, animal and clinical studies have shown that APOE genotype also predicts the timing and amount of cerebral amyloid-β (Aβ) peptide deposition and amyloid burden (E4>E3>E2) (Castellano et al., 2011, Raber et al., 2004, Bales et al., 2009, Holtzman et al., 2000, Reiman et al., 2009, Schmechel et al., 1993), i.e., APOE4 impairs Aβ clearance and increases amyloid formation (Liao et al., 2017, Hu et al., 2015). Mouse studies have shown that targeted deletion of the endogenous ApoE gene dramatically reduces fibrillar amyloid and total brain Aβ deposition in the PDAPP mouse model of AD (these mice express a mutant APP transgene that produces high brain levels of human Aβ) (Bales et al., 1997). By crossing PDAPP mice with human APOE-targeted replacement (TRE) mice, APOE isoform-dependent effects were observed on brain Aβ deposition and amyloid burden (E4>>E3>E2) in a manner recapitulating those observed in AD patients (Castellano et al., 2011; Bales et al., 2009; Holtzman et al., 2000; Fagan et al., 2002), suggesting that APOE isoforms play an important role in determining brain amyloid burden. Furthermore, studies in tau-associated P301S / E3, E2, and E4 mice indicate that ApoE influences neurodegeneration in the context of tau pathology independently of amyloid-β: ApoE4 exacerbated neurodegeneration, whereas the absence of ApoE was neuroprotective ( Shi et al., 2017 ).

[0121] APOE2 Gene Therapy for APOE4-Related CNS Lesions. Based on epidemiological data demonstrating that APOE2 is protective, gene therapy programs were developed to evaluate the hypothesis that transferring APOE2 to the CNS of APOE4 homozygotes could reduce E4 risk (Zhao et al., 2016; Rosenberg et al., 2018). Evidence in the literature for the feasibility of this approach came from a study by Dodart et al. (2005), which showed that administering APOE4 and APOE2 to the CNS of a mouse AD model using lentiviral vectors increased and decreased brain Aβ / amyloid burden, respectively. The finding that APOE2 reduced brain Aβ / amyloid burden was replicated by Hudry and colleagues (2013) using an AAV4 vector to deliver APOE2 to the lateral ventricles of mutant APP mice. Using an AAV8 vector administered into the CSF, Hu et al. (2015) found that increasing APOE2 expression in a TRE4 mouse model was an effective strategy for treating AD, while increasing APOE4 expression in TRE4 mice was detrimental. As described in detail below, an AAVrh.10 serotype vector encoding human APOE2 reduced amyloid burden in two mouse models (Zhao et al., 2016). Regardless of the molecular mechanisms by which these APOE alleles determine AD risk, given the fact that APOE2 is strongly protective and significantly reduces Aβ / amyloid burden when delivered to mouse brains via either gene therapy vector, CNS gene delivery of APOE2 via a viral vector represents a therapeutic strategy for APOE4 homozygous AD.

[0122] AAVrh.10. The AAVrh.10 serotype is derived from rhesus macaques. This serotype has been administered together with various expression cassettes to the CNS of experimental animals (mice, non-human primates), children with CLN2 disease, and adults with Alzheimer's disease (clinicaltrials.gov; NCT01414985, NCT01161576, NCT03634007). The AAVrh.10 vector is designed with a highly active constitutive promoter, an intron coding sequence, and rabbit β-globin polyA flanked by AAV2 inverted terminal repeats (see Figure 16 for details of the AAVrh.10hAPOE2 vector, for example). The AAVrh.10 capsid is highly effective at mediating gene expression within the CNS, comparable to AAV9, but with a lower potential for toxicity (Rosenberg et al., 2018, Rosenberg et al., 2014, Sondhi et al., 2012, Tardieu et al., 2014, Zerah et al., 2015). All vectors are characterized for multiple measures of identity, potency, and purity. Purity by polyacrylamide gel electrophoresis confirms the absence of contaminating proteins from the cells used in vector production (Figure 17). Demonstrating the efficacy of intracisternal administration of vectors encoding human APOE2 to non-human primates (NHPs), we provide widespread distribution of vector-derived proteins throughout the CNS, including in the cerebrospinal fluid (Figure 18). These observations confirm that CNS administration of AAVrh.10 vectors encoding human APOE isoforms allows for effective distribution of the encoded APOE isoforms throughout the CNS.

[0123] AAVrh.10hAPOE2 ameliorates pathology in an AD animal model. PDAPP mice are a well-characterized mouse model of cerebral amyloidosis that overexpress a human APP mutation and develop age-dependent Aβ and amyloid deposition in the hippocampus and cerebral cortex (Games et al., 1995; Schenk et al., 1999). At 9 months of age, there were abundant Aβ and amyloid plaques in the hippocampus. Expression of the AAVrh.10hAPOE2 vector significantly reduced the amyloid pathology observed in control animals treated with a similar viral vector expressing an unrelated gene (Figure 19) (Zhao et al., 2016). Expression of APOE2 using the AAVrh.10hAPOE2 vector resulted in a marked reduction in insoluble Aβ1-42 (70.4% reduction vs. mCherry control, p<0.001) and soluble Aβ1-42 (27.2% reduction vs. mCherry control, p<0.05) levels in the hippocampus of PDAPP mice. Insoluble Aβ1-40 was also dramatically reduced (61.2% reduction vs. control, p<0.001), while soluble Aβ1-40, which was already at relatively low levels, remained unchanged.

[0124] To further evaluate the role of APOE2 expression on brain Aβ burden, we also evaluated the effect of AAVrh.10hAPOE2 dose on Aβ / amyloid pathology in APP.PS1 / TRE4 triple transgenic mice. APP.PS1 / TRE4 mice carry the APP Swedish (APPswe) and PS1 mutations and the human APOE4 gene (Kim et al., 2011; Jankowsky et al., 2018). APP.PS1 / TRE4 mice develop robust age-dependent Aβ and amyloid pathology in multiple brain regions, including the cerebral cortex, hippocampus, and thalamus. Amyloid plaques are observed in the cortex of mice as early as 2 months of age, and the progression of brain Aβ / amyloid burden is dependent on APOE4 expression (Kim et al., 2011). Similar to the observations in PDAPP mice, a dose-dependent suppression of amyloid beta levels was observed in the hippocampus of APP.PS1 / TRE4 mice treated with intrahippocampal delivery of AAVrh.10hAPOE2 (FIG. 20).

[0125] Mouse model of APOE4-related AD pathology. Because the clinical goal is to suppress both amyloid and tau pathology in the CNS of APOE4 homozygotes, we selected mouse models with mouse ApoE knockout and human APOE4 knockin. One mouse model is for APOE4-related amyloid pathology, and the other is for APOE4-related tau pathology. APP.PS1 / TRE4 mice express human beta-amyloid (Aβ) precursor protein (APP) with the Swedish mutation (APPswe), mutant (L 166P) human presenilin 1, and the human APOE4 gene substituting for the endogenous mouse ApoE gene. These mice express high levels of human Aβ protein and human AD-like amyloid pathology (Kim et al., 2011) and were used in a previous study to demonstrate that AAVrh.10hAPOE2 suppresses AD amyloid-associated pathology (Zhao et al., 2016) (Figure 20). P301S / E4 mice carry human P301S 1N4R tau, a deletion of the mouse ApoE gene, and an addition of the human APOE4 gene (Shi et al., 2017; Allen et al., 2002). These mice have APOE4-associated tau pathology and will be used to evaluate the ability of gene therapy vectors to suppress tau pathology. These mice also have activated microglia and astrocytes, indicative of AD-associated inflammation (Shi et al., 2017).

[0126] Approach: The Christchurch variant (APOE3-R 136S) lies in the APOE112-158 "Alzheimer's risk region," which includes residues 112 and 118 that define APOE2, 3, and 4, the LDL-binding region (136-150), and the heparan sulfate proteoglycan-binding region (Figure 13). Demonstration that APOE4-associated Abeta and amyloid burden in an APOE4-associated mouse model can be prevented by CNS administration of AAVrh.10APOE2 supports the notion that APOE4-associated risk for AD can be mitigated by using gene therapy to deliver the APOE2 coding sequence to convert APOE4 homozygous brains to an APOE2-APOE4 heterozygous state (Zhao et al., 2016). The report of a woman from a PSEN1-E290A family with simultaneous homozygous inheritance of APOE3ChC, which abrogated PSEN1-E290A-driven AD-associated tau pathology, led to the focus of this proposal to develop second-generation gene therapy for APOE4 homozygotes by delivering the Christchurch mutation into either the APOE3 or APOE2 background using the AAVrh.10 vector. This will be tested in two human APOE4-associated mouse models: APP.PS1ffRE4, which displays primarily amyloid-related pathology, and P301S / E4, which displays primarily tau-related pathology (Shi et al., 2017; Kim et al., 2011). If the Christchurch variant in the APOE3 background is as effective in mouse models as the case reports of double inheritance of PSEN1-E290A and APOE3ChC, gene therapy with AAVrh.10APOE3ChC should suppress tau pathology in P301S / E4 mice but not amyloid pathology in APP.PS1ffRE4 mice. However, based on the efficacy of AAVrh.10hAPOE2 in suppressing amyloid pathology in APP.PS1ffRE4 mice (Zhao et al., 2016), we hypothesize that AAVrh.10hAPOE2ChC will suppress both amyloid pathology in APP.PS1ffRE4 mice and tau pathology in PSEN1-E290A mice.If this hypothesis is correct, AAVrh.10hAPOEChC would be an ideal second-generation candidate for clinical translation to treat APOE4 homozygotes. Everything is identical except for the different vectors (AAVrh.10hAPOE3ChC and AAVrh.10hAPOE3, or AAVrh.10hAPOE2ChC and AAVrh.10hAPOE2). Statistical analysis includes comparison of parameters within each experiment and separately to compare differences between AAVrh.10hAPOE3ChC and AAVrh.10hAPOE2ChC.

[0127] For each mouse model (APP.PS1ffRE4, P301S / E4), vectors [AAVrh.10hAPOE3ChC, AAVrh.10hAPOE3; AAVrh.10hAPOE2ChC, AAVrh.10hAPOE2; control AAVrh.10Null (no translatable transgene) or PBS for both] are administered bilaterally into the hippocampus at 2 months of age in 2 μl. 10 Genome copies (gc) or 1.0 × 10 10Administered gc. Evaluations are performed at 5 and 9 months. Ten males and ten females are used for every data point (mouse strain, vector or PBS, evaluation time point; Table I). Evaluations performed related to vector, general pathology, amyloid, tau, and inflammation are listed in Table II. Based on the findings that AAVrh.10hAPOE2 effectively suppresses amyloid-related pathology in APP.PS1ffRE4 mice (Zhao et al., 2016) and a case report in which homozygous inheritance of APOE3ChC resulted in amyloid pathology but prevented tau pathology in a female carrier of a PSEN1-E280A pedigree (Arboleda-Velasquez et al., 2019), the inventors hypothesize that both hAPOE3ChC and hAPOE2ChC vectors are likely to be more effective than hAPOE3 and hAPOE2 vectors lacking the ChC variant, and that the hAPOE2ChC construct is likely to be more effective than the hAPOE3ChC construct in preventing both amyloid and tau pathology. We will also test: (1) the efficacy of hAPOE3ChC and hAPOE2 vectors against more common pathologies (quantitative volumetric analysis of AD-related CNS structures, transcriptomic analysis of general cellular functions), and (2) AD-related inflammation (microglial and astrocyte activation, transcriptomic analysis of pro-inflammatory genes), as E4 variants are associated with greater innate immune-related inflammation than E2 and E3 variants ( Vitek et al., 2009 , Gale et al., 2014 ).

[0128] Table I. AAVrh.10-mediated treatment of APOE variants in the APP.PS1 / TRE4 and P301S / E4 mouse models of human APOE4-associated AD pathology. TIFF2025163078000006.tif63148

[0129] Each mouse model will be treated with PBS or an AAVrhlO vector encoding the listed transgene, all administered bilaterally into the hippocampus at 2 months at the listed doses of 2 μl each, with CNS assessments at 5 and 9 months. See Table II for a list of parameters assessed.

[0130] Table II. Evaluation of the efficacy of APOE variants in the APP.PS1 / TRE4 and P301S / E4 mouse models of human APOE4-associated AD pathology. TIFF2025163078000007.tif69148

[0131] Each mouse model was injected with AAVrh.10hAPOE3ChC, hAPOE3, hAPOE2ChC, hAPOE2, or Null (0.4 × 10 10 gc or 1×10 10 gc; bilateral, hippocampus), or PBS was administered and assessed at 5 and 9 months of age. For each vector, each dose, and each time point, 10 males and 10 females; PBS, 10 males and 10 females per time point; DNA, mRNA by qPCR, and APOE protein assessed by quantitative Western and quantitative immunohistochemistry; quantitative volumetric assessment of piriform / entorhinal cortex, hippocampus, posterior lateral ventricle, and dentate gyrus thickness; general cellular function-related quantitative transcriptome assessment by RNA-seq; soluble (RIPA) and insoluble Aβ-1-42 and Aβ-1-40 assessed by Aβ ELISA and immunohistochemistry (% positive area in hippocampus, Aβ plaque burden); tau and p-tau ELISA and immunohistochemistry (% positive area in hippocampus, quantification of tau tangles); quantification of microglia (CD68, lba1) and astrocytes (GFAP) assessed by cell counting and % hippocampal area, RNA-seq quantitative transcriptome of pro-inflammatory genes.

[0132] method. Vector-related. MVrh.10 is produced and purified as previously described (Sondhi et al., 2012; Sondhi et al., 2007). Briefly, the vector is produced by co-transfection of HEK293T cells with the expression cassette plasmid and an 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 grown in CellSTACKS (Corning) for 24 hours before transfection with the plasmid using the PElpro procedure. Cells are harvested after 3 days of incubation and then transfected five times. The cells are lysed by 1000 cycles of freeze / thaw. The resulting cell lysate is treated with 50 U / ml of benzonase for 30 minutes at 37°C. The cell lysate is purified by iodixanol density gradient followed by Q-HP ion exchange chromatography. The purified AAVrh.10 vector is concentrated in phosphate-buffered saline (PBS). The vector genome titer is determined by TaqMan 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.

[0133] Mouse Model. An APP.PS1 / TRE4 mouse colony has been established by crossing APP.PS1-21 mice with human APOE4 knock-in mice (Zhao et al., 2016). P301S / E4 mice are generated by crossing human tau mutant P301S mice with human APOE4 knock-in mice. The mouse genome is verified by PCR of the relevant knock-in gene. Intracerebral injection of AAV vectors is performed by stereotaxic surgery (Dodart et al., 2005). Briefly, under sterile conditions, mice are anesthetized with isoflurane and fixed in a stereotaxic frame (David Kopf Instruments). An incision is made on the top of the skull, and a burr hole the size of an injection needle is created using a high-speed drill. AAV preparations (2 μL at the indicated dose, gc) are injected bilaterally into the target brain region at a rate of 0.2 μL / min using a 33-gauge needle (Hamilton) and a syringe pump (KD Scientific). The stereotaxic coordinates used for injections are as follows: anterior-posterior ±1.7 mm from bregma, mediolateral ±1.2 mm from bregma, and dorsoventral ±1.7 mm below the dura. This is based on a diagram of the mouse brain. After each injection, the needle is left in place for 5 minutes to minimize reflux and then slowly withdrawn. Animals are housed individually and monitored until fully conscious. At 5 and 9 months of age, mice are deeply anesthetized with ketamine / xylazine and transcardially perfused with 0.3% heparinized saline (2500 IU / L). Brains are rapidly collected, each brain is divided along the sagittal plane, one hemisphere is processed for histology and immunohistochemistry (IHC) analysis, and the other hemisphere is microdissected into the hippocampus, thalamus, and entorhinal cortex, then rapidly frozen on dry ice and stored at -80°C for biochemical analysis.

[0134] Brains were postfixed in 4% paraformaldehyde for ≥48 hours, transferred to 30% sucrose for ≥48 hours, frozen, and sectioned using a CM3050-S cryostat (Leica). Serial coronal sections were prepared at 50 μm intervals. IHC analysis was performed using standard methods (Bales et al., 2009; Holtzman et al., 2000; Bales et al., 1997). Free-floating sections were washed and incubated with primary antibodies overnight at 4°C. Immunoreactivity (IR) was visualized using Alexa Fluor 488- or 594-conjugated secondary antibodies (1:200, Invitrogen) or the Vectastain ABC Elite kit (Vector) and 3,3-diaminobenzidine solution. Specificity of primary antibody staining was verified by the absence of IR signal when the primary antibody was omitted. Thioflavin-S staining was performed to detect amyloid plaques (Bales et al., 2009; Holtzman et al., 2000; Bales et al., 1997). Fixed brain sections were briefly incubated with freshly prepared 0.1% thioflavin-S solution (in 50% ethanol) for 8 min, then cleared with 80% ethanol for 1 min, followed by three 1-min washes with HO before coverslipping with mounting medium. Images were taken using an epifluorescence microscope (Nikon H550L).

[0135] Vector-derived APOE. For tissue homogenates processed for vector DNA, use the DNeasy Blood and Tissue Kit (Cat. No. 69506, Qiagen) with approximately 50 mg of homogenized tissue sample. For tissue homogenates processed for vector transgene mRNA, use the RNeasy Lipid Tissue Mini Kit (Qiagen) with approximately 50 mg of homogenized tissue sample. Quantitative analysis of vector DNA and transgene mRNA copies in brain sections is performed by PCR using TaqMan-based analysis (Applied Biosystems "Universal Master Mix II, no. UNG" reagent) and human-specific primer / probe sets (Applied Biosystems) to detect the 3' end of human APOE2, E3, or E4 cDNA from the viral genome in a background of mouse genomic DNA (forward primer: 5'GTGGAGAAGGTGCAGGCT-3' (SEQ ID NO: 10); reverse primer: 5'-AAGCGTAATCTGGAACATCGT-3' (SEQ ID NO: 11); probe, 5'CCCTGTGCCCAGCGACAATC-3' (SEQ ID NO: 12). PCR is performed using a QuantStudio6 Flex system (Applied Biosystems) (Rosenberg et al., 2018). APOE levels are measured in the brain using a sandwich ELISA (Bales et al., 2018). (2009). Tissue homogenates were diluted in sample dilution buffer (PBST containing 0.4% glycine). Samples were loaded onto a 96-well plate coated with anti-APOE antibody (1:2000, Millipore). After overnight incubation at 4°C, biotinylated anti-APOE antibody (1:10,000, Meridian Life Science) was used for detection. After incubation with HRP-conjugated streptavidin (Research Diagnostics), the IR signal was developed with TMB substrate (Thermo Scientific) and read on a Synergy H1 Hybrid plate reader (BioTek).APOE levels were calculated using a standard curve generated with recombinant human APOE (Meridian Life Science). APOE levels in brain homogenates were determined in triplicate, normalized to protein content, and expressed as mg APOE / mg protein. Examination of APOE and other protein levels in mouse brain tissue homogenates was also performed by Western analysis (Zhao et al., 2016; Sacramento et al., 2020). Total protein concentration in tissue homogenates was determined using a BCA protein assay kit (Thermo Scientific). For sodium dodecyl sulfate (SOS) polyacrylamide gel electrophoresis, equal amounts of protein (25 mg) samples were mixed with loading dye containing 2% SDS and 1% β-mercaptoethanol, incubated at 90°C for 10 minutes, and separated on a 10% Tris-glycine polyacrylamide gel. For non-denaturing PAGE, equal amounts of protein (25 μg) samples were mixed with non-denaturing loading dye to a final concentration of 0.04% bromophenol blue, 4.0% glycerol, and 100 mM Tris (pH 6.8) and separated on a 4–12% non-denaturing Tris-glycine polyacrylamide gel (Invitrogen). Proteins were transferred onto a polyvinylidene fluoride (Millipore) membrane at 100 V. After blocking with Superblock (Thermo Scientific) for 1 h at 23°C, the membrane was probed with primary antibodies overnight at 4°C, followed by species-specific HRP-conjugated secondary antibodies (1:8000, Invitrogen) for 1 h at 23°C. Bands were identified using enhanced chemiluminescence (GE Healthcare Biosciences). Protein molecular weights on denaturing gels were determined by comparison with Kaleidoscope molecular weight marker standards (Bio-Rad). Particle size on native gels is determined by comparison with native high molecular weight marker standards (GE Healthcare Biosciences). Signal quantification is performed using densitometric analysis of scanned autoradiograms with Image Lab software (Bio-Rad).Loading is normalized by stripping the blots and reprobing with β-actin antibody (Sigma-Aldrich).

[0136] General lesions. Volumetric analysis. Six coronal brain sections (300 μm between sections) were cryosectioned for each mouse, starting from bregma +2.1 mm rostral to the dorsal end of the hippocampus at bregma -3.9 mm. The mounted sections were stained with 0.1% Sudan Black in 70% ethanol for 20 minutes at room temperature, followed by three 1-minute washes in 70% ethanol. The sections were washed three times with Milli-Q water and coverslipped with Fluoromount. The stained slices were imaged with a NanoZoomer, and regions of interest were traced and measured in each slice using the NOP viewer. The volume was calculated using the formula: volume = (sum of areas) × 0.3 mm. For the hippocampus and posterior lateral ventricle, quantification began at bregma -1.1 and ended at bregma -3.9. For the piriform / entorhinal cortex, quantification begins at bregma -2.3 and ends at bregma -3.9.

[0137] Neuronal layer thickness measurements. Three sections from each mouse (at bregma -1.4, -1.7, and -2.0 mm) were mounted and stained in cresyl violet for 5 minutes at room temperature. Slices were then dehydrated sequentially for 1 minute in 50%, 70%, 95% (three times), and 100% ethanol (twice), followed by 4 minutes in xylene (twice) and coverslipping in Cytoseal (Coon et al., 2007) (Thermo Fisher Scientific, 8310-16). The thickness of the CA1 pyramidal cell layer and the dentate gyrus granule cell layer were measured by drawing a scale perpendicular to the cell layer at two spots in all three slices and averaging the thickness for each mouse.

[0138] RNASeq transcriptome analysis. Total RNA was extracted using the TRIzol method, followed by cleanup using RNeasy columns (Qiagen). RNA quantity was assessed using a Nanodrop ND-1000 (Thermo Scientific), and RNA quality was assessed using a Bioanalyzer (Agilent Technologies) (Raman et al., 2009; Tumor Analysis, 2004). Total RNA was purified, amplified, and loaded onto an Illumina flow cell for paired-end sequencing using an Illumina HiSeq 2500 (Illumina) (Ryan et al., 2014). Libraries were prepared using the TruSeq RNA Library Prep Kit v2 (0.5 μg total RNA). Illumina HiSeq paired-end reads were aligned to the GRCh37 / hg19 human reference genome and RefSeq gene definitions (2014-06-02) using STAR (2.3.1z13_r470). Aligned reads were converted to fragments per kilobase of exon per million fragments sequenced (FPKM) using RefSeq gene definitions using Cufflinks (2.2). Genes with an FPKM >0.125 were included in the analysis. Dysregulated genes were functionally annotated using Gene Ontology (GO) and the Human Protein Reference Data Base (www.hprd.org). Numerical data were compared using a two-tailed Student's t-test, and categorical data were compared using a chi-square test with Yates' correction. Files were processed with Partek Genomics Suite software version 6.6, 2012 (Partek). One-way ANOVA is used to compare matched groups, and two- or three-way ANOVA is used to compare groups with parameters identified as sources of variation. A p-value of less than 0.05 is considered significant (p-values ​​were corrected for multiple testing using the Benjamini-Hochberg correction).In relation to "general pathology," normal cell function genes and autophagy-related genes will be evaluated as detailed in the study by Shi et al. (2017).

[0139] Amyloid-related: Quantification of amyloid / Aβ burden is performed using standard methods (Zhao et al., 2016; Bales et al., 2009; Holtzman et al., 2000; Bales et al., 1997). Briefly, four brain sections per mouse, each 300 μm apart, are selected for quantification. These sections roughly correspond to sections at bregma -1.4, -1.7, -2.0, and -2.3 mm in the mouse brain diagram (Franklin et al., 2019). Quantification of brain Aβ burden is completed after immunostaining with biotinylated 3D6 antibody (Zhao et al., 2016). Images are thresholded to highlight plaques and then analyzed using the "Analyze Particles" function in ImageJ software (National Institutes of Health). After thresholding, identified objects are individually inspected to confirm that each quantified object is a plaque. The percentage of surface area covered by AβIR (Aβ burden) or thioflavin-S (amyloid burden) is determined for specific regions of the hippocampal formation, including the stratum oriens, stratum pyramidale, stratum radiatum, and dentate gyrus. Aβ or thioflavin-S burden is then expressed as a percentage of the total quantified area. An average of four tissue sections is used to quantify plaque burden for each mouse. All analyses are performed blind, with the examiner unaware of the treatment condition of any animal.

[0140] Aβ levels are assayed in brain homogenates from mice using a sandwich enzyme-linked immunosorbent assay (ELISA) using standard methods (Bales et al., 2009, Holtzman et al., 2000, Bales et al., 1997). Briefly, brain tissue is homogenized sequentially using RIPA and 5.5-M guanidine buffer (1:1000, Roche) containing a cocktail of protease inhibitors. Aβ measured after RIPA extraction represents the soluble pool of Aβ, while Aβ measured after guanidine extraction represents the insoluble pool. Homogenates are diluted with cold sample dilution buffer (1% bovine serum albumin-0.05% TWEEN 20 in phosphate-buffered saline [PBST]) prior to measurement of Aβ1-40 or Aβ1-42. Samples were loaded onto plates coated with antibodies specifically recognizing the C-terminal domain of Aβ (21F12) or Aβ (2G3) as capture antibodies, and biotinylated 3D6 was used for detection. After incubation with horseradish peroxidase (HRP)-conjugated streptavidin (Research Diagnostics), the IR signal was developed with TMB substrate (Thermo Scientific) and read on a Synergy H1 Hybrid plate reader (BioTek). Aβ levels were calculated using a standard curve generated with recombinant human Aβ (American Peptide Company). Aβ levels in brain homogenates were determined in triplicate, normalized to protein content, and expressed as the amount of Aβ / mg protein. Aβ oligomers were quantified using a 3D6 / biotin-3D6 sandwich ELISA (Immuno-Biological Laboratories), in which the same N-terminal (residues 1–16) antibody was used for both capture and detection.

[0141] Tau-related ELISA. Human total tau and p-tau were quantified using pS202 / T205-tau (ATS) and pT212 / pS214-tau (AT100) antibodies as sandwich ELISAs, as described by Chai et al. (2011). Briefly, 96-well plates were pre-coated with 5 μg / mL ATS or 2 μg / mL AT100 (Thermo Fisher Scientific) overnight at 4°C, followed by blocking with Starting Block blocking buffer (Thermo Fisher Scientific). Samples (S1 or P1) were diluted in Superblock buffer (Thermo Fisher Scientific) and loaded onto the plate along with biotinylated HT7 antibody (1:300, Thermo Fisher Scientific). After incubation for 1 hour at 23°C, samples were washed nine times with TBS / 0.5% Tween 20 wash buffer and subsequently incubated with streptavidin-HRP (Jackson Immunoresearch) for 30 minutes. Plates were then developed by incubation with one-step 3,3,5,5-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) for 30 minutes, stopped with 2N H2SO4, and read at 450 nm using a BioTek Synergy H1 Hybrid Reader. The amount of ATS or AT100-immunoreactive tau was determined using a standard curve derived from human AD brain homogenate.

[0142] Inflammation-related. The extent and characteristics of gene therapy responses to inflammation associated with the two mouse models will be evaluated as described by Shi et al. (2017). Immunohistochemistry will be performed to assess CD68 positivity and Iba-1 microglial and astrocyte cell lines (GFAP). In addition, RNA-seq data will be evaluated using the same pro-inflammatory genes used by Shi et al. (2017), with a particular focus on pro-inflammatory gene cluster 1 and astrocyte A1-specific (inflammation-only) genes (Shi et al., 2017).

[0143] Statistical Considerations. A multi-way ANOVA model was applied for each measure within the categories of vector-derived APOE, general pathology, amyloid-related, tau-related, and inflammation-related (Table II). The main ANOVA model was based on mouse model (APP.PS1 / TRE4, P301S / E4), vector (AAVrh.10-APOE3ChC, -APOE3, AAVrh.10APOE2ChC, -APOE2, AAVrh.10Null), dose (0.5 × 10 10 and 1 × 10 10Additional separate ANOVAs, including five factors (g / c), time (5 months and 9 months), and sex (male / female), are used to compare one vector at a time with untreated PBS (but otherwise the same factors) to explore effects relative to the PBS control. The central hypotheses are evaluated using a primary ANOVA model with the following a priori contrasts for amyloid- and tau-related measures: AA Vrh.1 0-APOE3ChC vs. AA Vrh.1 0 Null (Objective 1), AAVrh.10APOE2ChC vs. AAVrh.10 Null, and AAVrh.10-APOE3ChC vs. AAVrh.10APOE2ChC, each of which applies across mouse strains, across doses within each mouse strain, and across time points within each mouse strain. This includes 45 (= vector comparisons × factor comparisons × measures) planned trials, and a Bonferroni correction is used to assess significance, controlling for an overall type I error of 0.05. In the PDAPP mouse brain amyloidosis experiment, the 27.2% difference in soluble Aβ1-42 and the observed standard deviation for each group when comparing AAVrh.10hAPOE2 with control (Figure 8) provided a power of greater than 0.85, and the power was even greater when the empirical difference and standard deviation for insoluble Aβ1-42 were considered, indicating that the overall design had sufficient power to evaluate the central hypothesis. Similar to these a priori comparisons, an exploratory post hoc approach (with appropriate corrections) will additionally be applied to assess brain vector-derived APOE abundance for different mouse models, doses, and measurement times, explore the impact of a wider range of vectors (e.g., AAVrh.10-APOE3ChC and AAVrh.10APOE2ChC compared to each other and to AAVrh.10-APOE3 and AAVrh.10-APOE2) on general pathology and inflammation-related measurements (volume and RNA-Seq), and predict the contribution of mouse model, vector, dose, measurement time, and gender to these effects.

[0144] Example 2 overview Alzheimer's disease (AD), a degenerative brain disease and the most common cause of dementia, currently affects 5.8 million Americans and 50 million people worldwide. AD symptoms include a progressive decline in cognitive and functional abilities, as well as brain lesions, including extracellular beta-amyloid plaques, intracellular tau tangles, chronic inflammation, and brain atrophy. The strongest genetic risk factor for susceptibility to late-onset AD involves polymorphisms in the apolipoprotein E (APOE) allele. APOE4 is frequently found in AD patients, and homozygous inheritance is associated with a 14.5-fold increased risk of developing AD. In contrast, APOE2 reduces the risk of developing AD and delays disease onset, e.g., reducing the risk of developing AD by more than 50% and delaying the age of onset. Based on epidemiological data, APOE4 is associated with increased cerebral amyloid burden and greater memory impairment in AD, while APOE2 attenuates these effects. In humans, the odds ratio for developing AD for the APOE4 / 4 homozygous genotype is 14.5, while this is reduced to 2.6 for APOE2 / 4 heterozygotes. Thus, the presence of APOE4 still poses a risk, even when supplemented by protective APOE2. In addition, APOE4, in addition to its role in promoting β-amyloid production, is associated with increased innate immune activity, differential signaling in neurons, exacerbation of tau pathology, and abnormal brain function.

[0145] Using adeno-associated virus (AAV) vectors to directly deliver APOE2 to the CNS of AD mouse models can result in significant expression of APOE2, reducing the levels of soluble and insoluble amyloid-β peptides and amyloid burden. However, even with APOE2 replacement, APOE2 / 4 heterozygotes have a 2.6-fold increased risk of developing AD, so the presence of APOE4 still increases the risk. In one embodiment, AAV-based gene therapy is provided that reduces the harmful levels of endogenous APOE4 in APOE4 homozygotes while simultaneously introducing protective APOE2, modified APOE2, or APOE3 expression. In one embodiment, an artificial microRNA (miRNA) targeting endogenous APOE4 is introduced into an AAV expression cassette along with the cDNA of the human APOE2 gene (hAPOE2-mirAPOE4). In one embodiment, AAV9 serotype capsid is used to package the expression cassette, as it mediates efficient transduction of astrocytes, the primary producers of APOE, as well as microglia and neurons.The established P301S / E4 AD mouse model expresses mutant human tau and human APOE4, and has high phosphorylated tau load, chronic inflammation, and extensive neurodegeneration, to evaluate therapeutic efficacy.To evaluate the AAV9-hAPOE2-mirAPOE4 strategy, the AAV construct is tested for silencing endogenous APOE4 expression and delivering the APOE2 coding sequence in vitro.In addition, it is determined whether increasing APOE2 with reduced endogenous APOE4 protects against tau pathology, neurodegeneration, and neuroinflammation in vivo.

[0146] Delivery of the human APOE2 coding sequence into the central nervous system (CNS) of two mouse models of AD via intrahippocampal or intrathalamic routes using adeno-associated virus (AAV) serotypes rh.10 and 9 resulted in significant levels of human APOE2 expression throughout the brain. Furthermore, the amount of soluble and insoluble amyloid-β peptides and amyloid burden was reduced in certain parts of the brain. However, genetic modification of the brain from APOE4 / 4 to APOE2 / 4 by providing APOE2 alone does not completely mitigate the risk of APOE4. Using AAV gene therapy to deliver protective APOE2 and reduce endogenous APOE4 expression may attenuate the risk of APOE4 homozygotes more than gene therapy with APOE2 alone.

[0147] The coding sequences of human APOE2 and APOE4 differ by two nucleotides. We designed an artificial microRNA (miRNA) sequence that targets endogenous APOE4 mRNA for suppression. The artificial miRNAs were screened in vitro for their ability to silence APOE expression in human astrocytoma cell lines. The selected miRNAs were incorporated into AAV expression cassettes along with a modified human APOE2 cDNA that cannot be silenced by the miRNA. The selected AAV expression cassettes containing APOE2 and the artificial miRNA targeting APOE4 were packaged into AAV9 capsids to target expression to astrocytes, the primary source of APOE in the CNS.

[0148] The P301S / E4 AD mouse model expresses both human mutant tau and human APOE4 and develops high levels of phosphorylated tau, brain atrophy, and neuroinflammation due to microglial activation. Because APOE in the CNS is primarily expressed in astrocytes, this study uses AAV9 vectors to efficiently transduce astrocytes as well as microglia and neurons. AAV9-hAPOE2-mirAPOE4 and AAV9-hAPOE2 vectors are administered directly into the CNS of P301S / E4 mice via the intracisternal route. Mice are evaluated for changes in behavior and cognitive function over time. After sacrifice, mice are evaluated for expression of vector-derived hAPOE2 and endogenous hAPOE4 in the brain, as well as for β-amyloid, total and phosphorylated tau levels and pathology, microglial activation, and brain atrophy.

[0149] Strategy. Alzheimer's symptoms include a progressive decline in cognitive and functional abilities, including memory loss, confusion, personality and mood changes, and loss of the ability to perform basic activities.1 Brain pathology associated with AD includes the accumulation of extracellular beta-amyloid (Aβ) plaques and intracellular neurofibrillary tau tangles. In advanced AD brains, there is chronic neuroinflammation due to glial activation and cell loss leading to brain atrophy (Association As., 2019).

[0150] The strongest genetic risk factor for susceptibility to late-onset AD relates to polymorphisms in the apolipoprotein E (APOE) allele. APOE has three isoforms: APOE2, APOE3, and APOE4. APOE3 is the most common allele, while the allele frequency of APOE4 is approximately 15% and APOE2 is 8% (Farrer et al., 1997). However, the frequency of AD patients with APOE4 ranges from 40% to 65% (Farrer et al., 1997; Corder et al., 1993; Ward et al., 2012). APOE4 is associated with an accelerated rate and severity of cognitive decline and an earlier age at onset of AD. In contrast, APOE2 reduces the risk of developing AD by 50% and delays its onset by up to 10 years ( Corder et al., 1993 , Corder et al., 1997 , Sando et al., 2008 , Shinohara et al., 2016 ).

[0151] APOE is a secreted protein that plays a major role in lipid transport in both the peripheral and central nervous systems (CNS). APOE isoforms differ by only two amino acids, positions 112 and 158 (APOE2: Cys / Cys, APOE3: Cys / Arg, APOE4: Arg / Arg). However, these differences profoundly affect APOE structure and function (Hatters & Peters-Libeu, 2006). Serum and brain concentrations of APOE differ between isoforms (APOE2>APOE3>APOE4). There are differences in lipidation status between APOE2 and APOE3, which are typically associated with high-density lipoproteins (HDL), and APOE4, which is associated with low- and very-low-density lipoproteins (LDL, VLDL) (Hatters & Peters-Libeu, 2006; Dong et al., 1994). Additionally, APOE isoforms differentially bind to receptors including low-density lipoprotein receptor-related 1 (LRP1), heparan sulfate proteoglycans (HSPGs), and the LDL receptor (LDLR), with APOE2 binding with lower affinity than APOE3 or APOE4 (Bu, 2009, Arboleda-Velasquez et al., 2019).

[0152] APOE plays multiple roles in the pathology and neurodegeneration seen in AD. Extracellular Aβ plaques are a hallmark of AD pathology. Aβ deposits are more prevalent in APOE4 carriers (Dorey et al., 2014). APOE4 exhibits toxic gain-of-function effects by enhancing Aβ production, reducing Aβ clearance by impairing lysosomal degradation and transport across the blood-brain barrier, and promoting Aβ aggregation and oligomer stabilization (Jiang et al., 2008; Du et al., 2009; Nielsen et al., 2010; Rodriguez et al., 2014; Zekonyte et al., 2016; Lin et al., 2018; Wang et al., 2018). Hyperphosphorylated tau is a major component of neurofibrillary tangles seen in the later stages of AD disease (Hampel et al., 2010). APOE4 expression exacerbates tau pathology (Shi et al., 2017). APOE4 exhibits weaker binding to tau than other isoforms (Strittmatter et al., 1994; Fleming et al., 1996), and APOE4 expression in neurons can increase tau phosphorylation (Wang et al., 2018; Brecht et al., 2004; Harris et al., 2004). APOE also has immunoregulatory functions, and greater immune responses are associated with the APOE4 allele (Shi & Holtzman, 2018). Recent studies have shown that innate immune activation in the CNS plays an important role in neuroinflammation and subsequent neuronal loss and brain atrophy in AD, and that microglial activation is promoted by the presence of APOE4 (Rodriguez et al., 2014; Shi et al., 2017; Shi et al., 2019). APOE4 binds to receptors on neurons with higher affinity than APOE2 and stimulates enhanced activation of multiple signaling pathways, including synaptogenesis, and increased amyloid precursor protein (APP) transcription ( Ohkubo et al., 2001 , Huang et al., 2017 , Huang et al., 2019 ).APOE4 binding to these receptors on damaged neurons may also act as an opsonin promoting neuronal phagocytosis by microglia through interaction with the APOE-binding partner "trigger receptor expressed on myeloid cells-2" (TREM2), which is expressed by microglia, leading to enhanced neuronal loss and brain atrophy (Atagi et al., 2015, Bailey et al., 2015, Yeh et al., 2016, Jendresen et al., 2017). Furthermore, APOE4 disrupts normal astrocyte and microglial homeostatic function (Fernandez et al., 2019). Clinically, APOE4 is associated with accelerated cognitive decline, whereas APOE2 is protective (Shinohara et al., 2016, Helkala et al., 1996, Staehelin et al., 1999, Wilson et al., 2002). APOE4 therefore plays a central role in multiple processes linked to AD pathogenesis and is a prime target for intervention.

[0153] Human genetic data indicate that APOE4 and APOE2 alleles have semidominant inheritance (Corder et al., 1994; Genin et al., 2011). APOE4 homozygous individuals have a 14.5-fold increased risk of developing AD, whereas APOE2 / 4 individuals have only a 2.6-fold increased risk, suggesting that the presence of a protective APOE2 allele may partially mitigate the deleterious effects of the APOE4 allele. The risk of developing AD is substantially reduced in APOE2 / 4 heterozygotes, but remains significantly higher than individuals with the APOE2 / 3 or APOE2 / 2 genotypes (1.8- and 7.7-fold reduced risk of developing AD, respectively (Genin et al., 2011)). This genetic data suggests that reducing APOE4 levels in the CNS, along with increasing APOE2, may be beneficial for treating APOE4 carriers at high risk for developing AD.

[0154] Human induced pluripotent stem cell-derived neurons expressing APOE4 had increased Aβ production, increased levels of tau phosphorylation, and more degeneration that was corrected by converting APOE4 to APOE3 through gene editing (Lin et al., 2018, Wang et al., 2018). Previous studies from our group and others have demonstrated that delivery of APOE2 to the CNS by viral vectors both increased APOE2 expression and reduced brain Aβ and amyloid burden in a mouse model of AD41-43. The dose of delivered APOE2 and the amount of pre-existing Aβ1-42 deposits were important factors in determining the efficacy of treatment (Zhao et al., 2016). However, in these previous studies, endogenous APOE4 was still present.

[0155] The disclosed treatments can be delivered to APOE4 homozygotes long before the onset of symptoms to prevent or halt the disease process before it leads to AD.

[0156] Previous studies have tested the delivery of AAV serotypes rh.10 and 9 of the human APOE2 coding sequence into the CNS of two AD mouse models via intrahippocampal or intrathalamic routes. Human APOE2 was expressed at high levels throughout the brain using intrathalamic delivery of AAV9-APOE2 vectors in the APP.PS1 / TRE4 AD mouse model, which expresses human amyloid precursor protein (APP) and presenilin 1 (PS1) mutations and human APOE4 instead of mouse APOE43 (Figure 21A). Furthermore, APOE2 augmentation reduced Aβ and amyloid burden in these AD mice (Zhao et al., 2016) (Figure 21B). One strategy is to encode an artificial microRNA (miRNA) targeting endogenous APOE4 in an AAV expression cassette along with the cDNA for the human APOE2 gene.

[0157] Target sequence selection. APOE4 sequences were evaluated using multiple algorithms for the best targeting sequences for small interfering RNA (siRNA) and screened to minimize the possibility of off-target interactions. The knockdown ability of selected siRNAs was tested in vitro on endogenous APOE in the U87 human astrocytoma cell line (Figure 22). The siRNA sequences were not specific for APOE4 and would inhibit the expression of all APOE isoforms. The best siRNA sequences were selected for incorporation into artificial miRNA cassettes for expression in AAV vectors.

[0158] The effects of gene therapy focus on tau pathology, neuroinflammation, and neurodegeneration. An AAV vector delivers sustained expression of human APOE2 along with an artificial miRNA that targets endogenous APOE4 mRNA to downregulate its expression (hAPOE2-mirAPOE4). AAV9, which efficiently transduces astrocytes, the primary source of APOE in the brain, as well as microglia and neurons, is used in this study (Xu et al., 2006; Foust et al., 2009; Gray et al., 2011; Gong et al., 2015; Vagner et al., 2016; Zhang et al., 2014; Zhang et al., 2014). This AAV gene therapy provides enhanced APOE2 expression compared with APOE4 in APOE4 homozygotes, reduces neurofibrillary tau tangles, decreases microglial activity, reduces hippocampal and cortical pathology, and improves behavior.

[0159] AAV constructs that silence endogenous APOE4 expression and deliver APOE2 coding sequences in vitro were identified. We identified siRNA sequences that effectively knocked down APOE expression in U87 human astrocytoma cells. These sequences target a segment of APOE common to all allelic sequences. The most effective siRNA sequences were selected and incorporated into an enhanced mir155 artificial miRNA scaffold to yield mirAPOE4 (Fowler et al., 2016) (Figure 23A). Because the placement of mismatches in the hairpin stem and GC content can affect miRNA processing in cells (Fowler et al., 2016; Fang & Bartel, 2015), several repeats of mirAPOE4 were tested for their ability to knock down APOE in vitro to ensure the most effective design. Two to four tandem copies of the optimized mirAPOE4 were cloned into either an intron present in the AAV expression cassette promoter (hybrid CMV / chicken β-actin; CAG) or into the intervening sequence between the transgene (mCherry or hAPOE2-HA) stop codon and polyadenylation signal to determine the optimal number and location of mirAPOE4 in the expression cassette (Mueller et al., 2012) (Figure 23B). The AAV mirAPOE4 expression cassette plasmid was tested in vitro for its ability to reduce APOE expression by transfecting U87 cells for 72 hours before harvesting the media and cells. APOE mRNA expression was assessed by RT-qPCR, and secreted APOE protein by Western blot and ELISA.

[0160] In parallel, silent mutations were engineered in the coding sequence of the human APOE2 cDNA at the selected mirAPOE4 recognition site to ensure that vector-derived APOE2 expression could not be silenced. The vector-derived human APOE2 carries a C-terminal hemagglutinin (HA) tag for easy detection. Expression of vector-derived hAPOE2-HA in the presence of mirAPOE4 was confirmed by cotransfection and analysis of hAPOE2-HA expression by Western blot with an anti-HA antibody. The optimized hAPOE2-mirAPOE4 cassette was packaged into an AAV9 serotype capsid. AAV9 efficiently transduces astrocytes, the primary source of APOE in the brain, as well as microglia and neurons. The functionality of the AAV9 vectors expressing hAPOE2-HA and mirAPOE4 was confirmed in vitro before proceeding to in vivo experiments.

[0161] Increased APOE2 expression may correlate with reduced endogenous APOE4 expression, which protects against tau pathology, neurodegeneration, and neuroinflammation in vivo. CNS administration of AAV9-hAPOE2 increased APOE levels throughout the brain and reduced Aβ1-42 levels in the APP.PS1 / TRE4 AD mouse model of Aβ amyloidosis in the human APOE4 background (Zhao et al., 2016; Kim et al., 2011) (Figure 21). Given the importance of neurofibrillary tau tangles and the innate immune response in AD brain atrophy and cognitive impairment independent of Aβ burden (Arboleda-Velasquez et al., 2019, Shi et al., 2017, Shi et al., 2019, Josephs et al., 2008, Mattsson et al., 2018), we investigated the role of tau pathology, microglial activation, and neurodegeneration in AD. P301S / E4 mice overexpress 1N4R human tau, which contains the P301S mutation associated with frontotemporal dementia, and human APOE4, which substitutes for mouse APOE (Shi et al., 2017, Yoshiyama et al., 2007). P301S / E4 mice exhibit higher brain phosphorylated tau burden and more extensive neurodegeneration and neuroinflammation than mice expressing human APOE3 or APOE2 (Shi et al., 2017, Shi et al., 2019), but do not exhibit amyloid plaque formation (Shi et al., 2017, Yoshiyama et al., 2007, Haurigot et al., 2013). P301S1N4R tau B6 / C3 mice (Jackson Laboratories 008169) and human APOE4 knock-in mice (TRE4-Taconic1549:B6.129P2-Apoetm3(APOE*4)Mae N8) can reproduce published models by crossing P301S mice with TRE4 mice. C57B1 / 6 mice are used as wild-type controls.

[0162] The effects of vector-mediated delivery of AAV9-hAPOE2-mirAPOE4 expressing human APOE2, along with miRNA for endogenous APOE4, were evaluated in P301S / E4 mice after intracerebral (IC) administration. The IC route provides broad vector distribution throughout the brain with minimally invasive administration (Haurigot et al., 2013; Hinderer et al., 2018; Rosenberg et al., 2018; Markmann et al., 2018). AAV9-hAPOE2, AAV9-mCherry-mirAPOE4, AAV9-null, and no treatment (PBS) were used as controls. Mice were evaluated monthly for behavioral and neurological assessments. After sacrifice, mice will be evaluated for vector-derived hAPOE2 and endogenous APOE4 expression, Aβ peptide burden, total soluble and insoluble tau burden, hyperphosphorylated tau tangles, microglial activation, and brain neurodegeneration at 1 and 7 months post-injection (total age: 3 and 9 months, respectively) (Table 3).

[0163] Table 3. Mouse studies TIFF2025163078000008.tif148135

[0164] Eight-week-old adult mice were used per treatment cohort, n = 16 males and n = 16 females (n = 192 total). All mice were randomly assigned to cohort groups to avoid potential study bias, and animals were staggered for dosing across all groups. Cages were numbered rather than labeled with treatment descriptions to keep researchers blinded to treatment status. The treatment cohort will contain a double mutant mouse strain (P301S / E4) carrying human tau and APOE4 gene substitutions. A wild-type control C57B1 / 6 strain with the same genetic background will be used as a "no treatment" control. Vectors tested included: AAV9-hAPOE2, AAV9-hAPOE2-mirAPOE4, AAV9-mCherry-mirAPOE4, AAV9-null, or no treatment (PBS). All vectors, 10 11The dose is administered via the IC route in genome copies (gc) or PBS (10 μl) into the CSF. This dose is the dose that is safe to deliver in humans (10 14 gc) (Sharma & McNeill, 2009). Health examinations were performed three times per week for the first two weeks, then weekly thereafter on all surviving mice to observe any abnormal or altered behavior. As part of the weekly health examinations, mice were weighed and recorded by personnel blinded to treatment cohort. Mice were sacrificed at two time points (3 and 9 months of age, 1 and 7 months after vector administration), with half of each cohort sacrificed (n = 8 males / 8 females). During necropsy, mouse CSF was first sampled from the cisterna magna, and the mice were then perfused with cold PBS. The brains were collected, flash-frozen in liquid nitrogen, and stored at -80°C. One half of the brain was used for vector DNA, mRNA, and protein assays. The remaining hemisphere was fixed and sectioned coronally at 50 μM slices for immunohistochemistry (IHC), immunofluorescence (IF), or pathology. At monthly intervals, mice are subjected to behavioral and neurological evaluations. Locomotor activity and ability to stand / sit are assessed in an open-field chamber equipped with an infrared beam array to monitor mouse movement. Second, mice are tested for cognitive ability using novel object recognition 24 hours after the introduction of a new object. Testing is performed inside the open-field chamber, and the time spent exploring the new object is recorded (Webster et al., 2013). AD mice show a decline in performance on this test with age. All tests are videotaped by blinded personnel to examine altered behavior. hAPOE2-HA and hAPOE4 proteins are detected in brain tissue samples from multiple regions by Western blot / ELISA with antibodies specific for the hAPOE4 and hAPOE2 HA tags, and mRNA is detected by RTqPCR using allele-specific probes ( Zhong et al., 2016 ). Microdissected sections of the hippocampus, thalamus, and cerebral cortex were homogenized and sequentially extracted with RIPA (soluble Aβ) and 5.5 M guanidine (insoluble Aβ). Aβ1-42 / 1-40 levels were quantified by ELISA (Zhao et al., 2016). Brain lysates are probed by Western blot with antibodies against total tau (BT2) and phosphorylated tau (CP13) ( Sacramento et al., 2020 ). Brain slices are stained with anti-phospho-tau antibody AT8 for IHC / IF (Shi et al., 2017; Sacramento et al., 2020). Microglial activation is assessed in brain sections by staining for CD68-positive cells by IHC (Shi et al., 2017). Multiple brain slices, separated by 300 μM, were stained with 0.1% Sudan Black for brain volume analysis. Regions of interest, including the hippocampus, posterior lateral ventricle, and piriform / entorhinal cortex, were traced and measured using imaging software. Three sections were stained with cresyl violet for neuronal layer thickness measurements in the CA1 pyramidal cell layer and dentate granule cell layer for observational changes in hippocampal regions. Measurements were recorded and averaged using ImageJ software at all sites (Shi et al., 2017). The neurotoxicity of the AAV9 vector itself is assessed in cresyl violet-stained brain sections by assessing the percentage of positively stained cells for each of the three sections.

[0165] Statistics. Tests were performed in triplicate for three independent experiments, and data are presented as mean ± standard deviation (SD) unless otherwise specified. Differences between groups were analyzed using one-way analysis of variance (ANOVA) for multiple comparisons. Tests for Aim 2 were performed using n = 8 mice per cohort (8 males / 8 females). With a coefficient of variation of 35% for both groups, using n = 8 per group would result in a 2.8-fold difference between groups (p < 0.05, power = 0.95). Differences between groups for biochemical measurements were analyzed by ANOVA for multiple comparisons. Behavioral assessments were analyzed by two-way repeated measures ANOVA with multiple comparisons.

[0166] Exemplary modified APOE2 sequences: TIFF2025163078000009.tif192166TIFF2025163078000010.tif252165TIFF2025163078000011.tif252165TIFF2025163078000012.tif22165.

[0167] References TIFF2025163078000013.tif217145TIFF2025163078000014.tif253154TIFF2025163078000015.tif25399TIFF2025163078000016.tif210160

[0168] All publications, patents, and patent applications are incorporated herein by reference. While in the foregoing specification the invention has been described in connection with certain specific embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and that certain of the details described herein may be varied considerably without departing from the underlying principles of the invention.

[0169] Sequence information SEQUENCE LISTING <110> Cornell University <120> APOE GENE THERAPY <150> US 62 / 939,999 <151> 2019-11-25 <160> 34 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 317 <212> PRT <213> Homo sapiens <400> 1 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> 2 <211> 1098 <212> DNA <213> Homo sapiens <400> 2 ggaacttgat gctcagagag gacaagtcat ttgcccaagg tcacacagct ggcaactggc 60 agagccagga ttcacgccct ggcaatttga ctccagaatc ctaaccttaa cccagaagca 120 cggcttcaag cccctggaaa ccacaatacc tgtggcagcc agggggaggt gctggaatct 180 catttcacat gtggggaggg ggctcccctg tgctcaaggt cacaaccaaa gaggaagctg 240 tgattaaaac ccaggtccca tttgcaaagc ctcgactttt agcaggtgca tcatactgtt 300 cccaccccctc ccatcccact tctgtccagc cgcctagccc cactttcttt ttttcttttt 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> 3 <211> 1157 <212> DNA <213> Homo sapiens <400> 3 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 gccctggtg gaacagggcc gcgtgcgggc cgccactgtg ggctccctgg ccggccagcc 720 gctacaggag cgggcccagg cctggggcga gcggctgcgc gcgcggatgg aggagatggg 780 cagcggacc cgcgaccgcc tggacgaggt gaaggagcag gtggcggagg tgcgcgccaa 840 gctggaggag caggcccagc agatacgcct gcaggccgag gccttccagg cccgcctcaa 900 gagctggttc gagcccctgg tggaagacat gcagcgccag tgggccgggc tggtggagaa 960 ggtgcaggct gccgtgggca ccagcgccgc cctgtgccc agcgacaatc actgaacgcc 1020 gaagcctgca gccatgcgac cccacgccac cccgtgcctc ctgcctccgc gcagcctgca 1080 gcgggagacc ctgtccccgc cccagccgtc ctcctggggt ggaccctagt ttaataaaga 1140 ttcaccaagt ttcacgc 1157 <210> 4 <211> 317 <212> PRT <213> Homo sapiens <400> 4 Put Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys 1 5 10 15 Gln Wing Lys Val Glu Gln Wing Val Glue Thr Glue Pro Glue Pro Glue 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 Thr 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 Pro 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> 5 <211> 317 <212> PRT <213> Homo sapiens <400> 5 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> 6 <211> 317 <212> PRT <213> Homo sapiens <400> 6 With 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 With Asp Glu Thr With 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 With 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> 7 <211> 317 <212> PRT <213> Homo sapiens <400> 7 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> 8 <400> 8 000 <210> 9 <400> 9 000 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 10 gtggagaagg tgcaggct 18 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 11 aagcgtaatc tggaacatcg t 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 12 ccctgtgccc agcgacaatc 20 <210> 13 <400> 13 000 <210> 14 <400> 14 000 <210> 15 <400> 15 000 <210> 16 <400> 16 000 <210> 17 <400> 17 000 <210> 18 <400> 18 000 <210> 19 <400> 19 000 <210> 20 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 20 gguggagcaa gcgguggagu u 21 <210> 21 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 21 ggaguugaag gccuacaaau u 21 <210> 22 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 22 ggaagacaug cagcgccagu u 21 <210> 23 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 23 gcgcgcggau ggaggagauu u 21 <210> 24 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 24 ctggaggctt gctgaaggct gtatgctgat ttgtaggcct tcaactcctg ttttggccac 60 tgactgacag gagtgaggcc tacaaatcag gacacaaggc ctgttactag cactcacatg 120 gaacaaatgg cc 132 <210> 25 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 25 ctggaggctt gctttgggct gtatgctgat ttgtaggcct tcaactcctg ttttggccac 60 tgactgacag gagttgaagt cacaaatcag gacacaaggc cctttatcag cactcacatg 120 gaacaaatgg ccaccgtggg aggatgacaa 150 <210> 26 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 26 ctggaggctt gctttgggct gtatgctgtt ccgatttgta ggccttcaag ttttggccac 60 tgactgactt gaagtcacaa atcggaacag gacacaaggc cctttatcag cactcacatg 120 gaacaaatgg ccaccgtggg aggatgacaa 150 <210> 27 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <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 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> 28 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <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 240 300 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> 29 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <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 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> 30 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <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 240 300 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> 31 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <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 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> 32 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 32 atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60 gagcaagcgg tggagacaga gccgggagccc gaggctcgcc 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 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 33 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 33 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 ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840 agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900 gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca c 951 <210> 34 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide sequence <400> 34 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 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. An expression cassette encoding a mammalian apolipoprotein E having a residue other than arginine at at least one of positions 112, 136, or 158, but which is not a mammalian apolipoprotein E having R112, R136, and R158, or a mammalian apolipoprotein E having C112, R136, and C158, or encoding an antibody that binds to APOE4 or disrupts the binding of APOE to heparan sulfate proteoglycans. A gene therapy vector comprising:

2. The gene therapy vector of claim 1 , wherein the apolipoprotein E is human apolipoprotein E.

3. 3. The gene therapy vector of claim 1, wherein the residue other than arginine in the apolipoprotein E is serine, threonine, asparagine, cysteine, or glutamine.

4. The gene therapy vector according to any one of claims 1 to 3, wherein position 112 of the apolipoprotein E has a cysteine.

5. The gene therapy vector of any one of claims 1 to 4, wherein position 136 of the apolipoprotein E has arginine or serine.

6. The gene therapy vector of any one of claims 1 to 5, wherein position 158 of the apolipoprotein E has arginine or cysteine.

7. The gene therapy vector of any one of claims 1 to 3, wherein two of positions 112, 136, or 158 of the apolipoprotein E have arginine.

8. The gene therapy vector according to any one of claims 1 to 3, wherein position 112 of the apolipoprotein E does not contain arginine.

9. The gene therapy vector according to any one of claims 1 to 3, wherein position 136 of the apolipoprotein E does not contain arginine.

10. The gene therapy vector according to any one of claims 1 to 3, wherein position 158 of the apolipoprotein E does not have arginine.

11. The gene therapy vector according to any one of claims 1 to 10, which is a viral gene therapy vector.

12. 12. The gene therapy vector of claim 11, which is an adenoviral, adeno-associated viral (AAV), retroviral, or lentiviral vector.

13. The gene therapy vector of claim 12, wherein the viral gene therapy vector is an rAAV vector.

14. The gene therapy vector of claim 13 , wherein the AAV vector is pseudotyped.

15. 15. The gene therapy vector of claim 14, wherein the AAV vector is pseudotyped with an AAVrh.10, AAV8, AAV9, AAV5, AAVhu.37, AAVhu.20, AAVhu.43, AAVhu.8, AAVhu.2, or AAV7 capsid.

16. 16. The gene therapy vector of claim 15, wherein the AAV vector is pseudotyped with AAVrh.10, AAV8, or AAV5.

17. The gene therapy vector of any one of claims 13 to 16, wherein the AAV vector is AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh.

10.

18. The gene therapy vector of any one of claims 1 to 17, further comprising a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibiting APOE4 mRNA.

19. The gene therapy vector of claim 18, wherein the nucleotide sequence is linked to a second promoter.

20. 20. The gene therapy vector of claim 19, wherein the second promoter is a Pol III promoter.

21. The gene therapy vector of any one of claims 18 to 20, wherein the RNAi comprises a miRNA comprising multiple miRNA sequences.

22. The gene therapy vector of any one of claims 18 to 21, wherein the RNAi comprises an siRNA comprising multiple siRNA sequences.

23. A pharmaceutical composition comprising a gene therapy vector according to any one of claims 1 to 22.

24. The pharmaceutical composition of claim 23 , wherein the vector is a viral vector.

25. 25. The pharmaceutical composition of claim 24, wherein the vector is an rAAV vector.

26. The amount of the vector is about 1×10 １１ ~Approx. 1×10 １６ 26. The pharmaceutical composition of claim 24 or 25, which is a genome copy.

27. 23. 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 of any one of claims 1-22.

28. 23. A method for preventing or inhibiting cognitive decline in a mammal, comprising administering to the mammal an effective amount of a composition comprising a gene therapy vector of any one of claims 1 to 22.

29. 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 a gene therapy vector described in any one of claims 1 to 22.

30. 23. A method for preventing, inhibiting, or treating a lipid disorder in a mammal, comprising administering to the mammal an effective amount of a composition comprising a gene therapy vector of any one of claims 1 to 22.

31. 31. The method of claim 27, 28, 29, or 30, wherein the mammal is an E2 / E4 heterozygote, an E4 / E4 homozygote, or an E3 / E4 heterozygote.

32. The method of any one of claims 27 to 31, wherein the mammal is a human.

33. The method of any one of claims 27 to 31, wherein the composition is administered systemically.

34. The method of any one of claims 27 to 32, wherein the composition is injected.

35. 35. The method of any one of claims 27-32 or 34, wherein the composition is administered to the central nervous system.

36. 35. The method of any one of claims 27 to 32 or 34, wherein the composition is administered to the brain.

37. The method of any one of claims 27 to 36, wherein a catheter is used to administer the composition.

38. The method of any one of claims 27 to 37, wherein the composition is a sustained release composition.

39. The vector is Apolipoprotein E having C112, S136, and R158, or having C112, S136, and C158 The method according to any one of claims 27 to 38, wherein the method encodes

40. The method of any one of claims 27 to 38, wherein the vector comprises a nucleotide sequence having an RNAi sequence corresponding to APOE4 for inhibiting APOE4 mRNA.

41. The method of any one of claims 27 to 38, wherein the mammal is further administered a second composition comprising a nucleotide sequence having an RNAi sequence corresponding to APOE4 for the inhibition of APOE4 mRNA.

42. 42. The method of claim 41, wherein the second composition comprises a liposome.

43. 42. The method of claim 41, wherein the second composition comprises nanoparticles.

44. The method of any one of claims 27 to 38, wherein the mammal is further administered an anti-heparan antibody, or an antibody that binds to APOE4 or disrupts the binding of APOE to heparan sulfate proteoglycans, or a second composition comprising a nucleotide sequence encoding said antibody.

45. 45. The method of claim 44, wherein a viral vector comprises the nucleotide sequence encoding the antibody.

46. 46. ​​The method of any one of claims 41 to 45, wherein the second composition is administered systemically.

47. 46. ​​The method of any one of claims 41 to 45, wherein the second composition is administered topically.

48. The vector according to any one of claims 27 to 47, wherein the vector is an AAV vector.

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