ApoE gene therapy

Abstract

The present invention relates to polynucleotide constructs encoding ApoE3 associated proteins, optionally containing one or more introns. Potential uses of the different constructs include gene therapy targeting one or more diseases or disorders, such as diseases or disorders associated with cholesterol levels, atherosclerosis, coronary heart disease, dementia, cerebral amyloid angiopathy, or Alzheimer's disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 378,960, filed on October 10, 2022, the disclosure of which is incorporated herein by reference in its entirety. Reference to electronically submitted sequence listings

[0002] The contents of the electronic Sequence Listing (065830_11WO1.xml; size: 210,461 bytes; creation date: October 5, 2023) are incorporated herein by reference in its entirety. Background Art

[0003] ApoE is synthesized as a 317-amino acid precursor protein containing an 18-amino acid leader sequence. Cleavage of the leader sequence produces the mature form of ApoE, which contains 299 amino acids. (Khalil et al., Atherosclerosis (2021) 328:11-22 and Tudorache et al., (2017) Computational and Structural Biotechnology Journal 15:359-365.)

[0004] ApoE is primarily involved in lipid metabolism and has diverse activities, including mediating hepatic and extrahepatic uptake of plasma lipoproteins and cholesterol efflux from lipid-loaded macrophages. Processes implicated in ApoE include neuroprotection, antimicrobial defense, and oxidative stress. (Khalil et al., Atherosclerosis (2021) 328:11-22 and Tudorache et al., (2017) Computational and Structural Biotechnology Journal 15:359-365.)

[0005] There are three main ApoE isoforms in humans: ApoE2, ApoE3, and ApoE4. The different isoforms are distinguished by amino acids at two positions. Regarding mature ApoE, ApoE2 has a cysteine ​​at amino acid 112 and a cysteine ​​at amino acid 158, ApoE3 has a cysteine ​​at amino acid 112 and an arginine at amino acid 158, and ApoE4 has an arginine at amino acid 112 and an arginine at amino acid 158. (Khalil et al., Atherosclerosis (2021) 328: 11-22 and Tudorache et al., (2017) Computational and Structural Biotechnology Journal 15: 359-365.)

[0006] Different ApoE isoforms (including minor forms) are associated with increased risk of different diseases or disorders. (Zhou et al., (2021) Current Opinion is Neurobiology 69: 58-67.) ApoE genotypes (when ordered as ε2 / ε2, ε2 / ε3, ε2 / ε4, ε3 / ε3, ε3 / ε4, ε4 / ε4) were identified to have an approximately linear relationship with LDL-C and coronary artery risk. (Bennet et al., JAMA. (2007) 298(11): 1300-1311). Additional associations include that ApoE2 homozygosity often leads to type III hyperlipoproteinemia and that ApoE4 is associated with Alzheimer's disease. (Khalil et al., Atherosclerosis (2021) 328: 11-22 and Tudorache et al., (2017) Computational and Structural Biotechnology Journal 15: 359-365.)

[0007] The substitution identified as the Christchurch substitution provides an arginine to serine substitution at ApoE 136. ApoE2 containing the Christchurch substitution appears to significantly contribute to hyperlipidemia. (Wardel et al., J. Clin. Invest. (1987) 80(2): 483-490.) Patients with two copies of ApoE3 containing the Christchurch substitution ("ApoE3(ch)") are indicated to be resistant to autosomal dominant Alzheimer's disease and have elevated triglycerides and total cholesterol. (Arboleda-Velasquez et al., Nature Medicine (2019) 25: 1680-1683.)

[0008] International Patent Publication Nos. WO 2022 / 115535, WO 2021 / 108809, and WO 2020 / 243346 refer to treatments for Alzheimer's disease. Summary of the Invention

[0009] The present invention features a polynucleotide construct comprising a nucleotide sequence having a region having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 63-67 or nucleotides 55-951 of SEQ ID NOs: 95-105 or 124-130; optionally comprising one or more introns; and a CpG-reduced intron relative to any one of SEQ ID NOs: 119-121. Preferred constructs are CpG-reduced compared to the native ApoE3 sequence and may further comprise additional variants compared to the native sequence. The polynucleotide constructs can be used, for example, in gene therapy targeting one or more diseases or disorders (e.g., diseases or disorders associated with cholesterol levels, atherosclerosis, coronary heart disease, dementia (e.g., vascular dementia or frontotemporal dementia), cerebral amyloid angiopathy, or Alzheimer's disease).

[0010] Thus, a first aspect of the invention features a polynucleotide comprising a nucleotide sequence encoding ApoE having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 63-67 or nucleotides 55-951 of SEQ ID NOs: 95-105 or 124-130, wherein the polynucleotide encodes an ApoE3-related protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 32, wherein the protein comprises a cysteine ​​at a position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at a position corresponding to amino acid 158 of SEQ ID NO: 32, and wherein the nucleotide sequence encoding ApoE optionally comprises one or more introns. The at least 85% identity to any one of SEQ ID NOs: 63-67 or nucleotides 55-951 of SEQ ID NOs: 95-105 or 124-130 is independent of any introns that may be present in the ApoE-encoding nucleic acid.

[0011] Nucleotides 1-897 of SEQ ID NOs: 63-67 encode amino acids. Nucleotides 898-900 of SEQ ID NOs: 63-67 encode stop codons.

[0012] Reference to an indicated percent identity with two or more reference sequences and similar language throughout the specification providing an indicated percent identity with two or more reference sequences provides the indicated percent identity or percent identity range independently with each reference sequence. In determining percent identity of polynucleotides, in the absence of reference to whether the polynucleotide is RNA or DNA, the RNA and corresponding DNA are considered identical. The corresponding RNA and DNA include substitutions of uracil with thymine and ribose backbones with deoxyribose backbones.

[0013] Provided herein are various polynucleotide constructs, including polynucleotides encoding ApoE3-related proteins, polynucleotides comprising an expression cassette comprising a nucleic acid sequence encoding an ApoE3-related protein operably linked to one or more expression control elements, polynucleotides comprising a recombinant viral vector nucleic acid wherein the 5′ and / or 3′ ends of the polynucleotide have elements that provide for packaging into a viral vector and / or viral replication, and vector genome plasmids.

[0014] Reference to one or more expression control elements being "operably linked" or "operably coupled" to a nucleic acid encoding ApoE indicates that the one or more expression control elements affect ApoE3-related protein expression. ApoE3-related protein expression can be affected in various ways, such as increased production of ApoE3-related protein mRNA transcripts, increased nuclear transport and stability of mRNA transcripts, and increased mRNA translation.

[0015] Another aspect of the invention relates to administering the polynucleotide constructs described herein to achieve one or more of the following: lowering cholesterol, lowering LDL / VLDL, increasing HDL, or lowering the total cholesterol / HDL ratio; or treating or reducing the likelihood of hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia, post-stent restenosis, atherosclerosis, coronary heart disease, or Alzheimer's disease.

[0016] Additional aspects include the polynucleotide constructs described herein for use in medicine or to achieve one or more of the following: lowering cholesterol, lowering LDL / VLDL, increasing HDL, or lowering the total cholesterol / HDL ratio; or treating or reducing the likelihood of hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia, post-stent restenosis, atherosclerosis, coronary heart disease, or Alzheimer's disease; and the use of the constructs described herein in the preparation of a medicament for use in medicine or to achieve one or more of the following: lowering cholesterol, lowering LDL / VLDL, increasing HDL, or lowering the total cholesterol / HDL ratio; or treating or reducing the likelihood of hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia, post-stent restenosis, atherosclerosis, coronary heart disease, or Alzheimer's disease.

[0017] Additional aspects include AAV vector genomes; methods of producing rAAV vectors; methods of obtaining rAAV vectors; and polynucleotides related to any one of SEQ ID NOs: 119-121, wherein the polynucleotides have 0-5 CpGs.

[0018] Other features and advantages of the present invention will be apparent from the additional descriptions (including various examples) provided herein. The examples provided illustrate different components and methods useful in practicing the present invention. Such examples do not limit the claimed invention. Based on this disclosure, a skilled artisan can identify and employ other components and methods useful in practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic examples of recombinant adeno-associated virus (rAAV) polynucleotide cassettes are provided. The provided examples provide the locations of the 5' ITR, ApoE / hAAT promoter / enhancer, HBB2 intron, Kozak sequence, nucleic acid encoding ApoE3(ch), poly A sequence, and 3' ITR.

[0020] Figure 2A-2E The results show the expression of ApoE3 and ApoE3ch transgenes in ApoE knockout mice over the course of 36 weeks and the effect of transgene expression on cholesterol. Recombinant AAV containing the ApoE3 (ch) transgene was administered at a low dose of 3e12 vg / kg or a high dose of 1e13 vg / kg. Recombinant AAV containing the ApoE3 transgene was administered at a low dose of 3e12 vg / kg or a high dose of 6e12 vg / kg rAAV. Figure 2A hAPOE plasma levels are shown. Figure 2B Total cholesterol is shown, Figure 2CLDL / VLDL is shown, Figure 2D HDL is shown, and Figure 2E Total cholesterol / HDL ratios are shown. The week 0 time point indicates cholesterol levels before AAV administration.

[0021] Figure 3A-Figure 3E Shown are ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 3 weeks post-treatment. Figure 3A hAPOE levels are shown (**p<0.01, ***p<0.001, ***p<0.0001 compared to vehicle; +, equal to APOE3ch native expression), Figure 3B Total cholesterol is shown (#p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 compared to WT vehicle; *p<0.05, **p<0.01, **p<0.001, ****p<0.0001 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted on selection bars); Figure 3C LDL / VLDL is shown (##p<0.01, ###p<0.001, ####p<0.0001 compared to WT vehicle; *p<0.05, ***p<0.0001 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted on selection bars); Figure 3D HDL cholesterol is shown (#p<0.05, ##p<0.01, ###p<0.001 compared to WT vehicle); and Figure 3E Total cholesterol / HDL ratios are shown (##p<0.01 compared to WT vehicle; *p<0.05 compared to KO vehicle; percent increase relative to KO vehicle animals is depicted above selection bars).

[0022] Figures 4A-4E Shown are ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 4A hAPOE levels are shown (**p<0.01, ***p<0.001, ***p<0.0001 compared to vehicle; +, equal to APOE3ch native expression), Figure 4B Total cholesterol is shown (##p<0.01, ####p<0.0001 compared to WT vehicle; *p<0.05, ***p<0.001, ***p<0.0001 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted on selection bars); Figure 4CLDL / VLDL is shown (###p<0.001, ####p<0.0001 compared to WT vehicle; *p<0.01, ***p<0.0001 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted on selection bars); Figure 4D HDL cholesterol is shown (#p<0.05, ####p<0.0001 compared to WT vehicle; *p<0.05, **p<0.01 compared to KO vehicle; percent increase relative to KO vehicle animals is depicted on selection bars); and Figure 4E Total cholesterol / HDL ratios are shown (##p<0.01 compared to WT vehicle; *p<0.05 compared to KO vehicle).

[0023] Figures 5A-5E Shown are ApoE3 transgene expression from different constructs at 3 weeks and the effect of transgene expression on cholesterol. Figure 5A hAPOE levels are shown (***p<0.001, ***p<0.0001 compared to vehicle; unless indicated by brackets); Figure 5B Total cholesterol is shown (#p<0.05, ###p<0.01, ####p<0.0001 compared to WT vehicle; *p<0.05, ***p<0.0001 compared to vehicle; or comparisons are indicated by brackets; percent reduction relative to KO vehicle animals is depicted above selection bars); Figure 5C LDL / VLDL is shown (#p<0.05, ###p<0.01, ####p<0.0001 compared to WT vehicle; *p<0.05, ***p<0.0001 compared to KO vehicle; or comparisons are indicated by brackets; percent reduction relative to KO vehicle animals is depicted above selection bars); Figure 5D HDL cholesterol is shown (#p<0.05, ###p<0.01, ####p<0.0001 compared to WT vehicle; *p<0.05, ***p<0.0001 compared to KO vehicle; or comparisons are indicated by brackets; percent increase relative to KO vehicle animals is depicted on selection bars); and Figure 5E Total cholesterol / HDL ratios are shown (##p<0.01 compared to WT vehicle; *p<0.05 compared to KO vehicle).

[0024] Figures 6A-6E Shown are ApoE3 transgene expression from different constructs at 6 weeks and the effect of transgene expression on cholesterol. Figure 6A hAPOE levels are shown (***p<0.001, ***p<0.0001 compared to vehicle unless indicated by brackets); Figure 6B Total cholesterol is shown (#p<0.05, ####p<0.0001 compared to WT vehicle; ***p<0.0001 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted above selection bars). Figure 6C LDL / VLDL cholesterol is shown (#p<0.05, ###p<0.001, ####p<0.0001 compared to WT vehicle; ***p<0.0001 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted above selection bars). Figure 6D HDL cholesterol is shown (#p<0.05, ##p<0.01, ####p<0.0001 compared to WT vehicle; **p<0.01 compared to KO vehicle; percent reduction relative to KO vehicle animals is depicted above selection bars). Figure 6E Total cholesterol / HDL ratios are shown (##p<0.01 compared to WT vehicle; *p<0.05, **p<0.01 compared to KO vehicle).

[0025] Figure 7A and Figure 7B hAPOE / total protein produced by different constructs is shown ( Figure 7A ; p < 0.05 compared to E3 (ch) native construct) and vector genome copy number / μg gDNA ( Figure 7B ) in liver tissue. Figure 7B "Not significant compared to native" in the indicates that the differences between E3ch native and the different CpG reduced constructs were not significant (*p<0.05 for constructs E3-8, E3-9, E3-11, E3-12 and E3-15 compared to E3 native construct).

[0026] Figure 8 Black and white images of atherosclerotic plaque fatty content stained with Oil Red O are provided. WT vehicle refers to wild-type mice (C57BL / 6) administered vehicle. KO vehicle refers to ApoE knockout mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052) administered vehicle. APOE3ch(low) refers to KO mice administered a low dose of 3e12 vg / kg rAAV. APOE3ch(high) refers to KO mice administered a high dose of 1e13 vg / kg rAAV. APOE3(low) refers to KO mice administered a low dose of 3e12 vg / kg rAAV. APOE3(high) refers to KO mice administered a high dose of 6e12 vg / kg rAAV.

[0027] Figure 9 The percentage of aortic lesion area is shown. WT vehicle refers to wild-type mice (C57BL / 6) administered with vehicle. KO vehicle refers to ApoE knockout mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052) administered with vehicle. APOEch(low) refers to KO mice given a low dose of 3e12vg / kg rAAV. APOEch(high) refers to KO mice given a high dose of 1e13vg / kg rAAV. APOE3(low) refers to KO mice given a low dose of 3e12vg / kg rAAV. APOE3(high) refers to KO mice given a high dose of 6e12vg / kg rAAV. ***p<0.0001 compared to KO vehicle; or comparisons are indicated by brackets.

[0028] Figures 10A-10F : is a bar graph showing the anti-inflammatory effect of rAAV containing APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12 vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13 vg / kg rAAV. Anti-inflammatory effects were measured using the MesoScale Diagnostics Mouse Cytokine Panel. Figure 10A IL-5 levels are shown, Figure 10B IL-6 levels are shown, Figure 10C TNF-α levels are shown. Figure 10D IL-17A / F levels are shown, Figure 10E CCL2 levels are shown. Figure 10F CXCL2 is shown. One-way ANOVA, Dunnett's post hoc test. #p<0.05, ##p<0.01 compared to WT vehicle; *p<0.05, **p<0.01 compared to KO vehicle.

[0029] Figures 11A-11D: is a bar graph showing the effect of rAAV containing APOEch or APOE transgene on glial fibrillary acidic protein (GFAP) levels in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052). APOE3ch L and APOE3-L refer to KO mice given a low dose of 3e12 vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13 vg / kg rAAV. Figure 11A Shown are GFAP / total protein in the cortex as determined by JESS capillary electrophoresis (ProteinSimple). Figure 11B Shown are GFAP / total protein in the hippocampus as determined by JESS capillary electrophoresis (ProteinSimple). Figure 11C The % GFAP area in the whole brain determined by immunofluorescence staining with anti-GFAP antibody (AB5541, Millipore; 1:500) and the percentage of GFAP area in the whole brain determined by immunofluorescence staining with anti-GFAP antibody (AB5541, Millipore; 1:500) are shown. The percentage of GFAP area was quantified by image analysis software (IndicaLabs). Figure 11D Shows the use GFAP area% in the hippocampus determined by immunofluorescence quantification using image analysis software (IndicaLabs) One-way ANOVA followed by Dunn or Fisher post hoc test. #p<0.05 compared to WT, *p<0.05, **p<0.01 compared to KO vehicle.

[0030] Figures 12A-12D : is a bar graph showing the effects of rAAV containing APOE3ch or APOE3 transgene on presynaptic and postsynaptic proteins in the cortex and hippocampus in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052). APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12 vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13 vg / kg rAAV. Figure 12A Shown are synaptic vesicle protein / total protein in the hippocampus. Figure 12B PSD-95 / total protein in hippocampus is shown. Figure 12C Shown are synaptic vesicle protein / total protein in the cortex. Figure 12DPSD-95 / total protein in the cortex is shown. Synaptic proteins were determined by JESS capillary electrophoresis (ProteinSimple). Kruskal-Wallis one-way ANOVA followed by Dunn's post hoc test. #p<0.05 compared to WT, *p<0.05, **p<0.01 compared to KO vehicle

[0031] Figures 13A-13D is a bar graph showing the effect of rAAV containing APOE3ch or APOE3 transgene on reversal of pre-existing atherosclerosis in 1-year-old ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052). Figure 13A The results show that the expression of ApoE in KO mice and mice overexpressing ApoE(E3N(native ApoE3)2e 11 vg / kg、E3-3(ApoE3-3)2e 11 vg / kg and E3-3 (ApoE3-3)2e 12 % of aortic lesion area in mice with different transgenic sequences (vg / kg). Figure 13B Shown from Figure 13A The KO baseline neutralization from Figure 13A E3N (native ApoE3)2e 11 vg / kg and E3-3 (ApoE3-3)2e 11 % change in atherosclerotic lesions in the combined vg / kg groups. Figure 13C The results show that the KO mice were untreated and the mice were given the gene encoding ApoE3ch (E3N (native ApoE3ch) 2e 11 vg / kg、E3ch-9(ApoE3ch-9)2e 11 vg / kg and E3ch-9 (ApoE3ch-9) 2e 12 % of aortic lesion area in KO mice with different transgenic sequences (vg / kg). Figure 13D Shown from Figure 13C The KO baseline neutralization from Figure 13C E3N (natural ApoE3ch) 2e 11 vg / kg and E3ch-9 (ApoE3ch-9) 2e 11 % change in atherosclerotic lesions in the combined vg / kg groups.

[0032] Figures 14A-14DFigure 2 shows the results of cognitive deficits in the novel object recognition (NOR) memory test in mice administered with rAAV containing the APOE3(ch) transgene. rAAV was administered at a low dose of 2e11 vg / mouse or a high dose of 2e12 total vg / mouse. Figure 14A Shown are NOR results for 1-year-old C57BL / 6 and ApoE knockout mice (*p<0.05, unpaired two-tailed t-test), indicating cognitive impairment in 1-year-old ApoE knockout mice compared to age-matched C57BL / 6 mice. Figure 14B Shown are the NOR results using native ApoE3 and ApoE3(ch) sequences in the same mice before and after gene therapy treatment, as well as 5 weeks after treatment with gene therapy. Figure 14C Shown are NOR results with low doses of rAAV encoding CpG-0 ApoE3 (E3-3) and ApoE3ch (E3ch-9) rAAV. Figure 14D Shown are NOR results with high doses of rAAV encoding CpG-0 ApoE3 (E3-3) and ApoE3ch (E3ch-9) rAAV.

[0033] Figure 15A and Figure 15B ApoE expression from different transgenes is shown. Figure 15A A bar graph showing the performance of codon-optimized, CpG-reduced cDNA encoding ApoE3 is provided. Plasmids encoding wild-type (wt) ApoE3 or codon-optimized ApoE3 cDNA were transfected into AML-12 cells in triplicate, and antigen levels in the cell culture supernatant were measured 72 hours after transfection. ApoE3 levels were determined by ELISA and plotted as mean + / - standard deviation. Figure 15B A bar graph showing the performance of codon-optimized ApoE3 cDNA functionalized by the addition of intron sequences is provided. Plasmids encoding wild-type (wt) ApoE3 or intron-containing cDNA variants of ApoE3 were transfected into AML-12 cells in triplicate, and antigen levels in the cell culture supernatant were measured 72 hours after transfection. For comparison purposes, codon-optimized ApoE3-3 and H30 variants containing no introns were included as benchmarks. ApoE3 levels were determined by ELISA and plotted as mean + / - standard deviation. DETAILED DESCRIPTION

[0034] The invention features a polynucleotide construct comprising a nucleotide sequence encoding ApoE having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 63-67 or nucleotides 55-951 of SEQ ID NOs: 95-105 or 124-130, wherein the polynucleotide encodes an ApoE3-related protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 32, wherein the protein comprises a cysteine ​​at a position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at a position corresponding to amino acid 158 of SEQ ID NO: 32, and wherein the nucleotide sequence encoding ApoE optionally comprises one or more introns. ApoE3(ch) is an ApoE3-related protein comprising a serine at a position corresponding to amino acid 136 of SEQ ID NO: 32.

[0035] The constructs provided can be used in different methods, including gene therapy for different diseases or disorders related to cholesterol, lipoprotein levels, cardiovascular disease, dementia or Alzheimer's disease in a target subject. The examples provided below illustrate, for example, in a novel object recognition test in ApoE knockout mice, the ability of different transgenic expressions encoding ApoE3 (ch) and ApoE3 to reduce total cholesterol levels, increase HDL, reduce LDL / VLDL, reduce total cholesterol / HDL ratios, reduce atherosclerotic lesions (atherosclerotic plaques), reduce inflammatory proteins, increase synaptogenesis and improve cognitive function. Additional uses of the constructs include studying the effects of ApoE gene therapy in animal models.

[0036] Reference to a "subject" refers to mammals, including humans; non-human primates, such as apes, gibbons, gorillas, chimpanzees, orangutans, and macaques; domestic animals, such as dogs and cats; farm animals, such as poultry, ducks, horses, cattle, goats, sheep, and pigs; and laboratory animals, such as mice, rats, rabbits, and guinea pigs. A preferred subject is a human.

[0037] Polynucleotides encoding ApoE3-related proteins can be delivered using non-viral or viral delivery. Viral vectors that can be used for gene therapy include retroviral vectors, adenoviral vectors, AAV vectors, and herpes simplex virus vectors. Non-viral delivery includes naked DNA and the use of nanoparticles.

[0038] Reference to "identical," "percent identity," and similar terms refers to two sequences having maximum alignment over a specified region. The regions provided are with respect to the indicated reference sequence. For example, "identical" or "identical" to a sequence of human mature ApoE3 protein can be calculated by determining the number of identical amino acids in the aligned sequences, dividing by the total number of amino acids in SEQ ID NO: 32 (299 amino acids), and multiplying by 100. The percent "identical" or "identical" of a nucleic acid sequence can be determined in a similar manner by aligning the nucleotides to a reference sequence for maximum alignment, dividing by the total number of nucleotides in the reference sequence, and multiplying by 100. The percent "identical" or "identical" of an ApoE coding sequence determined independent of any introns indicates that one or more introns are removed prior to alignment for calculation purposes.

[0039] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in a 5' to 3' direction.

[0040] In certain embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA (polynucleotides and oligonucleotides), chromosomal DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA, inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA), locked nucleic acid analogs (LNA), single-stranded and double-stranded oligonucleotide DNA (ODN), immunostimulatory sequences (ISS), riboswitches, and ribozymes.

[0041] In certain embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single, doublets or triplets, quadruplets, linear or circular, and can be of any length.

[0042] According to certain embodiments, the polynucleotide is a single-stranded (ssDNA) or double-stranded DNA (dsDNA) molecule. According to certain embodiments, the dsDNA molecule is a minicircle, a nanoplasmid, an open linear duplex DNA, or a closed-end linear duplex DNA (CELiD / ceDNA / doggybone DNA). According to certain embodiments, the ssDNA molecule is a closed circular or open linear DNA.

[0043] "Transgene" refers to a nucleic acid that is intended to be or has been introduced into a cell and is operably linked to a promoter. A transgene includes a heterologous polynucleotide sequence, such as a nucleic acid encoding an ApoE3-related protein, and a heterologous promoter.

[0044] Preferred polynucleotide constructs are "CpG-reduced" or "CpG-depleted." "CpG-reduced" or "CpG-depleted" refers to a nucleotide sequence in which (i) one or more CpG dinucleotides (or motifs) are removed from a reference nucleic acid sequence; and / or (ii) the CpG percentage in the polynucleotide is between 0% and 15%. In various embodiments, the percentage of CpG is 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpG; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0%, at most about 5.0%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, or at most about 15% CpG.

[0045] In the nucleotide sequence encoding the ApoE3-related protein and other sequences present in a specific construct (e.g., expression cassette and viral vector), CpG motifs can be appropriately reduced or eliminated. Other sequences that may be present include non-coding sequences such as 5' and 3' untranslated regions (UTRs), stuffer sequences, promoters, enhancers, polyadenylation signals, ITRs, and one or more introns.

[0046] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] The connecting term "and / or" between multiple statement elements covers both individual options and combined options. For example, where two elements are combined by "and / or", the first option refers to the applicability of the first option without the second option, the second option refers to the applicability of the second option without the first option, and the third option refers to the applicability of the first option and the second option together. Any of the options is understood to fall within the meaning of the term "and / or" and therefore meets the requirements of the term "and / or". More than one of the options that are applicable at the same time is also understood to fall within the meaning of the term "and / or".

[0048] The terms "or" and "and" have the same meaning as "and / or" unless the context in which they are employed clearly indicates otherwise.

[0049] References to terms such as "including," "for example," "eg," "such as," followed by different members or examples are open-ended descriptions where the listed members or examples are illustrative and additional members or examples may be provided or used.

[0050] The terms "polypeptide," "protein," and "peptide" are used interchangeably to refer to an amino acid sequence without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acids. Provided amino acids include naturally occurring amino acids and amino acids provided by cellular modification.

[0051] References to "comprise," and variations such as "comprises" and "comprising," as used with respect to an element or a group of elements, are open ended and do not exclude additional, unrecited elements or method steps. Terms such as "including," "containing," and "characterized by" are synonymous with comprising. In the various aspects and embodiments described herein, references to open ended terms such as "comprising" may be replaced with "consisting of" or "consisting essentially of."

[0052] Reference to "consisting of" excludes any element, step or ingredient not specified in the listed claimed elements, where such element, step or ingredient is relevant to the claimed invention.

[0053] Reference to "consisting essentially of" limits the scope of a claim to the specified materials or steps and to those materials or steps that do not materially affect one or more basic and novel characteristics of the claimed invention.

[0054] The term "about" refers to a value that is within 10% (i.e., plus or minus 10%) of the underlying parameter. For example, "about 1:10" includes 1.1:10.1 or 0.9:9.9, and "about 5 hours" includes 4.5 hours or 5.5 hours. The term "about" at the beginning of a series of values ​​modifies each value by 10%.

[0055] Unless the context clearly indicates otherwise, all numerical values ​​or numerical ranges include the integers within such ranges and fractions of the integers within the stated values ​​or ranges. Thus, for illustration, reference to a 95% or greater reduction includes 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, and so on, and reference to a numerical range such as "1-4" includes 1, 2, 3, 4, as well as 1.1, 1.2, 1.3, 1.4, and so on. As a further illustration, "1 to 4 weeks" includes 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days.

[0056] Furthermore, reference to numerical ranges (e.g., "0.01 to 10") includes 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dose of about "0.01 mg / kg to about 10 mg / kg" of a subject's body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0057] Reference to an integer that is more than (greater than) or less than includes numbers greater than or less than the referenced number, respectively. Thus, for example, reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more; and reference to "two or more times" includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times.

[0058] Various references, including articles and patent publications, are cited or described in the Background and throughout the specification. Each of these references is incorporated herein by reference in its entirety. No reference is admitted to be prior art with respect to any invention disclosed or claimed. In some cases, specific references are indicated as incorporated herein by reference to highlight the incorporation.

[0059] The definitions provided herein, including those in this section and elsewhere in this application, apply throughout this application.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] The description has been divided into various parts and paragraphs, and examples of various embodiments are provided. These separations should not be considered as disconnecting the essence of one paragraph or part or embodiment from the essence of another paragraph or part or embodiment. The description provided has a wide range of applications and covers all combinations of conceivable parts, paragraphs and sentences. The discussion of any embodiment is only meant to be exemplary and is not intended to imply that the scope of this disclosure (including the claims (unless otherwise provided in the claims)) is limited to these examples.

[0062] In this article, affirmative language is used to disclose the present invention in general to describe numerous embodiments of the present invention. The present invention also specifically includes embodiments in which specific themes (such as substances or materials, method steps and conditions, schemes or procedures) are excluded in whole or in part. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Therefore, even if the present invention is not usually expressed in this article with the content that the present invention does not include, the embodiments that are not clearly excluded in the present invention are still disclosed in this article.

[0063] I. Polynucleotides encoding ApoE protein

[0064] The nucleotide sequence encoding ApoE encodes an ApoE3-related protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 32, wherein the protein contains a cysteine ​​at a position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at a position corresponding to amino acid 158 of SEQ ID NO: 32. If one or more introns are present, the percent identity is determined independently of the one or more introns. In certain embodiments, introns are absent from the nucleic acid encoding ApoE.

[0065] In various embodiments, the ApoE3-related protein is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 32; differs from SEQ ID NO: 32 by 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acids; or is provided by SEQ ID NO: 32.

[0066] In various embodiments, the ApoE3-related protein further comprises a serine at a position corresponding to amino acid 136 of SEQ ID NO: 32 and is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of SEQ ID NO: 32; differs from ApoE3(ch) of SEQ ID NO: 33 by 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acids; or is provided by SEQ ID NO: 32 or 33. The serine at the position corresponding to amino acid 136 of SEQ ID NO: 32 provides a Christchurch substitution.

[0067] Based on the existing knowledge of different ApoE domains and different ApoE sequences, additional ApoE3-related proteins can be obtained using the ApoE3 sequences of SEQ ID NO: 32 and SEQ ID NO: 33 as starting constructs. Examples of references describing different domains and sequences include Khalil et al., Atherosclerosis (2021) 328: 11-22 and Tudorache et al., (2017) Computational and Structural Biotechnology Journal 15: 359-365.

[0068] In certain embodiments, the ApoE coding sequence comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 63-67 and nucleotides 55-954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30 and SEQ ID NOs: 95-105 or 124-130. In further embodiments, the sequence identity provided is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or nucleotides 1-897 of any of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67; or SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 and SEQ ID Nucleotides 55-951 or 55-954 of any one of NO:130.

[0069] In certain embodiments, the ApoE coding sequence is terminated by one, two, or more than two stop codons.

[0070] References to sequences provided herein that comprise a stop codon include embodiments in which there is no stop codon, multiple stop codons are present, and different stop codons are present.

[0071] Mature ApoE3-related proteins can be formed intracellularly from mature ApoE sequences that further include a signal peptide. A signal peptide is a short N-terminal amino acid sequence that facilitates protein secretion. The signal peptide directs the protein to or through the endoplasmic reticulum secretory pathway and is typically cleaved within the endoplasmic reticulum prior to secretion. Thus, the signal peptide enhances the secretion of the polypeptide from the cell compared to the secretion level of the corresponding polypeptide lacking the signal peptide.

[0072] In certain embodiments, the ApoE3-related protein comprises a signal peptide. The signal peptide can be derived in whole or in part from the secretion signal of the secreted polypeptide and / or can be synthesized in whole or in part. Generally, the length of the known signal peptide is about 10-15 to 50-60 amino acids. In addition, the known secretion signal from the secreted polypeptide can be changed or modified (for example, by amino acid substitution, deletion, truncation or insertion), as long as the resulting secretion signal sequence plays a role in enhancing the secretion of the polypeptide.

[0073] In certain embodiments, the signal peptide comprises, consists essentially of, or consists of a naturally occurring secretory signal sequence or a modification thereof.Examples of synthetic or artificial secretory signal peptides are provided, for example, in Barash et al., Biochem. Biophys. Res. Comm. (2002).

[0074] In certain embodiments, the signal peptide is selected from the group consisting of ApoE3 signal peptide, human chymotrypsinogen B2 signal peptide ("sp7"; 18 amino acid signal peptide of NCBI reference sequence NP_001020371), alpha2-HS-glycoprotein (AHSG) signal peptide, CD300 signal peptide, lysosomal associated membrane glycoprotein 1 (LAMP1) signal peptide, Notch2 signal peptide, orosomucoid 1 (ORM1) signal peptide, transferrin (TF) signal peptide, secrecon (an artificial signal sequence described in Barash et al., Biochem Biophys Res Commun. 2002; 294: 835-842), mouse IgKVIII, human IgKVIII, CD33, tPA, alpha-1 antitrypsin signal peptide, and native secretory alkaline phosphatase (SEAP).

[0075] In certain embodiments, the signal peptide is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, and 56. In further embodiments, the signal peptide is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71; or differs from any one of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71 by any one of 1, 2, or 3 amino acids.

[0076] In various embodiments, the ApoE3-related protein comprises a signal peptide and is at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34 or 35; differs from SEQ ID NO: 34 or 35 by 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acids; is provided by SEQ ID NO: 34; or is provided by SEQ ID NO: 35. SEQ ID NO: 34 provides a native ApoE3 sequence comprising an ApoE3 signal peptide, while SEQ ID NO: 35 provides an ApoE3(ch) comprising an ApoE3 signal peptide.

[0077] In certain embodiments, the nucleotide sequence encoding ApoE comprises one or more introns. The introns are characterized by providing 5′ and 3′ splice consensus sequences that provide intron boundaries and adenosine at branch points. Adenosine participates in breaking the phosphodiester bond at the upstream exon-intron boundary. In certain embodiments, the 5′ and 3′ splice consensus sequences comprise 5′GU...AG 3′. In certain embodiments, the 5′ and 3′ splice consensus sequences comprise 5′AU...AC 3′. Consensus splice sites are described in, for example, Jurica and Royal, RNA Splicing, ed. Lennarz and Lane, Encyclopedia of Biological Chemistry (Second Edition), Academic Press, 2013, pp. 185-190; and Qu et al., Front Genet. 2017 Apr 11; 8:38; each of which is hereby incorporated herein by reference in its entirety.

[0078] The branch point is also typically located in a consensus sequence (e.g., a heptamer sequence) about 18-40 nucleotides upstream of the 3' acceptor site, and a polypyrimidine tract typically separates the branch point from the 3' splice site. In certain embodiments, the branch point comprising adenosine is about 18-40 nucleotides upstream of the 3' acceptor site, and there is a polypyrimidine tract comprising primarily C and U residues. RNA splicing is described, for example, in Jurica and Roybal, RNA Splicing, ed. Lennarz and Lane, Encyclopedia of Biological Chemistry (Second Edition), Academic Press, 2013, pp. 185-190; and Berger et al., Wiley Interdiscip Rev RNA. 2016 Jul; 7(4): 487-98; each of which is hereby incorporated herein by reference in its entirety.

[0079] In another embodiment, the nucleotide sequence encoding ApoE has one or two introns, and / or the introns are located in naturally occurring positions. The natural intron splice sites in the coding sequence are located at codon 15 (split Gly G / GC) between nucleotides 43 and 44 of SEQ ID NO: 1; and at codon 79 (split Arg AG / G) between nucleotides 236 and 237 of SEQ ID NO: 1.

[0080] In certain embodiments, the nucleotide sequence encoding ApoE (ApoE transgene) comprises, from 5′ to 3′: (a) a first exon corresponding to nucleotides 1-43 of SEQ ID NO: 1, wherein the first exon has at least 85% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130, with the proviso that the terminal 3′ nucleotide of the first exon is a G, and in additional embodiments involving the first exon, the first exon has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130; (b) the first intron at a position corresponding to between nucleotides 43 and 44 of SEQ ID NO: 1; (c) a second exon corresponding to nucleotides 44-236 of SEQ ID NO: 1; wherein the second exon has at least 85% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130, provided that the terminal 5' nucleotide of the second exon is G (in further embodiments, GC) and the terminal 3' nucleotide of the second exon is AG, and in further embodiments relating to the second exon, the second first exon has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130; (d) a second intron at a position corresponding to between nucleotides 236 and 237 of SEQ ID NO: 1, (e) a third exon corresponding to nucleotides 237-951 of SEQ ID NO: 1, wherein the third exon has at least 85% sequence identity to nucleotides 237-951 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130, with the proviso that the terminal 5' nucleotide of the third exon is a G, and in further embodiments involving the third exon, the third exon has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 237-951 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130; wherein the first exon, the second exon, and the third exon together encode an ApoE3-related protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 35, and in further embodiments, the ApoE-related protein is identical to SEQ ID NO: NO:35 comprises at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity and differs from SEQ ID NO:35 by 1, 2, 3, 4 or 5 amino acids, or comprises SEQ ID NO:34 or SEQ ID NO:35.

[0081] For each provided sequence and sequence identity, each possibility within (a), (b), (c), (d) and (e) can be combined independently. For example, (1) the first exon can, independently of the second exon and the third exon, have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130; (2) the second exon, independently of the first exon and the third exon, can have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130; and (3) the third exon, independent of the first exon and the second exon, can have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130. Nucleotides 44-236 of any of NOs: 3-31, 95-105, or 124-130 have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0082] Reference to a "corresponding" nucleotide or nucleotide region to a reference sequence (e.g., SEQ ID NO: 1) indicates that the corresponding nucleotide position or nucleotide region (e.g., an exon or intron) is that position or region that matches the indicated position or region of the reference sequence when maximum alignment occurs. Maximum alignment takes into account any additions, deletions, and / or substitutions. Preferably, only substitutions are present.

[0083] In certain embodiments, the first intron comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 119-122, and the second intron independently comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 119-122.

[0084] In certain embodiments, the first intron consists of the sequence of any one of SEQ ID NOs: 119-121, or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides, and the second intron independently consists of the sequence of any one of SEQ ID NOs: 119-121, or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides.

[0085] In certain embodiments, the first exon has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 11, 15, 20, 26, 31, or 95-105; The second exon has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 11, 15, 20, 26, 31, or 95-105; and The third exon has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to nucleotides 237-951 of any one of SEQ ID NOs: 11, 15, 20, 26, 31 or 95-105, wherein the ApoE-related protein comprises at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 35, differs from SEQ ID NO: 35 by 1, 2, 3, 4 or 5 amino acids, or comprises SEQ ID NO: 34 or SEQ ID NO: 35.

[0086] In certain embodiments, the nucleotide sequence encoding ApoE comprises a sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 106-115.

[0087] In certain embodiments, the one or more introns are selected from a rabbit β-globin intron with a splice donor / splice acceptor, an SV40 intron with a splice donor / splice acceptor, a human β-globin intron, intron 2 of the human hemoglobin β gene, hFIXint1 (intron 1 of the human coagulation factor IX gene), CBA-rHHB (a synthetic intron derived from a fusion of intron 1 of the chicken β-actin gene and intron 2 of the rabbit hemoglobin β), CBA (intron 1 of the chicken β-actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (a synthetic intron derived from a different part of the human coagulation factor IX gene and present in the pLIVE vector), Mirus Bio, Madison, WI); human hemoglobin subunit β (HBB2) synthetic introns and optimized HBB2; and chimeric introns, such as an intron consisting of the 5′-splice donor of the first human β-globin intron and the branch and 3′-acceptor sites of the intron located between the leader sequence and the body of the immunoglobulin gene heavy chain variable region. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti et al. Mol. Ther. Methods Clin Dev. (2016) Jul 20; 3: 16049; and pCMVNT TM The HBB-IGG intron is provided by the vector.)

[0088] In certain embodiments, an intron is 50 bases to 1,500 bases.

[0089] In certain embodiments, any of the nucleotide sequences encoding ApoE and introns provided herein contain 0-5, 0-10, or 0-15 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 5.0% CpGs; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0% or at most about 5.0% CpGs; preferably 0 CpGs.

[0090] II expression cassette

[0091] The polynucleotide expression cassette contains a nucleic acid encoding ApoE operably linked to one or more expression control elements. An "expression control element" is a nucleic acid sequence that affects the expression of the nucleic acid encoding ApoE. For example, expression control can be affected at the levels of transcription, translation, splicing, and message stability. Expression control elements are typically located 5' ("upstream") or 3' ("downstream") of the transcribed nucleic acid. Expression control elements can also be located within the transcript (e.g., in an intron). Expression control elements can be located near the transcribed sequence or at a distance from the transcribed sequence. One or more expression control elements may be present. Examples of expression control elements include promoters, enhancers, introns, polyadenylation signals, Kozak sequences, post-transcriptional regulatory elements, and termination sequences.

[0092] A promoter is a region of DNA where transcription begins. Typically, the transcribed nucleic acid is located 3' to the promoter sequence. In certain embodiments, the promoter sequence is coupled to an enhancer. An enhancer is a region of DNA that increases transcription from a promoter. The enhancer can be adjacent to the promoter or distal to it. Typically, the enhancer is located upstream of the promoter, but can also be located downstream or within the promoter sequence.

[0093] Expression control elements such as promoters and enhancers can be selected to preferentially drive expression in specific cell or tissue types. Expression control elements are generally active in specific cells, tissues, or organs because they are recognized by transcriptional activators or other transcriptional regulators that are unique to the specific cell, tissue, or organ type. (See, for example, Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th edition, Volume II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al., (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0094] Incorporation of tissue-specific regulatory elements into the expression construct provides at least partial tissue tropism for the expression of ApoE3-related proteins. Reference to a promoter or enhancer as being specific for a particular cell type of a tissue indicates that the promoter or enhancer provides for higher levels of expression and / or secretion in the specified cell or tissue type. Examples of promoters that are specific for the liver are the transthyretin (TTR) gene promoter; the human α1-antitrypsin (hAAT) promoter; the apolipoprotein AI promoter; albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); the hepatitis B virus core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (Aep), Arbuthnot et al., Hum. Gene Ther, 7:1503-14 (1996); the human factor IX promoter; the thyroxine-binding globulin (TBG) promoter; the TTR minimal enhancer / promoter; the α-antitrypsin promoter; LSP (845 nt) (required for intronless scAAV); and the LSP1 promoter. Examples of enhancers active in the liver are apolipoprotein E (ApoE) HCR-I and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0095] Expression control elements also include ubiquitous or ubiquitous promoters and promoter / enhancers that can drive polynucleotide expression in many different cell types. Such elements include cytomegalovirus (CMV) immediate early promoter / enhancer sequence, Rous sarcoma virus (RSV) promoter / enhancer sequence, phosphoglycerate kinase (PKG) promoter, CAG (a complex of CMV enhancer, chicken beta actin promoter (CBA) and rabbit beta globin intron) (see, e.g., Boshart et al., (1985) Cell, 41: 521-530), SV40 promoter, dihydrofolate reductase promoter, and cytoplasmic b-actin promoter.

[0096] Examples of CNS-specific promoters include neuron-specific promoters such as NSE (neuron-specific enolase), synapsin or NeuN, platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), methyl-CpG binding protein 2 (MeCP2), Ca 2 / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or neurofilament heavy chain (NFH), β-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte-specific promoters such as glial fibrillary acidic protein (GFAP) and EAAT2 promoters; oligodendrocyte-specific promoters such as myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoters; neuron / hypothalamus-specific promoters such as proopiomelanocortin (POMC) promoter; and neuron / spinal cord-specific promoters. (See, e.g., U.S. Patent Publication No. 2021 / 214749 and Adeno-Associated Virus Vectors (2019), ed. Castle., 1st ed., Springer New York, New York, NY.; both of which are hereby incorporated by reference in their entirety.)

[0097] Additional promoters include the SV40 early promoter, the superoxide dismutase 1 (SOD1) promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the SFFV promoter, the rat insulin promoter, the TBG promoter, the desmin promoter and similar muscle-specific promoters, the EF1-alpha promoter, synthetic promoters, hybrid promoters, and promoters with multiple tissue specificities.

[0098] Expression control elements can also affect expression in a manner that can be regulated by a signal or stimulus that increases or decreases expression. Regulatory elements that increase expression of a transcribed nucleic acid in response to a signal or stimulus are also referred to as "inducible elements" (i.e., induced by a signal). Typically, the amount of increase or decrease imparted by such elements is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionein (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (International Patent Publication No. WO 1998 / 10088); the tetracycline repressor system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline inducible system (Gossen et al., Science 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486 inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4: 432-441 (1997); and the rapamycin inducible system (Magari et al., J. Clin. Invest. 100: 2865-2872 (1997); and Rivera et al., Nat. Medicine. 2: 1028-1032 (1996)). Other examples of regulatable control elements include those that are regulated by specific physiological states (such as temperature, acute phase, or development).

[0099] In certain embodiments, the expression cassette further comprises one or more introns independent of the nucleotides encoding ApoE. A variety of different introns can be used to enhance gene expression. Examples of introns that can be used include rabbit β-globin introns with splice donors / splice acceptors, SV40 introns with splice donors / splice acceptors, human β-globin introns, intron 2 of the human hemoglobin β gene, hFIXint1 (intron 1 of the human coagulation factor IX gene), CBA-rHHB (a synthetic intron derived from a fusion of intron 1 of the chicken β-actin gene and intron 2 of the rabbit hemoglobin β), CBA (intron 1 of the chicken β-actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (a synthetic intron derived from different parts of the human coagulation factor IX gene and present in the pLIVE vector, Mirus Bio, Madison, WI); human hemoglobin subunit β (HBB2) synthetic introns and optimized HBB2; and chimeric introns, such as an intron consisting of the 5′-splice donor of the first human β-globin intron and the branch and 3′-acceptor sites of the intron located between the leader sequence and the body of the immunoglobulin gene heavy chain variable region. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti et al. Mol. Ther. Methods Clin Dev. (2016) Jul 20; 3: 16049; and pCMVNT TM The HBB-IGG intron is provided by the vector.)

[0100] In certain embodiments, the expression cassette includes a post-transcriptional regulatory element. Post-translational regulatory elements such as the woodchuck post-transcriptional regulatory element (WPRE) and the hepatitis B regulatory element can increase gene expression (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).

[0101] The polyadenylation signal sequence provides the formation of a poly A tail that promotes nuclear export, translation and / or mRNA stability, and can also participate in transcription termination. Examples of polyadenylation signal sequences include SV40 late polyadenylation signal, bovine growth hormone poly A (bGHpA) signal sequence, synthetic poly A, mouse β-globin pA, rabbit β-globin pA, and H4-based pA (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).

[0102] In certain embodiments, the expression cassette comprises a Kozak consensus sequence or a variant thereof. The Kozak consensus sequence plays a role in translation initiation. Kozak consensus sequences and variants are provided, for example, in McClements et al., (2021) Molecular Vision, 27, 233-242.

[0103] In certain embodiments, the expression cassette comprises, operably coupled from 5' to 3' to the ApoE coding sequence: a promoter or promoter / enhancer, an intron, a Kozak sequence, the ApoE coding sequence, and a polyadenylation signal.

[0104] In certain embodiments, the expression cassette further comprises a miRNA target sequence, and in other embodiments, the miRNA target sequence is incorporated into the 3′UTR of the expression cassette. The miRNA target sequence is recognized by miRNAs present in specific cells or tissues, resulting in degradation of the mRNA transcript. Incorporation of miRNA target sequences can be used to reduce expression in certain cell or tissue types based on the presence of certain miRNAs in specific cells. Multiple tandem repeats of the miRNA target sequence can be used to increase degradation. (Geisle et al. (2016) World Journal of Experimental Medicine 6(2): 37-54.)

[0105] In certain embodiments, the expression cassette comprises a miRNA target sequence for dorsal root ganglion, liver, or immune cells.

[0106] In certain embodiments, the nucleotide sequence encoding the expression cassette contains any of: 0-5, 0-10, 0-15, 0-50, or 0-100 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10% , about 11%, about 12%, about 13%, about 14% or about 15% CpG; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0%, at most about 5.0%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or at most about 15% CpG.

[0107] III. Recombinant Viral Vector Nucleic Acid

[0108] The polynucleotide recombinant viral nucleic acid contains 5′ and / or 3′ viral elements that provide viral packaging and may provide additional activities such as self-priming, DNA replication, promoter activity, genomic integration, or episomal concatemerization. The 5′ and 3′ elements are typically located at or near the 5′ and 3′ ends of the recombinant viral nucleic acid and may be naturally occurring or modified forms of naturally occurring sequences. Examples of 5′ and 3′ elements include adenoviral ITRs, adeno-associated viral ITRs, and packaging sequences; and retroviral 5′ and 3′ long terminal repeats (LTRs) and packaging sequences. (Naso et al. (2017) BioDrugs, 31(4), 317-334; Bulcha et al. (2021) Sig. Transduct. Target Ther. 6:53(2021); and Liu and Seol (2020) BMB Reports; 53(11):565-575.)

[0109] The term "recombinant" as a modification of a nucleic acid or vector indicates a combination of elements that do not occur in nature. For example, a recombinant viral vector nucleic acid provides a 5' and / or 3' viral element and an expression cassette containing one or more elements that are not naturally associated with the 5' and / or 3' elements. Similarly, a viral vector (such as an rAAV vector) can contain a naturally occurring or modified capsid that encapsidates the recombinant viral vector nucleic acid.

[0110] The polynucleotides, expression cassettes, and viral vector nucleic acids are compatible with the particular viral vector. For example, rAAV contains ssDNA, adenoviral vectors contain dsDNA, and retroviral vectors contain ssRNA.

[0111] In certain embodiments, the viral vector nucleic acid contains any of: 0-5, 0-10, 0-15, 0-50, 0-100, or 0 to 150 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10 at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or about 15% CpG; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0%, at most about 5.0%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or at most about 15% CpG.

[0112] IV. Viral Vectors

[0113] In certain embodiments, the gene delivery vehicle is a viral vector. The viral vector comprises a protein capsid that encapsidates the recombinant viral nucleic acid and can deliver the nucleic acid to cells or tissues. Depending on the specific vector, the viral vector may further comprise a viral envelope. Examples of viral vectors that can be used for gene therapy include adenoviral vectors, rAAV, retroviral vectors, and herpes simplex vectors.

[0114] There are different serotypes in different types of viruses. Different serotypes can provide different activities, such as cell or tissue tropism and the possibility of generating a host immune response. The term "serotype" broadly refers to both serologically different viruses and non-serologically different viruses that may be within a subgroup or variant of a given serotype. Serological distinctiveness can be determined based on the lack of cross-reactivity between antibodies against one capsid compared to another capsid. Such cross-reactivity differences are typically due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0115] As more naturally occurring virus isolates are discovered or capsid mutants are generated, there may or may not be serological differences from any currently existing serotype. Thus, in the event that a new virus is not serologically different, the new virus will be a subgroup or variant of the corresponding serotype.

[0116] IV.A. Adenoviral Vectors

[0117] Adenovirus is a non-enveloped double-stranded DNA virus. Recombinant adenoviral vectors contain recombinant adenoviral nucleic acid lacking one or more proteins involved in viral replication, and further contain an adenoviral capsid. Recombinant adenoviral vectors containing different amounts of adenoviral DNA can be produced. The Ad genome is flanked by hairpin-like inverted terminal repeats (ITRs), the length of which varies between 30-371 bp at the ends. ITRs serve as self-priming structures that promote DNA replication independent of primers. A packaging signal located on the left arm of the genome is required for viral genome packaging. (Liu and Seol (2020) BMB Reports; 53(11): 565-575; and Bulcha et al. (2021) Sig. Transduct. Target Ther. 6: 53.)

[0118] In certain embodiments, the recombinant adenoviral vector is a third generation vector, which is also referred to as "gutless" or "helper-dependent." Gutless vectors can be generated from recombinant adenoviral nucleic acids in which all or substantially all viral sequences are absent, except for the ITRs and packaging signals. Gutless adenoviral vectors are high-capacity vectors capable of accommodating DNA inserts of up to about 36 kb. Preferred recombinant adenoviral nucleic acids are about 27 kb to about 37 kb. Stuffer sequences can be added to the recombinant adenoviral nucleic acid to increase nucleic acid size and capsid incorporation. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences. (Liu and Seol (2020) BMB Reports; 53(11): 565-575; Bulcha et al. (2021) Sig. Transduct. Target Ther. 6: 53; and Sandig et al. PNAS (2000) 97(3): 1002-1007, each of which is hereby incorporated by reference in its entirety.)

[0119] In certain embodiments, recombinant adenoviral vectors can be generated based on rare human serotypes or chimpanzee serotypes. The use of chimpanzee serotypes and rare human serotypes may help reduce the host immune response to recombinant adenoviral vectors due to pre-existing immunity. (Guo et al., (2018) Human vaccines & immunotherapeutics, 14(7): 1679-1685 and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: 53)

[0120] Adenoviral vectors can be produced in trans by providing the viral proteins required for vector production, using, for example, appropriate helper viruses or plasmids and cell lines. (Liu and Seol (2020) BMB Reports; 53(11): 565-575; and Bulcha et al. (2021) Sig. Transduct. Target Ther. 6: 53.)

[0121] IV.B. AAV Vectors

[0122] Recombinant adeno-associated viral vectors (also referred to herein as "rAAV") are based on adeno-associated viruses. Adeno-associated viruses are single-stranded DNA viruses containing a 4.7-kb genome flanked by 145-nt ITRs at both ends of the genome. ITR activity is important for self-priming and packaging and can also provide additional activities, such as promoter activity.

[0123] rAAV contains AAV recombinant nucleic acid and viral capsid. rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. In certain embodiments, rAAV nucleic acid is at least about 2.5kb. In certain embodiments, rAVV nucleic acid has a size range of about 4kb to about 5.2kb. If necessary, filler sequences can be used to increase rAAV nucleic acid size and packaging efficiency. In different embodiments, the rAAV nucleic acid including filler is 4-5.2kb, 3.0-5.5kb, 4.0-5.0kb, 4.3-4.8kb, about 4.2kb, about 4.3kb, about 4.4kb about 4.5kb, about 4.6kb or about 4.7kb. Preferred filler sequences avoid coding sequences, repetitive sequences, recombinant sequences and immunogenic sequences.

[0124] In certain embodiments, the rAAV nucleic acid comprises a 5' ITR and / or a 3' ITR independently selected from the 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3 BITR. In other embodiments, both 5' and 3' ITRs are present and are derived from the same serotype genome.

[0125] In additional embodiments, the 5′ ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 81, 88, 90, 92, and 94, and the 3′ ITR independently comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 82, 89, 91, and 93.

[0126] In certain embodiments, rAAV is a self-complementary adeno-associated viral vector (scAAV) or a short hairpin adeno-associated viral vector (shAAV). scAAV and shAAV provide double-stranded rAAV nucleic acids that can be incorporated into AAV capsids. scAAV and shAAV contain reverse dimer repeats that provide intramolecular double-stranded DNA. scAAV can be produced by mutating the ITR end resolution site so that rep cannot produce a nick at the end resolution site. shAAV can utilize short hairpins to produce double-stranded AAV nucleic acids. scAAV and shAAV, as double-stranded DNA, provide the advantage of avoiding the DNA synthesis step necessary for single-stranded rAAV nucleic acids to enter cells. A potential disadvantage of scAAV and shAAV is that the size of the DNA insert that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acids. (U.S. Patent No. 10,457,940; Xie et al., Mol Ther. (2017) 25(6): 1363-1374; and McCarty Mol. Ther. (2008) 16(10): 1648-1656; each of which is hereby incorporated by reference in its entirety.)

[0127] Naturally occurring AAV capsids contain the viral proteins VP1, VP2, and VP3 in a ratio of approximately 1:1: 10. AAV vectors can be produced where all three viral proteins are based on a particular serotype, or where one, two, or all three viral proteins are based on different serotypes or variants thereof.

[0128] In certain embodiments, the AAV capsid is based on a VP1, VP2, or VP3 having at least 80% sequence identity to VP1, VP2, or VP3 of any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-Bl, AAV-AS, AAV1 / rh.10, SEQ ID NO:83, and SEQ ID NO:84; and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions, and deletions). (See, e.g., U.S. Patent Nos. 9,909,142 and 9,840,719, which disclose RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6; U.S. Patent No. 2013 / 0059732 and U.S. Patent No. 9,169,299, which disclose LK01, LK02, and LK03; and U.S. Patent No. 11,110,153; the disclosures of which are incorporated herein in their entireties.)

[0129] The recombinant AAV capsid and nucleic acid can be based on the same serotype (or subgroup or variant), or can be different from each other. In certain embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the encapsidated capsid protein.

[0130] In various embodiments, the rAAV capsid comprises a VP1, VP2, or VP3 having the same sequence as any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10; and SEQ ID NO: 83 or SEQ ID NO: 84. NO: 84 has a protein with a sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9% or 100% identical to VP1.

[0131] In certain embodiments, the rAAV capsid comprises VP1 comprising SEQ ID NO:83, VP2 comprising SEQ ID NO:122, and VP3 comprising SEQ ID NO:123.

[0132] In certain embodiments related to the treatment of CNS diseases or disorders, capsid provides CNS expression. The example of such rAAV capsid and the design of rAAV capsid capable of providing CNS expression are provided in the following documents: Chen et al., (2021) Journal of Controlled Release 333, 129-138 (e.g., AAV9, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS and AAV1 / rh.10), U.S. Patent No. 9,585,971, Goertsen et al., Nat.Neurosci.25, 106-115 (2022), and Ittner et al., Br.J.Pharmacol. (2019) 176: 3649-3655; Each of the documents is incorporated herein by reference in its entirety.

[0133] Recombinant AAV can be produced in trans by providing the viral proteins required for vector production using, for example, an appropriate helper virus or plasmid and a cell line. In certain embodiments, rAAV is produced using an rAAV vector genome plasmid. The plasmid comprises that portion of the rAAV nucleic acid that is ultimately packaged or encapsidated to form viral (e.g., rAAV) particles. "Plasmid backbone" contains elements that are important for propagation and recombinant virus production. Except for possible 3' ITR and / or 5' ITR clone remnants, the plasmid backbone itself is not packaged or encapsidated into viral (e.g., AAV) particles.

[0134] Recombinant AAV can be produced by different types of cell lines. In certain embodiments, human HEK293 cells (American Type Culture Collection Accession No. ATCC CRL1573) are used. Other host cell lines suitable for rAAV production are described, for example, in Robert et al. (2017) Biotechnol. J., 12: 1600193; and in International Application PCT / US2017 / 024951, the disclosures of which are incorporated herein in their entirety.

[0135] The AAV genome contains two main genes: rep and cap. Transcription from the rep gene is initiated from two different promoters, resulting in the production of nonstructural proteins designated Rep78, Rep68, Rep52, and Rep40. The rep protein plays a role in genome replication and / or encapsidation. The cap gene encodes the structural proteins (VP1, VP2, and Vp3) that make up the capsid; the nonstructural assembly activating protein (APP), which performs functions related to capsid assembly; and membrane-associated accessory proteins that may be associated with the production phase of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther. 31(9-10): 499-511, hereby incorporated by reference in its entirety.)

[0136] AAV requires helper virus function to complete its replication cycle. Helper virus function can be provided by different viruses in a permissive cell line. A permissive cell line is a cell line that can support viral replication. Examples of helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HBoV1, which can be used in combination with, for example, permissive primate cells; and baculovirus, which can be used in combination with, for example, permissive insect cells such as sf9. (Maurer and Weitzman (2020) Hum. Gene Ther. (2020) 31(9-10): 499-511 and Meier et al., (2020) Viruses 19; 12(6): 662, both of which are incorporated herein by reference in their entirety.)

[0137] Using, for example, appropriate helper viruses or plasmids and cell lines, recombinant AAV can be produced in trans by providing the viral proteins required for vector production. In certain embodiments, rAAV is produced using rAAV vector genome plasmids. The plasmid comprises that portion of the rAAV nucleic acid that is ultimately packaged or encapsidated to form a viral (e.g., rAAV) vector. "Plasmid backbone" contains elements that are important for propagation and recombinant virus production. Except for possible 3'ITR and / or 5'ITR clone remnants, the plasmid backbone itself is not packaged or encapsidated into viral particles.

[0138] The vector genome plasmid may contain regions such as an origin of replication and a selectable marker. Additional sites that may be present include cloning sites.

[0139] Recombinant AAV can be produced by different types of cell lines, including HeLa, A549, BHK, Vero, and HEK293 or their derivatives. Other host cell lines suitable for rAAV vector production are described, for example, in Robert et al., (2017) Biotechnol. J. (2017) 12(3), 1600193; and International Application No. PCT / US2017 / 024951, the disclosures of which are incorporated herein in their entirety.

[0140] Recombinant AAV can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV production include Clément and Grieger (2016) Mol. Ther. Methods Clin. Dev. 16; 3: 16002; Robert et al. (2017) Biotechnol. J. 12 (3), 1600193; and Adeno-Associated Virus Vectors (2019), ed. Castle., 1st edition, Springer New York, New York, NY.; each of which is hereby incorporated herein by reference in its entirety.)

[0141] In certain embodiments, the rAAV vector is produced by a rAAV producer cell comprising rAAV helper virus activity. The genome of the rAAV producer cell comprises rAAV nucleic acid, rep gene, and cap gene.

[0142] In certain embodiments, rAAV vectors are produced by culturing rAAV-licensed cells comprising an AAV genome plasmid, wherein the rAAV-licensed cells further comprise rep and cap genes provided as part of the cell genome and / or provided by one or more separate plasmids; and helper virus activity provided as part of the cell genome and / or provided by one or more separate plasmids. In further embodiments, (a) the rAAV-licensed cell line is a packaging cell, wherein the genome of the packaging cell comprises a cap gene and a rep gene; (b) the rep gene, cap gene, and helper activity are provided by the same plasmid; or (c) the rep gene and cap gene are provided by a rep / cap plasmid and the helper activity is provided by a helper plasmid.

[0143] In certain embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding at least UL5, UL8, UL52, and ICP8.

[0144] In certain embodiments involving the use of adenoviral helper functions, the helper functions are provided by genes encoding at least E1A, E1B19K, E1B55K, E2A, E4orf6, and VA RNA. In certain embodiments, E1, E2A, and VR RNA functions are provided by a helper plasmid, with additional helper functions being provided by the host strain.

[0145] In certain embodiments, rAAV vectors are obtained by producing rAAV and purifying rAAV using the methods described herein. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based methods, and combined methods. (See, for example, Ayuso et al., (2010), Curr Gene Ther. (2010) 10(6):423-36, which is hereby incorporated by reference in its entirety.)

[0146] In certain embodiments, AAV helper functions are introduced into host cells by transfecting the host cells with an AAV helper construct prior to or in parallel with transfection of an AAV expression vector. Host cells with AAV helper functions may be referred to as "helper cells" or "packaging helper cells." Thus, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the missing AAV functions necessary for productive AAV transduction. AAV helper constructs typically lack AAV ITRs and are neither self-replicating nor self-packaging capable. These constructs may be in the form of, for example, plasmids, phages, transposons, cosmids, viruses, or virions. Many AAV helper constructs have been described, such as the plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known. For example, recombinant AAV can be produced as described in U.S. Patent 9,408,904; and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein in their entireties.

[0147] IV.C. Retroviral Vectors

[0148] Retroviruses are enveloped single-stranded RNA viruses that contain 5' and 3' LTRs and a signal packaging sequence just outside the LTR. Different types of retroviral vectors can contain different amounts of viral genomes. In certain embodiments, the retroviral vector is an HIV-based lentiviral vector that retains all cis-acting sequences required for viral RNA packaging, reverse transcription, and proviral DNA integration, while removing all HIV protein-coding genes. Lentiviral vectors have a packaging capacity of up to about 9 kb. If necessary, stuffer sequences can be used to increase the size and packaging efficiency of rAAV nucleic acid. Appropriate plasmids and cell lines can be used to produce lentiviral vectors in trans by providing the viral proteins required for vector production. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: 53.) V. Non-viral vectors

[0149] In certain embodiments, the gene delivery vehicle is a non-viral vector. Non-viral vectors include nanoparticles and naked nucleic acids. Preferred non-viral vectors are nanoparticles. A variety of different nanoparticles can be used, including lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, e.g., Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) Nanomaterials 20, 100261; Neshat et al. (2020) Current Opin. Biotechnol. 66: 1-10; Ouranidis et al., (2022) Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21; 7(1): 166; each of which is hereby incorporated by reference in its entirety.)

[0150] If desired, nanoparticles can be targeted to cell types using, for example, targeting ligands that recognize target cell receptors. Examples of targeting ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies, and proteins / peptides (e.g., RGD, epidermal growth factor, and low-density lipoprotein) and peptides. (e.g., Teo et al., Advanced Drug Delivery Reviews (2016), 98, 41.)

[0151] Nanoparticles can be used to deliver the polynucleotide constructs encoding ApoE described herein to cells. In various embodiments, the nanoparticles can deliver additional therapeutic compounds; and one or more additional compounds are provided in various nanoparticles. Mention of compounds includes small molecules and macromolecules (e.g., therapeutic proteins and antibodies).

[0152] The production of different nanoparticles and the incorporation of nucleic acids and other compounds are well known in the art. Examples of publications describing the incorporation of nucleic acids into specific nanoparticles (such as LPNPs and LNPs) include Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12 (1), 65; and Teixeira et al., (2017) Prog. Lipid Res. 10; 68: 1-11 (each of which is hereby incorporated herein by reference in its entirety). Factors that may affect the incorporation of small molecules into nanoparticles include the presence of hydrophobicity and ionizable moieties. (See, for example, Nii and Ishii International Journal of Pharmaceutics (2005) 298, 198; and Chen et al., Journal of Controlled Release (2018) 286, 46.) VA lipid-based delivery system

[0153] Lipid-based delivery systems include the use of lipids as components. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0154] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles that can be used to deliver nucleic acid molecules and have nanoscale dimensions. In various embodiments, the nanoparticles are about 10 nm to about 1000 nm, about 50 nm to about 500 nm, or about 50 nm to about 200 nm.

[0155] DNA is negatively charged. Therefore, it may be beneficial for LNP to include cationic lipids (e.g., amino lipids). Exemplary amino lipids are described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651 and U.S. Patent Nos. In patent publication numbers 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411 and 2010 / 0117125, all of which are incorporated herein in their entirety. In certain embodiments, LNPs comprise the amino lipids described in U.S. Patent No. 9,512,073, which is hereby incorporated herein in its entirety.

[0156] The terms "cationic lipid" and "amino lipid" are used interchangeably herein and include lipids and salts thereof having one, two, three or more fatty acid or fatty alkyl chains and pH titratable amino groups (e.g., alkylamino or dialkylamino groups). Cationic lipids are typically protonated (i.e., positively charged) at a pH lower than the pKa of the cationic lipid and are substantially neutral at a pH higher than the pKa. Cationic lipids can also be titratable cationic lipids. In certain embodiments, the cationic lipid comprises a protonable tertiary amine (e.g., pH titratable) group; a C18 alkyl chain, wherein each alkyl chain independently can have one or more double bonds, one or more triple bonds; and an ether, ester, or ketal connection between the head group and the alkyl chain.

[0157] Cationic lipids include 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenoyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA, also known as DLin-C2K-DMA), , XTC2 and C2K), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleoylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-triacontac-6,9,28,31-tetraen-19-yl ester (DLin-M-C3-DMA, also known as MC3), its salts and mixtures thereof. Other cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleoyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleoyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA and (DLin-MP-DMA) (also known as 1-B11).

[0158] Still other cationic lipids include 2,2-dilinoleoyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3- Morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.CI), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.CI), 1,2-dilinoleoyloxy-3-(N-methylpiperazine)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N-(1 -(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl bromide ammonium (DMRIE), 2,3-dioleoyloxy-N-[2(spermine-formamido)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propylammonium (DOSPA), octadecanoylglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-β-oxy)-3′-oxopentyloxy)-3-dimethylamino]-2-(cholest-5-en-3-β-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), Methyl-1-(cis, cis-9', 1-2'-octadecadienyloxy) propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0159] Many commercial preparations of cationic lipids are available, such as (including DOTMA and DOPE, available from GIBCO / BRL) and (comprising DOSPA and DOPE, available from GIBCO / BRL).

[0160] Additional ionizable lipids that can be used include C12-200, 306Oi10, MC3, cKK-E12, bCKK-E12, lipid 5, lipid 9, ATX-002, ATX-003, and Merck-32. Merck-32 is described in U.S. Patent Application Publication No. 2017 / 0367988.

[0161] In further embodiments, the cationic lipid can be present in an amount from about 10% by mole of LNP to about 85% by mole of LNP, or from about 50% by mole of LNP to about 75% by mole of LNP.

[0162] LNP can comprise neutral lipid.Neutral lipid can be included in the lipid material that exists with uncharged or neutral zwitterion form under physiological pH.This type of lipid comprises diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.The selection of neutral lipid is guided by the consideration factor that comprises granularity and stability usually.In certain embodiments, neutral lipid component can be the lipid (for example, diacylphosphatidylcholine and diacylphosphatidylethanolamine) with two acyl groups.

[0163] The lipids of various acyl chain groups with different chain lengths and saturation are available, or can be separated or synthesized. In certain embodiments, lipids containing saturated fatty acids with carbon chain lengths in the range of C14 to C22 can be used. In certain embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 are used. In addition, lipids with a mixture of saturated fatty acid chains and unsaturated fatty acid chains can be used. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycerol-3-phosphatidyl-ethanolamine (DOPE), 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) or phosphatidylcholine. Neutral lipids can also be made of sphingomyelin, dihydrosphingomyelin or phospholipids with other head groups (such as serine and inositol).

[0164] In additional embodiments, a neutral lipid is provided and can be present in an amount from about 0.1% by weight of the LNP to about 99% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0165] LNP can contain other components, such as sterol and polyethylene glycol. Sterol can give mobility to LNP. As used herein, " sterol " refers to the naturally occurring sterol of plant (phytosterol) or animal (zoosterol) source and the synthetic sterol of non-natural occurrence, all of which are characterized by the presence of hydroxyl groups at the 3 positions of the steroid A ring. Suitable sterol is included in those conventionally used in the field of liposome, lipid vesicle or lipid particle preparation, and the most common is cholesterol. Phytosterol includes campesterol, sitosterol and stigmasterol. Sterol also includes lipid modified by sterol, such as those described in U.S. Patent Application Publication No. 2011 / 0177156. In the different embodiments providing sterol, sterol is present in an amount of about 1% by weight of LNP to about 80% by weight of LNP or about 10% by weight of LNP to about 25% by weight of LNP.

[0166] Polyethylene glycol (PEG) is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG 2000 has an average molecular weight of approximately 2,000 daltons, and PEG 5000 has an average molecular weight of approximately 5,000 daltons. Commercially available PEGs from Sigma Chemical Co. and other companies include monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-toluenesulfonate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0167] In certain embodiments involving PEG, PEG has an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In other embodiments, PEG is substituted with a methyl group at the terminal hydroxyl position. In other embodiments, PEG has an average molecular weight of from about 750 to about 5,000 daltons, or from about 1,000 to about 5,000 daltons, or from about 1,500 to about 3,000 daltons, or from about 2,000 daltons, or from about 750 daltons.

[0168] The lipid modified through PEG is included in the PEG-dialkoxypropyl conjugate (PEG-DAA) described in U.S. Patent numbers 8,936,942 and 7,803,397. The lipid modified through PEG (or lipid-polyoxyethylene conjugate) can have a variety of " anchoring " lipid moieties to fix the PEG moiety to the surface of the lipid vesicle. The example of suitable lipid modified through PEG includes phosphatidylethanolamine and phosphatidic acid modified through PEG, the PEG-ceramide conjugate (for example, PEG-CerC14 or PEG-CerC20) described in U.S. Patent number 5,820,873, PEG-modified dialkylamine and 1,2-diacyloxypropane-3-amine modified through PEG. In certain embodiments, the lipid modified through PEG can be diacylglycerol and dialkyl glycerol modified through PEG. In certain embodiments, the amount of PEG can be about 0.1% by weight of LNP to about 50% by weight of LNP or about 5% by weight of LNP to about 15% by weight of LNP.

[0169] In additional embodiments involving LNP size, prior to encapsulation of the nucleic acid, the LNP has a size range of about 10 nm to 500 nm, or about 50 nm to about 200 nm, or 75 nm to about 125 nm.

[0170] In certain embodiments involving LNPs, LNPs are described by Billingsley et al., Nano Lett. 2020, 20, 1578 or Billingsley et al., International Patent Publication No. WO 2021 / 077066 (both documents are hereby incorporated herein by reference in their entirety). Billingsley et al. and WO 2021 / 077066 describe LNPs containing lipid-anchored PEG, cholesterol, phospholipids, and ionizable lipids. In certain embodiments, LNPs contain a C14-4 polyamine core and / or have a particle size of about 70 nm. C14-4 has the following structure.

[0171] In certain embodiments, the LNP is composed of a cationic lipid or lipopeptide as described in U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374, or WO2021 / 077066 (each of which is hereby incorporated by reference in its entirety). In certain embodiments, the LNP contains a cationic lipid, a cholesterol-based lipid, and / or one or more PEG-modified lipids. In certain embodiments, the LNP contains cKK-E12 (Dong et al., PNAS (2014) 111 (11), 3955).

[0172] In certain embodiments, the LNP comprises a modified form of cKK-E12, referred to herein as "bCKK-E12," which has the following structure:

[0173] In certain embodiments, the LNP comprises lipids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described in Sabnis et al., Molecular Therapy 2018, 26: 6, 1509-1519 (hereby incorporated herein by reference in its entirety). In certain embodiments, the LNP comprises lipids 5, 8, 9, 10, or 11 as described in Sabnis et al.

[0174] Lipid 5 of Sabnis et al. has the following structure:

[0175] Lipid 9 of Sabnis et al. has the following structure:

[0176] Additional lipids that may be utilized include those described by Roces et al., Pharmaceutics, 2020, 12, 1095; Jayaraman et al., Angew. Chem. Int. Ed., 2012, 51, 8529-8533; Maier et al., www.moleculartherapy.org, 2013, Vol. 21, No. 8, 1570-1578; Liu et al., Adv. Mater. 2019, 31, 190257 5, e.g., BAMEA-O16B; Cheng et al., Adv. Mater., 2018, 30, 1805308, e.g., 5A2-SC8; Hajj and Ball, Small, 201915, 1805097, e.g., 306Oi10; Du et al., U.S. Patent Application Publication No. 20160376224; and Tanaka et al., Adv. Funct. Mater., 2020, 30, 1910575; each of which is hereby incorporated herein by reference in its entirety.

[0177] In further embodiments, the nanoparticle is an LNP. In further embodiments, the LPN comprises, consists essentially of, or consists of, in mole %: (1) about 20% to about 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipid lipids, about 0.1% to about 10% of one or more PEG-conjugated lipids, and about 0% to about 70% of cholesterol; and (2) about 20% to about 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipid lipids, about 0.1% to about 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol. In further embodiments, the phospholipid lipid is a neutral lipid; and the phospholipid lipid is DOPE or DSPC.

[0178] In other embodiments, the LNP comprises, consists essentially of, or consists of the following components, in mole %: (1) cKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (2) bCKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (3) lipid 9 (further described in Sabnis et al.), about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; or (4) Lipid 5 (further described in Sabnis et al.), about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; and (5) Ionizable Lipid, about 50%; DSPC, about 10%; cholesterol, about 37.5%; and stabilizer (PEG-lipid), about 2.5%; or (6) GenVoy-ILM TM LNP (Precision NanoSystems). VB polymer-based nanoparticles

[0179] Polymer-based delivery systems can be made from a variety of different natural and synthetic materials. DNA and other compounds can be embedded in the polymer matrix of polymer nanoparticles, or can be adsorbed or conjugated to the surface of nanoparticles. Examples of commonly used polymers for nucleic acid delivery include poly (lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly (ethyleneimine) (PEI) and PEI derivatives, chitosan, dendrimers, polyanhydrides, polycaprolactone, polymethacrylate, poly-L-lysine, pullulan, dextran and hyaluronic acid, poly-β-amino esters. (Thomas et al., (2019) Molecules 24, 3744.)

[0180] The polymer-based nanoparticles can have different sizes ranging from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, and about 150 nm or less. VC lipid polymer nanoparticles

[0181] Lipopolymer nanoparticles are hybrid nanoparticles that provide both a lipid component and a polymer component and can therefore be considered LNPs or LPNPs. LPNP configurations can provide an outer polymer and an inner lipid or an outer lipid and an inner polymer. The presence of two different types of materials facilitates the design of nanoparticles to provide delayed release of the components. Different lipid and polymer components can be selected, taking into account the material to be delivered. (See, e.g., Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct; 68: 1-11.) VD protein- and peptide-based nanoparticles

[0182] Protein and peptide-based systems can employ a variety of different proteins and peptides. Examples of proteins that can be employed include gelatin and elastin. Peptide-based systems can employ, for example, CPP.

[0183] CPPs are short peptides (6-30 amino acid residues) that are potentially capable of intracellular penetration to deliver therapeutic molecules. Most CPPs are composed primarily of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphiphilic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., HIV-1 Tat protein) or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018; 25(1): 1996-2006). Examples of CPPs include cationic CPPs (highly positively charged) (e.g., Tat peptide, penetratin, protamine, poly-L-lysine, and polyarginine); amphipathic CPPs (chimeric or fusion peptides, constructed from different sources, containing both positively and negatively charged amino acid sequences) (e.g., transportan, VT5, bactenecin 7 (Bac7), proline-rich peptide (PPR), SAP (VRLPPP) 3, TP10, pep-1, and MPG); membrane-philic CPPs (exhibiting both hydrophobic and amphipathic properties and containing both large aromatic residues and small residues) (e.g., H625, SPIONs-PEG-CPP, and NP); and hydrophobic CPPs (containing only nonpolar motifs or residues) (e.g., SG3, PFVYLI, pep-7, and fibroblast growth factor).

[0184] Protein and peptide nanoparticles can be provided in various sizes, for example, ranging from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm or less. VE peptide cage nanoparticles

[0185] Peptide cage-based delivery systems can be produced from protein materials that can assemble into cage-like structures to form a confined internal environment. The peptide cage can comprise a protein shell that self-assembles to form a protein cage (e.g., a structure with an internal cavity that is naturally accessible to solvents or can be made by changing solvent concentration, pH, or equilibrium ratios). The monomers of the protein cage can be in naturally occurring form or in variant form, including amino acid substitutions, insertions, and deletions (e.g., fragments).

[0186] Different types of protein "shells" can be assembled and loaded with different types of materials. Protein cages can be produced using one or more viral coat proteins (e.g., protein coat from Cowpea Chlorotic Mottle Virus) as well as non-viral proteins (e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Publication No. 20040028694, and U.S. Patent Publication No. 20090035389, each of which is incorporated herein by reference in its entirety).

[0187] Examples of protein cages derived from non-viral proteins include: ferritins and apoferritins of eukaryotic or prokaryotic origin, such as 12- and 24-subunit ferritins; and heat shock proteins (HSPs), such as the class of 24-subunit HSPs that form the inner core space, the small HSPs of Methanococcus jannaschii, the dodecameric Dsp HSPs of Escherichia coli (E. coli), and the MrgA protein.

[0188] Protein cages can have different core sizes, for example, ranging from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm or less. VF exosomes

[0189] Exosomes are small biofilm vesicles that have been used to deliver a variety of cargoes, including small molecules, peptides, proteins, and nucleic acids. Exosomes typically range in size from approximately 30 nm to 100 nm and can be taken up by cells and deliver their cargo. The cargo can be associated with exosome surface structures or encapsulated within the exosome bilayer.

[0190] Various modifications can be made to exosomes to promote cargo delivery and cell targeting. Modifications for promoting cargo delivery include structures for associating with cargo, such as protein scaffolds and polymers. Modifications for cell targeting include targeting ligands and modified surface charges. Publications describing the production, modification, and use of exosomes for delivering different cargoes include Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) Nanobody 20, 100261; and Dooley et al., (2021) Molecular Therapy 29 (5), 1729 (each of which is hereby incorporated herein by reference). VI. Pharmaceutical Compositions

[0191] The pharmaceutical composition comprises a pharmaceutically acceptable carrier that facilitates the administration and / or storage of the polynucleotide constructs, viral vectors, and non-viral vectors described herein. Reference to "pharmaceutically acceptable" indicates that the component does not cause substantially undesirable biological effects in the amount used. Pharmaceutically acceptable carriers can contain different components, such as one or more pharmaceutically acceptable excipients, such as salts, sugars, buffers, solvents, preservatives, proteins, and surfactants. A particular excipient can have more than one function. Examples of pharmaceutically acceptable excipients and carriers that can be used for viral vectors are provided, for example, in International Patent Publication No. WO 2021 / 071835.

[0192] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery. Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions, which are generally sterile and can be isotonic with the blood of the intended recipient. Illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, glucose, fructose, ethanol, animal oils, vegetable oils, and synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0193] In one embodiment, the pharmaceutical composition comprises a formulation that can be injected into a subject. Examples of injectable formulation components include isotonic sterile saline solutions, salts (e.g., sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and mixtures of such salts), buffered saline, sugars (e.g., glucose), and water for injection. Pharmaceutical compositions include dried (e.g., freeze-dried) compositions that, upon addition of sterile water or physiological saline, allow for the formation of a solution suitable for administration.

[0194] Alternatively, the suspension may be prepared as an appropriate oil injection suspension. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides or liposomes). Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound, thereby facilitating the preparation of a concentrated solution.

[0195] "Effective amount" or "sufficient amount" refers to an amount that provides the indicated or desired effect. An effective amount can be administered in single or multiple doses, alone or in combination with one or more other compositions (e.g., additional therapeutic agents or immunosuppressants), treatments, regimens, or therapeutic regimens; and provide long-term or short-term responses.

[0196] A pharmaceutical composition comprising a transgene encoding an ApoE3-related protein can be delivered to a subject to allow production of the encoded protein. Delivery can be in vivo or ex vivo. In certain embodiments, the pharmaceutical composition comprises sufficient genetic material to enable the recipient to produce a therapeutically effective amount of the protein in the subject.

[0197] A "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired or indicated biological or medical response in a subject. A therapeutically effective amount can be determined based on observed symptoms and / or by using biomarkers associated with a particular disease or disorder. The selection of a specific effective dose can be optimized taking into account various factors, including the disease to be treated or prevented, the symptoms involved, safety and efficacy in animal models, the patient's weight, and the patient's immune status. The optimal dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and can be evaluated based on the patient's condition. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0198] In certain embodiments, the pharmaceutical composition comprising the rAAV vector comprises an empty AAV capsid. In certain embodiments, in the pharmaceutical composition comprising the rAAV vector and the empty AAV capsid, the ratio of the empty AAV capsid to the rAAV vector is within or between about 100:1-50:1, about 50:1-25:1, about 25:1-10:1, about 10:1-1:1, about 1:1-1:10, about 1:10-1:25, about 1:25-1:50, or about 1:50-1:100. In certain embodiments, the ratio of the empty AAV capsid to the rAAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0199] Additional guidance and examples of pharmaceutical compositions and delivery systems are provided in, for example, Remington: The Science and Practice of Pharmacy (2020) 23rd edition, University of the Sciences in Philadelphia, published by Elsevier; The Merck Index (2013) 15th edition, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th edition, Lippincott Williams & Wilkins, Baltimore, MD.

[0200] VII Administration and Treatment

[0201] The polynucleotide constructs, viral vectors, and non-viral vectors described herein can be administered to a subject, preferably a human subject, to provide prophylactic treatment, reduce the likelihood or severity of a disease or disorder, and / or treat a diagnosed disease or disorder. In certain embodiments, the polynucleotide component, vector selection, route of administration, and / or specific pharmaceutical composition are selected with consideration given to the specific disease or disorder being treated.

[0202] Subjects who have or are at increased risk for a particular disease or disorder can be identified, for example, based on symptoms, biomarkers, and genetic markers. Examples of treatment include one or more of: lowering cholesterol, lowering LDL / VLDL, increasing HDL, or lowering the total cholesterol / HDL ratio; or treating or reducing the likelihood of hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia, post-stent restenosis, atherosclerosis, coronary heart disease, or Alzheimer's disease.

[0203] In certain embodiments, treatment results in a reduction in atherosclerotic lesions.

[0204] In certain embodiments, the subject is a human subject having one or two ApoE4 alleles or two ApoE2 alleles.

[0205] In certain embodiments, treatment is performed by providing peripheral ApoE3-related expression (e.g., liver expression). For example, a polynucleotide expression cassette comprising a promoter or promoter / enhancer providing high liver expression can be used; a viral vector providing liver tropism; and nanoparticles targeting the liver to achieve high liver expression. In different embodiments, the promoter is a human α1-antitrypsin (hAAT) promoter, apolipoprotein AI promoter, an albumin promoter, a hepatitis B virus core promoter, alpha-fetoprotein (Aep), a human factor IX promoter, a thyroxine-binding globulin promoter, a TTR minimal enhancer / promoter, an α-antitrypsin promoter, or an LSP1 promoter; and / or apolipoprotein E (apoE) HCR-I and HCR-2 enhancers are provided. In additional embodiments, rAAV has a serotype based on AAV2, AAV3B or a VP1 based on SEQ ID NO: 83 or SEQ ID NO: 84.

[0206] In certain embodiments, Alzheimer's disease is treated using nucleic acid expression components, vector components, and / or techniques that provide CNS expression of an ApoE3-related protein; the subject is selected based on a biomarker or genetic marker associated with Alzheimer's disease; and / or the subject is diagnosed with Alzheimer's disease. In other embodiments, the ApoE3-related protein contains a Christchurch substitution.

[0207] In certain embodiments, nucleic acid expression components, vector components, and / or techniques for providing peripheral ApoE3-related protein expression are used to treat Alzheimer's disease. Peripheral ApoE isoforms are separated from isoforms in the brain by the blood-brain barrier and have been shown to differentially affect the pathogenesis and cognition of Alzheimer's disease. (Liu et al., (2022), Nature Neuroscience 25: 1020-1033.) In other embodiments, the ApoE3-related protein contains a Christchurch substitution.

[0208] Alzheimer's disease subjects can be identified based on an abnormal decline in cognitive ability, which can be combined with measuring amyloid plaques (e.g., PET scan or Lumipulse G β-amyloid ratio (1-42 / 1-40) test). Subjects at greater risk of developing Alzheimer's disease can be identified based on genetic markers associated with Alzheimer's disease. In various embodiments, the subject is a human subject with one or two ApoE4 alleles, or a PSEN1 (presenilin 1) mutation carrier.

[0209] Depending on the disease or disorder being targeted, administration can be carried out by different routes, such as subcutaneous, epidermal, intradermal, intrathecal, intraorbital, intramucosal, intranasal, intraperitoneal, intravenous, intrapleural, intraarterial, intracavitary, oral, intrahepatic, via the portal vein, intramuscular, intraparenchymal, intracisternal or intraventricular administration. In certain embodiments, viral or non-viral vectors are administered to the patient via infusion in a pharmaceutical carrier.

[0210] CNS administration can also be performed using techniques that promote transport across the blood-brain barrier, including disrupting the blood-brain barrier and using blood-brain barrier carriers. (Chen et al., (2021) Journal of Controlled Release 333, 129-138; and Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44 (Suppl 2): ​​131; Haumann et al., (2020) CNS Drugs 34, 1121-1131; and Cammalleri et al., J. Clin. Neurophysiol. (2020) March; 37 (2): 104-117, each of which is hereby incorporated herein by reference in its entirety.) Techniques that promote crossing the blood-brain barrier can be used on gene delivery vehicles and / or ApoE3-related proteins.

[0211] In certain embodiments, treatment of a CNS disease or disorder, such as Alzheimer's disease, is performed using expression systems that provide expression outside the CNS (eg, liver expression) in combination with techniques that facilitate transport of ApoE-related proteins across the blood-brain barrier.

[0212] In certain embodiments, techniques that facilitate the passage of gene delivery vectors across the blood-brain barrier are used to treat CNS diseases or disorders, such as Alzheimer's disease. In other embodiments, focused ultrasound combined with microbubbles is used to facilitate passage across the blood-brain barrier. (Cammalleri et al., J Clin Neurophysiol. (2020) Mar; 37(2): 104-117, which is hereby incorporated by reference in its entirety.)

[0213] In certain embodiments, the component providing CNS expression comprises a PGK promoter, a CBh promoter, or an EF1α promoter.

[0214] In certain embodiments, AAV capsids that provide CNS entry are used. Examples of such capsids are provided in Chen et al., (2021) Journal of Controlled Release 333, 129-138 (e.g., AAV9, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, and U.S. Patent Publication No. US 202 / 1214749, each of which is hereby incorporated herein by reference in its entirety.

[0215] CNS administration can also be performed, for example, by direct administration to the brain using a needle or catheter. (For example, International Publication No. WO 2021 / 108809, Cohen-Pferrer et al., Pediatric Neurology 67 (2017) 23-35; and U.S. Patent No. 10,369,329; each of which is hereby incorporated by reference in its entirety.)

[0216] Another example of a technique for CNS administration is convection-enhanced delivery. Convection-enhanced delivery involves surgical exposure of the brain followed by placement of a catheter directly into the target area, followed by infusion of the therapeutic agent. (U.S. Patent Publication No. 2022 / 010001; and Debinski et al. (2009) Expert Rev Neurother. 9(10): 1519-27; both of which are hereby incorporated by reference in their entirety.)

[0217] CNS delivery devices, systems and techniques also include those described in, for example, U.S. Patent No. 8,128,600, U.S. Patent Publication No. 2020 / 0324089, U.S. Patent No. 1,112,9643, U.S. Patent No. 1,115,4377, U.S. Patent Publication No. 2021 / 0343397, U.S. Patent Publication No. 2021 / 0282866, U.S. Patent No. 9,572,928, U.S. Patent No. 8,337,458, U.S. Patent No. 10,722,265 and U.S. Patent Publication No. 2021 / 214,749, each of which is incorporated herein by reference in its entirety.

[0218] The optimal dosage may vary depending on various factors (such as the specific therapeutic agent and the desired endpoint). Taking into account adverse side effects, complications or other risk factors of treatment or therapy and the state of the subject, the dosage amount, quantity, frequency or duration may be proportionally increased or decreased.

[0219] "Unit dosage form" refers to a physically discrete unit containing a predetermined effective amount of an active ingredient in combination with a pharmaceutically acceptable carrier. Unit dosage forms can be provided, for example, in ampoules and vials, which can include a pharmaceutically acceptable carrier, or in a freeze-dried or lyophilized state. In the case of a freeze-dried or lyophilized state, a sterile liquid carrier can be added prior to administration. Separate unit dosage forms can be included in multiple-dose kits or containers.

[0220] An "effective amount" achieves the desired or indicated effect. For example, a therapeutically effective amount reduces one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces the progression of a disease or disorder. Preferred therapeutically effective amounts are effective to reduce multiple or all adverse symptoms.

[0221] In certain embodiments, a pharmaceutical composition comprising a viral or non-viral vector is administered to a subject at a dose suitable for lowering cholesterol, lowering LDL / VLDL, increasing HDL, or lowering the total cholesterol / HDL ratio; or treating hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia (e.g., vascular dementia or frontotemporal dementia), post-stent restenosis, atherosclerosis, coronary heart disease, or Alzheimer's disease or reducing the likelihood of these diseases. In various embodiments, a suitable dose is from about 0.01 mg / kg to about 10 mg / kg of vector per kg of subject body weight, from about 0.01 mg / kg to about 0.1 mg / kg of vector per kg of subject body weight, from about 0.1 mg / kg to about 1.0 mg / kg of vector per kg of subject body weight, or from about 1.0 mg / kg to about 10 mg / kg of vector per kg of subject body weight.

[0222] Typically, rAAV doses range from at least 1x10 8 vector genomes / kg (vg / kg) of subject body weight, or more, e.g., 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x10 13 or 1x10 14 or more vector genomes / kg (vg / kg) of subject body weight to achieve a therapeutic effect. In various embodiments, the rAAV dose is about 5x10 11 rAAV vg / kg or greater than about 5x10 11 rAAV vg / kg; approximately 1x10 12 rAAV vg / kg or greater than about 1x10 12 rAAV vg / kg; approximately 2x10 12 rAAV vg / kg or greater than about 2x1012 rAAV vg / kg; approximately 3x10 12 rAAV vg / kg or greater than approximately 3x10 12 rAAV vg / kg; approximately 4x10 12 rAAV vg / kg or greater than approximately 4x10 12 rAAV vg / kg; approximately 5x10 12 rAAV vg / kg or greater than approximately 5x10 12 rAAV vg / kg; approximately 1x10 13 rAAV vg / kg or greater than about 1x10 13 rAAV vg / kg; approximately 2x10 13 rAAV vg / kg or greater than about 2x10 13 rAAV vg / kg; approximately 3x10 13 rAAV vg / kg or greater than approximately 3x10 13 rAAV vg / kg; approximately 4x10 13 rAAV vg / kg or greater than approximately 4x10 13 rAAV vg / kg; approximately 5x10 13 rAAV vg / kg or greater than approximately 5x10 13 rAAV vg / kg; approximately 6x10 13 rAAV vg / kg or greater than approximately 6x10 13 rAAV vg / kg.

[0223] Examples of dosage ranges for rAAV vg / kg include approximately 5x10 11 to about 6x10 13 rAAV dose range of vg / kg; approximately 5x10 11 to about 5.5x10 11 The dose range of rAAV vg / kg is approximately 5.5x10 11 to about 6x10 11 The dose range of rAAV vg / kg is approximately 6x10 11 to about 6.5x10 11 The dose range of rAAV vg / kg is approximately 6.5x10 11 to about 7x10 11 rAAV dose range of vg / kg; approximately 7x10 11 to about 7.5x10 11 rAAV vg / kg dose range; approximately 7.5x10 11 to about 8x10 11 rAAV vg / kg dose range; approximately 8x1011 to about 8.5x10 11 The dose range of rAAV vg / kg is approximately 8.5x10 11 to about 9x10 11 The dose range of rAAV vg / kg is approximately 9x10 11 to about 9.5x10 11 The dose range of rAAV vg / kg is approximately 9.5x10 11 to about 1x10 12 The dose range of rAAV vg / kg is approximately 1x10 12 to about 1.5x10 12 The dose range of rAAV vg / kg is approximately 1.5x10 12 to about 2x10 12 The dose range of rAAV vg / kg is approximately 2x10 12 to about 2.5x10 12 The dose range of rAAV vg / kg is approximately 2.5x10 12 to about 3x10 12 The dose range of rAAV vg / kg is approximately 3x10 12 to about 3.5x10 12 The dose range of rAAV vg / kg is approximately 3.5x10 12 to about 4x10 12 rAAV dose range of vg / kg; approximately 4x10 12 to about 4.5x10 12 The dose range of rAAV vg / kg is approximately 4.5x10 12 to about 5x10 12 rAAV dose range of vg / kg; approximately 5x10 12 to about 5.5x10 12 The dose range of rAAV vg / kg is approximately 5.5x10 12 to about 6x10 12 The dose range of rAAV vg / kg is approximately 6x10 12 to about 6.5x10 12 The dose range of rAAV vg / kg is approximately 6.5x10 12 to about 7x10 12 rAAV dose range of vg / kg; approximately 7x10 12 to about 7.5x10 12 rAAV vg / kg dose range; approximately 7.5x10 12 to about 8x10 12 rAAV vg / kg dose range; approximately 8x10 12to about 8.5x10 12 The dose range of rAAV vg / kg is approximately 8.5x10 12 to about 9x10 12 The dose range of rAAV vg / kg is approximately 9x10 12 to about 9.5x10 12 The dose range of rAAV vg / kg is approximately 9.5x10 12 to about 1x10 13 The dose range of rAAV vg / kg is approximately 1x10 13 to about 1.5x10 13 The dose range of rAAV vg / kg is approximately 1.5x10 13 to about 2x10 13 The dose range of rAAV vg / kg is approximately 2x10 13 to about 2.5x10 13 The dose range of rAAV vg / kg is approximately 2.5x10 13 to about 3x10 13 The dose range of rAAV vg / kg is approximately 3x10 13 to about 3.5x10 13 The dose range of rAAV vg / kg is approximately 3.5x10 13 to about 4x10 13 rAAV dose range of vg / kg; approximately 4x10 13 to about 4.5x10 13 The dose range of rAAV vg / kg is approximately 4.5x10 13 to about 5x10 13 The dose range of rAAV vg / kg is approximately 5x10 13 to about 5.5x10 13 The dose range of rAAV vg / kg is approximately 5.5x10 13 to about 6x10 13 The dose range of rAAV vg / kg is approximately 6x10 13 to about 1x10 14 rAAV dose range of vg / kg.

[0224] In certain embodiments, rAAV vg / kg is at about 5x10 11 vg / kg, about 6x10 11 vg / kg, about 7x10 11 vg / kg, about 8x10 11 vg / kg, about 9x10 11 vg / kg, about 1x10 12 vg / kg, about 2x1012 vg / kg, about 3x10 12 vg / kg, about 4x10 12 vg / kg, about 5x10 12 vg / kg, about 6x10 12 vg / kg, about 7x10 12 vg / kg, about 8x10 12 vg / kg, about 9x10 12 vg / kg, about 1x10 13 vg / kg, about 2x10 13 vg / kg, about 3x10 13 vg / kg, about 4x10 13 vg / kg, about 5x10 13 vg / kg or about 6x10 13 vg / kg dose was administered.

[0225] In certain embodiments, the dosages and dosage ranges of other viral vectors are as provided herein for rAAV. For example, in certain embodiments, recombinant adenoviral vectors, recombinant retroviral vectors (e.g., lentivirus), and recombinant herpes simplex virus vectors are the same as described above for rAAV.

[0226] In certain embodiments, the polynucleotide constructs, viral vectors, and non-viral vectors described herein are administered in combination with additional compounds or treatments for a specific disease or disorder; and / or are administered in combination with compounds that reduce the immune response to the polynucleotides, delivery vehicles, and / or produced proteins. The additional compounds or treatments can be provided in different ways, such as administered separately; and administered or performed before, substantially simultaneously with, or after the administration of the polynucleotide constructs, viral vectors, and non-viral vectors described herein.

[0227] In certain embodiments, the administration of the polynucleotide constructs, viral vectors and non-viral vectors described herein is carried out in combination with an immunosuppressant or regimen. Such agents and regimens can be used as needed to achieve immune tolerance to the ApoE3-related proteins produced, the polynucleotides provided, or the delivery vehicles provided, or to reduce the immune response to the ApoE3-related proteins produced, the polynucleotides provided, or the delivery vehicles provided. Examples of immunosuppressants and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, (Synthetic Vaccine Particles (SVP)-Rapamycin (Rapamycin encapsulated in biodegradable nanoparticles)), ImmTOR-IL TM(ImmTOR with a Treg-selective IL-2 agonist), B cell depletion, immunoadsorption, and plasmapheresis.

[0228] In certain embodiments, viral vectors or non-viral vectors are co-administered with one or more immunosuppressants, wherein one or more immunosuppressants are administered before, substantially simultaneously with, or after administration of the vector or non-viral vector. In certain embodiments, one or more immunosuppressants are administered simultaneously with the vector or non-viral vector. In certain embodiments, one or more immunosuppressants are administered 1-12 hours, 12-24 hours, or 24-48 hours before viral or non-viral vector administration; or 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, 30-50 days, or more than 50 days. In certain embodiments, 1-12 hours, 12-24 hours or 24-48 hours after viral or non-viral vector administration;Or 2-4 days, 4-6 days, 6-8 days, 8-10 days, 10-14 days, 14-20 days, 20-25 days, 25-30 days, 30-50 days or more than 50 days administer one or more immunosuppressants.For example, if a period of time after carrier or non-viral vector administration (for example, 20-25 days, 25-30 days, 30-50 days, 50-75 days, 75-100 days, 100-150 days, 150-200 days or more than 200 days) the encoded protein decreases after the initial expression level, then the administration of immunosuppressants can be carried out after a period of time after carrier or non-viral vector administration.

[0229] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid, such as a corticosteroid. In certain embodiments, the immunosuppressant is prednisone, prednisolone, a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g., ), CD52 inhibitors (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, belatacept), anti-CD3 mAb, anti-LFA-1 mAb (e.g., efalizumab), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epratuzumab), anti-CD20 mAb (e.g., rituximab, ocrelizumab, ofatumumab, veltuzumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., atacicept), anti-C5 mAb (e.g., eculizumab), mycophenolate mofetil, azathioprine, sirolimus, everolimus, TNFR-Ig, anti-TNF mAb, tofacitinib, anti-IL-2R (e.g., basiliximab), anti-IL-17 mAb (e.g., secukinumab), anti-IL-6 mAb (e.g., anti-IL-6 antibody sirukumab), anti-IL-6 receptor antibody tocilizumab IL-10 inhibitors, TGF-β inhibitors, B cell targeting antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVPs), TM )-rapamycin (rapamycin encapsulated in biodegradable nanoparticles), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g., ibrutinib), cyclophosphamide, fingolimod, B-cell activating factor (BAFF) inhibitors (e.g., anti-BAFF mAbs, e.g., belimumab), proliferation-inducing ligand (APRIL) inhibitors, anti-IL-1b mAbs (e.g., canakinumab), ), C3a inhibitors, Tregitope (see, e.g., U.S. Patent No. 10,213,496), or combinations and / or derivatives thereof.

[0230] Immunosuppressive regimens (including rapamycin alone or in combination with IL-10) can be used to reduce, diminish, inhibit, prevent or block humoral and cellular immune responses to ApoE3-related proteins. Liver gene transfer using viral vectors (e.g., rAAV) and non-viral vectors can be used to induce immune tolerance to ApoE3-related proteins by inducing regulatory T cells (Tregs).

[0231] Strategies to reduce (overcome) or avoid humoral immunity to viral vectors (such as rAAV) in systemic gene transfer include: administering high vector doses; using AAV empty capsids as bait to adsorb anti-AAV antibodies; administering immunosuppressive drugs to reduce, lower, inhibit, prevent or eradicate the humoral immune response to rAAV; changing the rAAV capsid serotype or engineering the rAAV capsid to make it less sensitive to neutralizing antibodies; using plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers; and using delivery techniques such as balloon catheters followed by saline flushing. Such strategies are described in Mingozzi et al., (2013) Blood, 122: 23-36. Additional strategies include using AAV-specific plasma apheresis columns to selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool in plasma, as described in Bertin et al., 2020, Sci. Rep. 10: 864. Similar techniques and strategies can be used for other types of viral vectors.

[0232] Empty capsids used as bait probes can be provided at different ratios to viral vectors. The amount of the empty capsids administered can be calibrated based on the amount (titer) of antibodies produced in a particular subject. In certain embodiments, the ratio of empty AAV capsids to rAAV vectors is within or between about 100: 1-50: 1, about 50: 1 to 25 to 1, about 25: 1 to 10: 1, about 10: 1 to 1: 1, about 1: 1 to 1: 10, about 1: 10 to 1: 25, about 1: 25 to 1: 50, or about 1: 50 to 1: 100. In certain aspects, the ratio of the empty AAV capsids administered to the rAAV vector is about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1. Preferably, the serotype of the empty capsid is the same as the rAAV serotype.

[0233] In certain embodiments, viral vectors are delivered using methods that bypass the bloodstream and viral antibodies. Examples of such techniques include delivery to the liver via the hepatic artery; and delivery to the liver via endoscopic retrograde cholangiopancreatography (ERCP). Delivery to the CNS is via the carotid artery. Other ductal systems (such as the duct of the submandibular gland) can also be used as a portal for delivering viral vectors to subjects who produce or have pre-existing anti-antibodies to the viral vector.

[0234] Other strategies for reducing humoral immunity to rAAV (which can be applied to other viral vectors) include methods of removing, depleting, capturing and / or inactivating AAV antibodies, commonly referred to as apheresis, more specifically, plasmapheresis of blood products. Apheresis or plasmapheresis is a process in which the plasma of a human subject is circulated ex vivo (in vitro) by a device that alters the plasma by adding, removing and / or replacing components before the plasma is returned to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from a blood product (e.g., plasma). This procedure can be used to deplete, capture, inactivate, reduce or remove immunoglobulins (antibodies) that bind to AAV, thereby reducing the titer of AAV antibodies in the treated subject that may contribute to rAAV neutralization. One example is the use of a device consisting of an AAV capsid affinity matrix column and passing a blood product (e.g., plasma) through the AAV capsid affinity matrix, resulting in the binding of AAV antibodies of different isotypes. (See, e.g., Bertin et al., 2020, Sci. Rep. 10, 864, which is hereby incorporated by reference in its entirety.)

[0235] In certain embodiments, polynucleotide constructs, viral vectors, and non-viral vectors can be used in combination with agents that block, inhibit, or reduce the interaction of IgG with the neonatal Fc receptor (FcRn), such as anti-FcRn antibodies, to reduce IgG recycling and enhance IgG clearance in vivo; and / or agents that reduce circulating antibodies that bind to recombinant viral vectors or to nucleic acids or polypeptides, proteins, or peptides encoded by polynucleotides encapsidated by recombinant viral vectors. In certain embodiments, antibody binding to viral vectors is reduced or inhibited by agents that reduce the interaction of IgG with FcRn, proteases, or glycosidases.

[0236] In certain embodiments, the polynucleotide constructs, viral vectors, and non-viral vectors described herein can be combined with endopeptidases (e.g., Ides from Streptococcus pyogenes) or modified variants thereof, or endoglycosidases (e.g., Streptococcus pyogenes EndoS) or modified variants thereof. For example, such treatments can be performed to reduce or eliminate neutralizing antibodies against gene delivery vehicles (e.g., viral vector capsids) and can treat patients who were previously considered ineligible for gene therapy or who produce antibodies produced by gene therapy. Such strategies are described, for example, in Leborgne et al., (2020) Nat. Med., 26: 1096-1101.

[0237] In certain embodiments, the treatment method of the subject is combined with a compound that reduces the expression of natural subject ApoE. Natural ApoE expression can be inhibited, for example, using inhibitory nucleic acids that selectively target natural ApoE coding sequences. Reference to "selectively targeting" natural ApoE sequences indicates that the expression of polynucleotides encoding ApoE3-related proteins is not significantly affected. The inhibitory nucleic acid can be provided on the same polynucleotide and / or vector encoding the ApoE3-related protein, or provided using a separate viral or non-viral vector. Examples of inhibitory nucleic acids include short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), RNAi, ribozymes, and antisense RNA.

[0238] In certain embodiments, the expression cassette further comprises an inhibitory nucleic acid that selectively targets one or both of the nucleic acids encoding naturally occurring ApoE2, ApoE3, and ApoE4. In other embodiments, ApoE2 is targeted or ApoE4 is targeted. In certain embodiments, the subject has one ApoE4 allele or two ApoE4 alleles, and the inhibitory nucleic acid targets the ApoE4 encoding nucleic acid; and the subject has two ApoE2 alleles and the inhibitory nucleic acid targets ApoE2. In certain embodiments, an ApoE3-related protein comprising the Christchurch mutation is used in combination to target natural ApoE3.

[0239] In certain embodiments, the polynucleotide constructs, viral vectors, and non-viral vectors described herein are used in combination with one or more additional treatments (e.g., for the treatment of hyperlipidemia, atherosclerosis, cardiovascular disease, and / or dementia). In various embodiments, additional treatments include statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin), PCSK9 inhibitors, and / or ezetimibe. In certain embodiments, additional treatments include donepezil, galantamine, rivastigmine, or memantine. In certain embodiments, additional dementia (e.g., Alzheimer's disease) treatments include statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin), PCSK9 inhibitors, and / or ezetimibe.

[0240] VIII. Kit

[0241] The present invention includes kits comprising packaging materials and one or more components thereof. The kits typically include a label or package insert that includes a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. The kits may contain a collection of such components, for example, a viral or non-viral vector, and optionally a second active substance, such as another compound, agent, medicament, or composition.

[0242] A kit refers to a physical structure that houses one or more components. The packaging material can sterilely maintain the components and can be made of materials commonly used for such purposes, such as paper, corrugated fiber, glass, plastic, foil, ampoules, vials, and tubes.

[0243] The label or insert may include identification information for one or more components, dosage amounts, clinical pharmacology (including mechanism of action), pharmacokinetics, and pharmacodynamics of one or more active ingredients. The label or insert may include information identifying the manufacturer, batch number, manufacturer location, and date, as well as expiration date. The label or insert may include information about the disease for which the kit components can be used. The label or insert may include instructions for a clinician or subject to use one or more kit components in a method, use, or treatment protocol. Instructions may include dosage amounts, frequency, or duration, and instructions for implementing any of the methods, uses, treatment protocols, or preventive or therapeutic regimens described herein.

[0244] The label or insert can include information about one or more benefits (such as preventive or therapeutic benefits) that the component can provide. The label or insert can include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which a particular composition would not be suitable for use. Adverse side effects or complications may also occur when the subject has, will, or is taking one or more other drugs that may be incompatible with the composition, or when the subject has, will, or is currently experiencing another treatment protocol or therapeutic regimen that is incompatible with the composition, so instructions can include information about such incompatibilities.

[0245] The label or insert includes "printed matter", for example, paper or cardboard, either separately or fixed to a component, kit or packaging material (e.g., box), or attached to an ampoule, tube or vial containing a kit component. The label or insert may additionally be included in a computer-readable medium such as a barcode printed label, a magnetic disk, an optical disk (e.g., CD-ROM / RAM or DVD-ROM / RAM, DVD), an MP3, a magnetic tape or an electronic storage medium (e.g., RAM and ROM) or a hybrid of these (e.g., magnetic / optical storage medium, flash memory medium or memory-type card).

[0246] IX. mRNA therapeutics

[0247] In certain embodiments, an RNA form of a nucleic acid encoding an ApoE-related protein as described herein is provided as an mRNA construct capable of expressing the encoded protein inside a cell. The mRNA construct comprises a 5′-cap, a 5′UTR, a coding RNA, a 3′UTR, and a poly(A) tail. The UTR and the poly(a) tail can provide different functions, such as participating in mRNA subcellular localization, regulating translation efficiency, and mRNA stability. The design and production of mRNA constructs (including different modifications) are described in different publications: Ouranidis et al., (2022) Biomedicines, 10, 50; Qin et al., Signal Transduct Target Ther. (2022) May 21; 7(1): 166, and U.S. Patent Publication No. US2013 / 0259924, each of which is hereby incorporated herein by reference in its entirety.

[0248] In certain embodiments, nanoparticles are used to deliver the mRNA construct to a cell or subject. Examples of nanoparticles include those provided in sections VA-VE, supra, Ouranidis et al., (2022) Biomedicines, 10, 50, and U.S. Patent Publication No. US2013 / 0259924.

[0249] Guidance regarding therapeutic administration and targeting of diseases or disorders is provided, for example, in Section VII, supra.

[0250] X. Additional Aspects and Embodiments

[0251] Additional aspects, embodiments, and combinations thereof include the following:

[0252] A first aspect relates to a polynucleotide comprising a nucleotide sequence encoding ApoE having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 63-67 or nucleotides 55-951 of any one of SEQ ID NOs: 95-105 and 124-130, wherein the polynucleotide encodes an ApoE3-related protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 32, wherein the protein comprises a cysteine ​​at a position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at a position corresponding to amino acid 158 of SEQ ID NO: 32, wherein the nucleotide sequence encoding ApoE optionally comprises one or more introns. The percent identity is determined independently of any introns present in the nucleotide sequence encoding ApoE.

[0253] Embodiment E1a further describes the first aspect, wherein the nucleic acid sequence encoding ApoE comprises one or more introns.

[0254] Embodiment E1b further describes the first aspect, wherein the nucleic acid sequence encoding ApoE does not contain any introns. Introns may be present in other parts of the polynucleotide.

[0255] Embodiment E2a further describes the first aspect, embodiments E1a and E1b, wherein the nucleotide sequence encoding ApoE has at least 95% sequence identity to nucleotides 55-951 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, and 27-30. In additional embodiments, the nucleic acid sequence encoding ApoE is at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to nucleotides 55-951 or 55-954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25 and 27-30; or differs from any one of nucleotides 55-951 or 55-954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25 and 27-30 by 1-40 nucleotides, 1-20 nucleotides or 1-10 nucleotides.

[0256] Embodiment E2b further describes the first aspect and embodiments E1a and E1b, wherein the nucleotide sequence encoding ApoE has at least 95% sequence identity with nucleotides 55-951 of any one of SEQ ID NOs: 95-105 and 124-130. In further embodiments, the nucleic acid sequence encoding ApoE has at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with nucleotides 55-951 or 55-954 of any one of SEQ ID NOs: 95-105 and 124-130; or differs from any one of nucleotides 55-951 or 55-954 of any one of SEQ ID NOs: 95-105 and 124-130 by 1-40 nucleotides, 1-20 nucleotides, or 1-10 nucleotides. Preferably, the sequence identity is with respect to any one of SEQ ID NOs: 95-105.

[0257] Embodiment E3 further describes the first aspect, embodiments E1a and E1b, wherein the nucleotide sequence encoding ApoE has at least 95% sequence identity to any one of SEQ ID NOs: 63-67. In further embodiments, the nucleic acid sequence encoding ApoE has at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 63-67 or to nucleotides 1-897 of any one of SEQ ID NOs: 63-67; or differs from any one of SEQ ID NOs: 63-67 by 1-40 nucleotides, 1-20 nucleotides, or 1-10 nucleotides.

[0258] Embodiment E4 further describes the first aspect, embodiments Ela, Elb, E2a, E2b and E3, wherein the ApoE3-related protein comprises a serine at the position corresponding to amino acid 136 of SEQ ID NO:32.

[0259] Embodiment E5 further describes the first aspect, embodiments E1a, E1b, E2a, E2b, and E3, wherein the ApoE3-related protein is at least 95% identical to the sequence of SEQ ID NO: 32. In further embodiments, the protein is at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 32; or differs from SEQ ID NO: 32 by 1-10 amino acids, or any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0260] Embodiment E6 further describes the first aspect, embodiments E1a, E1b, E2a, E2b, and E3, wherein the ApoE3-related protein is at least 95% identical to the sequence of SEQ ID NO: 33. In further embodiments, the protein is at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 33, or differs from SEQ ID NO: 33 by 1-10 amino acids, or any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0261] Embodiment E7 further describes the first aspect, embodiments E1a, E1b, E2a, E2b, E3, E4, E5 and E6, wherein the ApoE3-related protein further comprises a 5′ signal peptide, and the nucleic acid sequence encoding ApoE3 further comprises a nucleotide sequence encoding the signal peptide.

[0262] Embodiment E8 further describes embodiment E7, wherein the signal peptide is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71. In further embodiments, the signal peptide is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71; or differs from any one of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71 by any one of 1, 2, or 3 amino acids.

[0263] Embodiment E9 further describes embodiment E8, wherein the nucleotide sequence encoding the signal peptide sequence comprises at least 90% identity to a sequence selected from SEQ ID NOs: 37-41, 43, 45, 47, 49, 51, 53, 55, 57, and 72-75. In further embodiments, the nucleotide sequence encoding the signal peptide is at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 37-41, 43, 45, 47, 49, 51, 53, 55, 57, and 72-75; or differs from any one of SEQ ID NOs: 37-41, 43, 45, 47, 49, 51, 53, 55, 57, and 72-75 by 1-10 nucleotides, or any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0264] Embodiment E10a further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, and E9, wherein the nucleic acid sequence encoding ApoE has at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 3-31. In further embodiments, the nucleic acid sequence encoding ApoE has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 3-31; or differs from any one of SEQ ID NOs: 3-31 by 1-40 nucleotides, 1-20 nucleotides, or 1-10 nucleotides. In further embodiments, the percent identity is with respect to SEQ ID NOs: 20, 26, 31, 11, or 15; or with respect to SEQ NOs: 11 and 20.

[0265] Embodiment E10b further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, and E9, wherein the nucleic acid sequence encoding ApoE has at least 90% sequence identity to any one of SEQ ID NOs: 95-105 and 124-130. In further embodiments, the nucleic acid sequence encoding ApoE has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 95-105 and 124-130; or differs from any one of SEQ ID NOs: 95-105 and 124-130 by 1-40 nucleotides, 1-20 nucleotides, or 1-10 nucleotides. In further embodiments, the percent identity is with respect to SEQ ID NOs: 95-105.

[0266] Embodiment E11 further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, E9, E10a and E10b, wherein the ApoE3-related sequence comprises the sequence of SEQ ID NO: 34 or 35.

[0267] Embodiment E12a further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, E9, E10a, E10b, and E11, wherein the nucleotide sequence encoding ApoE comprises the following sequence: (i) nucleotides 55 to 951 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, and 27-30; (ii) nucleotides 55 to 954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, and 27-30; (iii) nucleotides 1 to 897 of any one of SEQ ID NOs: 63-67; (iv) any one of SEQ ID NOs: 63-67; (v) nucleotides 1 to 951 of any one of SEQ ID NOs: 3-31; or (vi) nucleotides 1 to 951 of any one of SEQ ID NOs: 3-32. NO: Any one from 3 to 31.

[0268] Embodiment E12b further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, E9, E10a, E10b and E11, wherein the nucleotide sequence encoding ApoE comprises the following sequence: (i) nucleotides 55 to 951 of any one of SEQ ID NOs: 95-105 and 124-130; (ii) nucleotides 55 to 954 of any one of SEQ ID NOs: 95-105 and 124-130; (iii) nucleotides 1 to 951 of any one of SEQ ID NOs: 95-105 and 124-130; or (iv) any one of SEQ ID NOs: 95-105 and 124-130.

[0269] Embodiment 12c further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, E9, E10a, E10b, and E11, wherein the nucleotide sequence encoding ApoE comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to: (i) nucleotides 55 to 951 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (ii) nucleotides 55 to 954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (iii) nucleotides 55 to 956 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; NO: nucleotides 1-897 of any one of SEQ ID NO: 63-67; (iv) any one of SEQ ID NO: 63-67; (v) nucleotides 1 to 951 of any one of SEQ ID NO: 3-31, 95-105 and 124-130; or (vi) any one of SEQ ID NO: 3-31, 95-105 and 124-130; and the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO: 34 or 35.

[0270] Embodiment 12d further describes embodiment 1a, wherein the nucleotide sequence encoding ApoE comprises from 5′ to 3′: (a) a first exon corresponding to nucleotides 1-43 of SEQ ID NO: 1, wherein the first exon has at least 85% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105, or 124-130, with the proviso that the terminal 3' nucleotide of the first exon is a G, and in further embodiments directed to the first exon, the first exon has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130; and in further embodiments, the sequence identity is with respect to nucleotides 1-43 of any one of SEQ ID NOs: 11, 20, and 95-105; (b) the first intron at a position corresponding to between nucleotides 43 and 44 of SEQ ID NO: 1; (c) a second exon corresponding to nucleotides 44-236 of SEQ ID NO: 1; wherein the second exon has at least 85% sequence identity with nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the terminal 5' nucleotide of the second exon is G (in further embodiments, GC) and the terminal 3' nucleotide of the second exon is AG. In further embodiments involving the second exon, the second exon has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130; and in further embodiments, the sequence identity is with respect to nucleotides 44-236 of any one of SEQ ID NOs: 11, 20, 95-105, and 124-130; (d) the second intron at a position corresponding to between nucleotides 236 and 237 of SEQ ID NO: 1; (e) a third exon corresponding to nucleotides 237-951 of SEQ ID NO: 1, wherein the third exon has at least 85% sequence identity with nucleotides 237-951 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the terminal 5' nucleotide of the third exon is G, wherein the first exon, the second exon, and the third exon together encode the ApoE3-related protein. In additional embodiments involving the third exon, the third exon has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to nucleotides 237-951 of any one of SEQ ID NOs: 3-31, 95-105 and 124-130; and in additional embodiments, the sequence identity is to nucleotides 237-951 of any one of SEQ ID NOs: 11, 20 and 95-105; wherein the ApoE-related protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 35, and in additional embodiments, the ApoE-related protein comprises an amino acid sequence at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO: 35, differs from SEQ ID NO: 35 by 1, 2, 3, 4 or 5 amino acids, or comprises SEQ ID NO: 34 or SEQ ID NO: 35.

[0271] For each sequence, each possibility with (a), (b), (c), (d) and (e) can be combined independently. For example, (1) the first exon can have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105 and 124-130 independently of the second exon and the third exon; (2) the second exon can have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105 and 124-130 independently of the first exon and the third exon; and (3) the third exon can have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105 and 124-130 independently of the first exon and the second exon. Nucleotides 44-236 of any of ID NOs: 3-31, 95-105, and 124-130 have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0272] Embodiment 12e further describes embodiment 12d, wherein the first intron comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 119-122, and the second intron independently comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 119-122.

[0273] Embodiment 12f further describes embodiment 12e, wherein the first intron consists of a sequence of any one of SEQ ID NOs: 119-121 or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides, and the second intron independently consists of a sequence of any one of SEQ ID NOs: 119-121 or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides.

[0274] Embodiment 12g further describes embodiments 12d, 12e and 12f, wherein The first exon has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 11, 20 and 95-105; the second exon has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 11, 20 and 95-105; and the third exon has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to nucleotides 237-951 of any one of SEQ ID NOs: 11, 20 and 95-105, wherein the ApoE-related protein has at least 98% sequence identity to SEQ ID NO: 35.

[0275] Embodiment 12h further describes embodiment 12g, wherein the nucleotide sequence encoding ApoE comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 106-115.

[0276] Embodiment 12i further describes embodiments E12d, E12e, E12f, E12g and E12h, wherein the ApoE-related protein comprises the sequence of SEQ ID NO: 34 or 35.

[0277] Embodiment E13 further describes the first aspect, Embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, E9, E10a, E10b, E11, E12a, E12b, E12c, E12d, E12e, E12f, E12g, E12h, and E12i, wherein the nucleic acid sequence encoding ApoE contains any one of: -5, 0-10, or 0-15 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpGs; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0%, or at most about 5.0% CpGs. Preferably, 0-10, 0-5, or 0 CpGs.

[0278] Embodiment E14 further describes embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, E9, E10a, E10b, E11, E12a, E12b, E12c, E12d, E12e, E12f, E12g, E12h, E12i, and E13, wherein the polynucleotide is an expression cassette further comprising one or more expression control elements operably linked to the nucleic acid sequence encoding ApoE. In additional embodiments, one or more expression control elements are present selected from the group consisting of a promoter, a promoter / enhancer, an intron, a polyadenylation signal, and a Kozak sequence. In another embodiment, the expression cassette comprises a promoter operably linked to the nucleic acid sequence encoding ApoE at 5′ and a polyadenylation site operably linked to the nucleic acid sequence encoding ApoE at 3′; or the expression cassette comprises, from 5′ to 3′, a promoter operably linked to the nucleic acid sequence encoding ApoE, an intron, a Kozak sequence, the nucleic acid sequence encoding ApoE and a polyadenylation signal.

[0279] Embodiment E15 further describes embodiment E14, wherein the promoter is a liver-specific promoter. In additional embodiments, the promoter is hAAT.

[0280] Embodiment E16 further describes embodiments E14 and E15, wherein the promoter is operably coupled to an HCR1-based enhancer having at least 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO:58.

[0281] Embodiment E17 further describes embodiment E14, wherein the promoter provides expression in CNS cells. In another embodiment, the promoter is a CAG promoter, a CBh promoter, an EF1α promoter, or a human synapsin (hSynapsin) promoter. In another embodiment, the expression cassette further comprises a miRNA target sequence that inhibits expression in dorsal root ganglia, liver, and / or immune cells.

[0282] Embodiment E18 further describes embodiments E14, E15, E16, and E17, wherein the intron comprises the sequence of SEQ ID NO: 60. In further embodiments, the polyadenylation signal comprises the sequence of SEQ ID NO: 61 or SEQ ID NO: 62.

[0283] Embodiment E19 further describes embodiments E14, E15, E16, E17, and E18, wherein the expression cassette further comprises an inhibitory nucleic acid that selectively targets one or both of the nucleic acids encoding ApoE2, ApoE3, and ApoE4. In further embodiments, ApoE2 is targeted or ApoE4 is targeted.

[0284] Embodiment E20 further describes embodiments E14, E15, E16, E17, E18, and E19, wherein the expression cassette contains any of: 0-5, 0-10, 0-15, 0-50, or 0-100 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or about 15% CpG; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0%, at most about 5.0%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or at most about 15% CpG.

[0285] Embodiment E21 further describes embodiments E14, E15, E16, E17, E18, E19, and E20, wherein the expression cassette comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 76-80; comprises a sequence that differs from any one of SEQ ID NOs: 76-80 by 1-40 nucleotides, 1-20 nucleotides, or 1-10 nucleotides; or comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a modified SEQ ID NO: 76, wherein SEQ ID NO: 76 is modified by replacing SEQ ID NO: 76 with SEQ ID NO: 76. The sequence of any one of NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-115, and 124-130 is modified by replacing nucleotides 1295-2248.

[0286] Embodiment E22 further describes E14, E15, E16, E17, E18, E19, E20, and E21, wherein the polynucleotide is a recombinant viral vector nucleic acid comprising an expression cassette and 5' and / or 3' viral element-associated elements that provide viral packaging and / or replication. In additional embodiments, the recombinant viral vector nucleic acid is DNA; and / or is based on the AAV genome and comprises AAV 5' and 3' ITRs; the recombinant viral vector nucleic acid is DNA and is based on an adenovirus genome and comprises 5' and 3' ITRs and a packaging signal; and the recombinant viral vector nucleic acid is RNA and is based on a retroviral genome (e.g., a lentivirus) and comprises 5' and 3' LTRs and a packaging signal. Reference to "based on" a viral genome indicates the ability to replicate and be packaged into the capsid of a reference virus.

[0287] Embodiment E23 further describes E22, wherein the polynucleotide is a rAAV nucleic acid comprising ITRs flanking the 5' end of the polynucleotide and / or the 3' end of the polynucleotide. In further embodiments, the ITRs flank both the 5' and 3' ends of the polynucleotide.

[0288] Embodiment E24 further describes E23, wherein the 5′ and / or 3′ viral elements are each selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B.

[0289] Embodiment E25 further describes E23, wherein the 5′ ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 81, 88, 90, 92 and 94, and the 3′ ITR independently comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NOs: 82, 89, 91 and 93.

[0290] Embodiment E26 further describes E22, E23, E24, and E25, wherein the recombinant viral vector nucleic acid contains any of: 0-5, 0-10, 0-15, 0-50, 0-100, or 0 to 150 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or about 15% CpG; and / or at most about 0.5%, at most about 1.0%, at most about 2.0%, at most about 3.0%, at most about 4.0%, at most about 5.0%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14% or at most about 15% CpG.

[0291] Embodiment E27 concerns a gene delivery vector, wherein the gene delivery vector is a viral or non-viral vector comprising a polynucleotide according to the first aspect, any one of E1-E21, or a recombinant viral vector according to any one of E22-E26.

[0292] Embodiment E28 further describes E27, wherein the gene delivery vehicle is a viral vector. In another embodiment, the vehicle is a rAAV vector, a recombinant retrovirus (e.g., lentivirus) vector, or a recombinant adenovirus vector.

[0293] Embodiment E29 further describes E28, wherein the viral vector is rAAV, and the rAAV vector comprises a capsid comprising VP1, VP2 or VP3 having at least 90% sequence identity to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 83 and SEQ ID NO: 84. In further embodiments, the recombinant AAV vector capsid comprises a VP1, VP2, or VP3 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-Bl, AAV-AS, AAV1 / rh.10, SEQ ID NO: 83, and SEQ ID NO: 84. In further embodiments, the capsid comprises a VP1 comprising the sequence of SEQ ID NO: 83, a VP2 comprising the sequence of SEQ ID NO: 122, and a VP3 comprising the sequence of SEQ ID NO: 123.

[0294] Embodiment E30 further describes E27, wherein the gene delivery vehicle is a non-viral vector. In another embodiment, the non-viral vector is a nanoparticle; is a nanoparticle selected from the group consisting of lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein or peptide-based nanoparticles, DNA dendrimers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes; is LPN; or is LPNP.

[0295] Embodiment E31 relates to a pharmaceutical composition comprising the polynucleotide according to the first aspect, any one of E1-E21, the recombinant viral vector according to any one of E22-E26, or the gene delivery vehicle according to any one of E27-E30; and a pharmaceutically acceptable carrier.

[0296] Embodiment E32 further describes E31, wherein the composition comprises rAAV and empty AAV capsids, and the ratio of the empty AAV capsids to the rAAV is between 100:1 and 1:100. In further embodiments, the ratio of the empty AAV capsids to the rAAV is between about 100:1 and 50:1, about 50:1 and 25:1, about 25:1 and 10:1, about 10:1 and 1:1, about 1:1 and 1:10, about 1:10 and 1:25, about 1:25 and 1:50, or about 1:50 and 1:100. In further embodiments, the ratio of the empty AAV capsids to the rAAV is between about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0297] The second aspect of the present invention relates to a method for lowering cholesterol, lowering LDL / VLDL, increasing HDL or lowering the total cholesterol / HDL ratio; or treating hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia, post-stent restenosis, atherosclerosis, coronary heart disease or Alzheimer's disease or reducing the likelihood of these diseases; the method comprises administering to a subject an effective amount of a polynucleotide according to any one of the first aspects, any one of E1-E21, a recombinant viral vector according to any one of E22-E26, or a gene delivery vector according to any one of E27-E30 or a pharmaceutical composition according to E31 or E32.

[0298] Embodiment 33 further describes the second aspect, wherein the method lowers cholesterol, lowers LDL / VLDL, increases HDL, or lowers the total cholesterol / HDL ratio in a subject in need thereof.

[0299] Embodiment 34 further describes the second aspect or embodiment E33, wherein the subject has hypercholesterolemia.

[0300] Embodiment 35 further describes the second aspect, embodiments E33 and E34, wherein the method treats or reduces the likelihood of hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, cerebral amyloid angiopathy, dementia, post-stent restenosis, atherosclerosis, coronary artery disease, or Alzheimer's disease in the subject.

[0301] Embodiment 36 further describes the second aspect, embodiments E33, E34 and E35, wherein the subject is a statin low responder or is statin intolerant.

[0302] Embodiment 37 further describes the second aspect, wherein the method relates to treating or reducing the likelihood of Alzheimer's disease in a subject.

[0303] Embodiment 38 further describes the second aspect, wherein the method relates to treating or reducing the likelihood of vascular dementia or frontotemporal dementia in a subject.

[0304] Embodiment 39a further describes embodiment E37, wherein administering comprises intraparenchymal, intracisternal, or intraventricular administration.

[0305] Embodiment 39b further describes the second aspect and embodiments E37, E36, E37 and E38, wherein administering comprises intravenous administration.

[0306] Embodiment 40 further describes this second aspect, embodiments E33, E34, E35, E37, E38, E39a and E39b, wherein the subject has at least one ApoE4 allele, is homozygous for EpoE4, or is a PSEN1 mutation carrier.

[0307] Embodiment 41 further describes the second aspect, embodiments E33, E34, E35, E36, E37, E38, E39a, E39b, and E40, wherein native ApoE expression is inhibited. In further embodiments, native ApoE expression is inhibited using an inhibitory nucleic acid; and the inhibitory nucleic acid is selected from the group consisting of short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), RNAi, ribozymes, and antisense RNA.

[0308] Embodiment 42 further describes this second aspect, Embodiments E33, E34, E35, E36, E37, E38, E39a, E39b, E40 and E41, wherein the subject is a human.

[0309] The third aspect relates to a polynucleotide according to any one of the first aspect, any one of embodiments E1-E21, a recombinant viral vector according to any one of embodiments E22-E26, a gene delivery vehicle according to any one of embodiments E27-E30, or a pharmaceutical composition according to embodiment E31 or E32, for use in medicine and for use in the methods provided in the second aspect and any one of embodiments E33-E42.

[0310] The fourth aspect relates to the use of a polypeptide according to any one of the first aspect, any one of embodiments E1-E21, a recombinant viral vector according to any one of embodiments E22-E26, a gene delivery vector according to any one of embodiments E27-E30, or a pharmaceutical composition according to embodiment E31 or E32 for the preparation of a medicament for use in medicine or as provided in any one of the second aspect and embodiments E33-42.

[0311] The fifth aspect relates to an AAV vector genome plasmid comprising the recombinant viral nucleic acid according to any one of embodiments E22-26.

[0312] Embodiment 43 further describes the fifth aspect, wherein the plasmid lacks the rep gene and the cap gene.

[0313] The sixth aspect relates to a method for producing an rAAV vector, the method comprising the step of culturing an rAAV producer cell line comprising rAAV helper virus activity, wherein the genome of the producer cell comprises the recombinant viral vector nucleic acid, rep gene and cap gene according to any one of embodiments E22-26, wherein the rAAV vector is produced.

[0314] Embodiment 44 relates to a method for producing an rAAV vector, the method comprising the step of culturing an rAAV-permissive cell comprising the AAV genome plasmid according to the sixth aspect or embodiment 43, wherein the rAAV-permissive cell further comprises (a) a rep gene and a cap gene provided as part of the cell genome and / or provided by one or more separate plasmids, and (b) helper virus activity provided by the cell genome and / or provided by one or more separate plasmids.

[0315] Embodiment 45 further describes embodiment 44, wherein the rAAV-permissive cell is a packaging cell, wherein the packaged genome comprises a cap gene and a rep gene.

[0316] Embodiment 46 further describes embodiment 44, wherein (a) the rep gene, the cap gene and the auxiliary activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided by a rep / cap plasmid and the auxiliary activity is provided by a helper plasmid.

[0317] The seventh aspect relates to a method for obtaining an rAAV vector, comprising the steps of: (a) producing the rAAV using the method according to any one of the sixth aspect or embodiments 44-46 and (b) purifying the rAAV.

[0318] An eighth aspect relates to a polynucleotide comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 119-121, wherein the polynucleotide has 0-5 CpGs. Preferably, the polynucleotide is an intron that can be spliceosome-mediated excision from the pre-mRNA transcript.

[0319] Embodiment 47 further describes the eighth aspect, wherein the polynucleotide comprises the sequence of any one of SEQ ID NOs: 119-121, or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1-10 nucleotides, wherein the polynucleotide does not have a CpG. In another embodiment, the polynucleotide consists of the sequence of any one of SEQ ID NOs: 119-121.

[0320] XI. Sequence

[0321] Various nucleic acid and amino acid sequences are provided in Table 1. Sequences indicated in bold provide the codons.

[0322] In various embodiments, (1) the polynucleotide comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the nucleic acid sequences provided in Table 1; (2) the polynucleotide comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the nucleic acid sequences provided in Table 1, wherein the stop codon shown in bold is absent and / or replaced by a different stop codon; or (3) the polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the amino acid sequences provided in Table 1.

[0323] Table 1 Example

[0324] The following examples are provided to further illustrate various features of the invention and methods of practicing the invention. The examples provided do not limit the claimed invention.

[0325] Example 1: Liver-mediated expression of ApoE3(ch) and ApoE3 transgene

[0326] The effects of ApoE3(ch) and ApoE3 transgene expression on cholesterol were evaluated using wild-type C57BL / 6 and ApoE knockout (KO) mice and rAAV containing a transgene expressing ApoE3(ch) or ApoE3. The recombinant AAV nucleic acid consists of a rAAV polynucleotide containing the ApoE / hAAT promoter / enhancer functionally coupled to a nucleic acid encoding ApoE3 or ApoE(ch), along with other expression vector components and ITRs, such as Figure 1 As shown in .

[0327] Female wild-type C57BL / 6 and ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). ApoE knockout mice are hyperlipidemic and develop spontaneous atherosclerosis at six months of age. Baseline plasma was collected at 8 weeks of age. At 9 weeks of age, 200 mL of vehicle diluent (PBS180 / 0.001% Pluronic F66, pH 7.3) was administered intravenously via the tail vein as a control or 200 mL of diluent containing low or high doses of rAAV (transgene and capsid). Recombinant AAV containing the ApoE3 (ch) transgene was provided at a low dose of 3e12 vg / kg or a high dose of 1e13 vg / kg. Recombinant AAV containing the ApoE3 transgene was provided at a low dose of 3e12 vg / kg or a high dose of 6e12 vg / kg rAAV. The overall design is provided in Table 2. The viral capsid contained VP1 of SEQ ID NO: 83 (U.S. Patent No. 9,169,299), VP2 of SEQ ID NO: 122, and VP3 of SEQ ID NO: 123.

[0328] Table 2

[0329] Plasma was collected weekly for the first 8 weeks after AAV injection and monthly thereafter for use with EnzyChrom TM Total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein / very-low-density lipoprotein (LDL / VLDL) cholesterol were measured using AF HDL and LDL / VLDL assay kits (Bioassay Systems, Hayward, CA, USA), and ApoE3 protein was measured by ELISA (Mabtech Ab, Sweden).

[0330] Figures 2A-2EThe expression of ApoE3 and ApoE3(ch) transgenes in ApoE knockout mice over the course of 36 weeks and the effect of transgene expression on cholesterol are shown. Recombinant AAV containing the ApoE(ch) transgene was given at a low dose of 3e12 vg / kg or a high dose of 1e13 vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12 vg / kg or a high dose of 6e12 vg / kg rAAV. Figure 2A hAPOE production is shown, Figure 2B Total cholesterol is shown, Figure 2C shows HDL, Figure 2D The LDL / VLDL cholesterol ratio is shown, and Figure 2E The total / HDL cholesterol ratio is shown. Plasma hAPOE levels were significantly negatively correlated with total cholesterol (p < 0.0001; Spearman r = -0.6214), LDL / VLDL cholesterol (p < 0.0001; Spearman r = -0.6854), and the total cholesterol / HDL ratio (p < 0.0001; Spearman r = -0.6792), but were significantly positively correlated with HDL cholesterol (p < 0.0001, Spearman r = 0.4584).

[0331] Example 2: CpG-depleted ApoE3 (ch) and ApoE3 transgene expression

[0332] Wild-type C57BL / 6 and ApoE knockout (KO) mice were used along with rAAV containing different CpG-depleted ApoE3(ch) and ApoE3 transgenes to evaluate the effects of different CpG-depleted ApoE(ch) and ApoE constructs on cholesterol. The recombinant AAV nucleic acid contained the ApoE / hAAT promoter / enhancer functionally coupled to the nucleic acid encoding ApoE3 or ApoE(ch), along with other expression vector components and ITRs, such as Figure 1 As shown in .

[0333] Female wild-type C57BL / 6 and ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Baseline plasma was collected at 8 weeks of age. At 9 weeks of age, 200 mL of vehicle diluent (PBS180 / 0.001% Pluronic F66, pH 7.3) was administered intravenously via the tail vein as a control or 200 mL of diluent containing 1e13 vg / kg (2e11 total vg) of rAAV (transgene and capsid). The viral capsid contained VP1 of SEQ ID NO: 83, VP2 of SEQ ID NO: 122, and VP3 of SEQ ID NO: 123.

[0334] Plasma was collected at 3 and 6 weeks after rAAV injection and used for EnzyChrom TM Total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein / very-low-density lipoprotein (LDL / VLDL) cholesterol were measured using AF HDL and LDL / VLDL assay kits (Bioassay Systems, Hayward, CA, USA), and ApoE3 protein was measured by ELISA (Mabtech Ab, Sweden). The results are shown in Figures 3A-3E , 4A-4E, 5A-5E and 6A-6E. Table 3 provides the SEQ ID NOs for the different constructs.

[0335] Table 3 ApoE3(ch) SEQ ID NO: ApoE3 SEQ ID NO: APOE3ch-N 1 APOE3-N 2 APOE3ch-4 6 APOE3-3 20 APOE3ch-6 8 APOE3-8 25 APOE3ch-7 9 APOE3-9 26 APOE3ch-9 11 APOE3-11 28 APOE3ch-10 12 APOE3-12 29 APOE3ch-11 13 APOE3-15 31 APOE3ch-13 15 APOE3ch-15 17

[0336] Figures 3A-3E Shown are ApoE3(ch) transgene expression from different constructs at 3 weeks and the effect of transgene expression on cholesterol. Figure 3A hAPOE levels are shown. Figure 3B Total cholesterol is shown, Figure 3C The LDL / VLDL cholesterol ratio is shown. Figure 3D HDL cholesterol is shown, and Figure 3E Total cholesterol / HDL ratios are shown.

[0337] Figures 4A-4E Shown are ApoE3(ch) transgene expression from different constructs at 6 weeks and the effect of transgene expression on cholesterol. Figure 4A hAPOE levels are shown. Figure 4B Total cholesterol is shown, Figure 4CThe LDL / VLDL cholesterol ratio is shown. Figure 4D HDL cholesterol is shown, and Figure 4E Total cholesterol / HDL ratios are shown.

[0338] Figures 5A-5E Shown are ApoE3 transgene expression from different constructs at 3 weeks and the effect of transgene expression on cholesterol. Figure 5A hAPOE levels are shown. Figure 5B Total cholesterol is shown, Figure 5C The LDL / VLDL cholesterol ratio is shown. Figure 5D HDL cholesterol is shown, and Figure 5E Total cholesterol / HDL ratios are shown.

[0339] Figures 6A-6E Shown are ApoE3 transgene expression from different constructs at 6 weeks and the effect of transgene expression on cholesterol. Figure 6A hAPOE levels are shown. Figure 6B Total cholesterol is shown, Figure 6C The LDL / VLDL cholesterol ratio is shown. Figure 6D HDL cholesterol is shown, and Figure 6E Total cholesterol / HDL ratios are shown.

[0340] Example 3: Liver expression

[0341] Liver tissue samples were obtained from mice treated in Example 2 at six weeks and the hAPOE / total protein ( Figure 7A ) and vector genome copy number (VCGN) / ug gDNA ( Figure 7B hAPOE levels were determined by JESS. VCGN per microgram of gDNA was assessed by qPCR. No immune infiltration of liver tissue was observed by H&E pathological evaluation (data not shown).

[0342] Example 4: Atherosclerotic plaque lesions

[0343] The effects of ApoE3(ch) and ApoE3 transgene expression on atherosclerotic plaques were evaluated using wild-type and ApoE knockout mice and rAAV containing a transgene expressing ApoE3(ch) or ApoE3. Atherosclerotic plaques were evaluated in the aorta by visualizing and quantifying atherosclerotic plaque fatty content 40 weeks after AAV injection in mice used in Example 1.

[0344] Aortas were collected 40 weeks after AAV injection by immersion fixation in paraformaldehyde (4%) for 24 hours and then transferred to sucrose gradients. Aortas were washed in dPBS, equilibrated in 60% isopropanol, and atherosclerotic plaque fatty content was stained with Oil Red O. Aortas were dissected whole in the above preparation for imaging. Figure 8 Representative black and white images from different groups are provided in . Figure 9 Quantification of percent lesion area is shown, where each point on the graph is data from a mouse.

[0345] Example 5: Anti-inflammatory Effects of ApoE3 and ApoE3ch Transgenic Expression

[0346] The mice treated in Example 1 were further evaluated to determine the anti-inflammatory effects of rAAV containing APOE3ch or APOE transgene. Plasma was collected monthly from APOE KO mice treated with rAAV. Plasma from weeks 20, 24, and 28 (n=5 / group) was combined to provide sufficient volume to run a mouse cytokine detection panel manufactured by MesoScale Diagnostics to measure inflammatory markers. The MesoScale Diagnostics mouse cytokine detection panel measures 19 inflammatory markers (V-PLEX Mouse Cytokine 19-Plex Kit). Six inflammatory plasma proteins were elevated in APOE KO mice treated with excipient (vehicle control) compared to WT mice and normalized with APOE3 (ch) gene therapy.

[0347] The results are shown in Figures 10A-10F APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12 vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13 vg / kg rAAV. Figure 10A IL-5 levels are shown, Figure 10B IL-6 levels are shown, Figure 10C TNF-α levels are shown. Figure 10D IL-17A / F levels are shown, Figure 10E CCL2 levels are shown, and Figure 10F CXCL2 is shown. One-way ANOVA, Dunnett's post hoc test. #p<0.05, ##p<0.01 compared to WT vehicle; *p<0.05, **p<0.01 compared to KO vehicle.

[0348] Example 6: Effects of ApoE3 and ApoE3ch Transgenic Expression on GFAP

[0349] The mice treated in Example 1 were further evaluated to determine the effect of rAAV containing APOEch or APOE transgene on glial fibrillary acidic protein (GFAP) levels. GFAP levels in different brain regions were measured by protein quantification using capillary electrophoresis (JESS, Protein Simple) or immunofluorescence quantification. Brain samples were collected 40 weeks after APOE3(ch) gene therapy treatment of APOE KO mice.

[0350] The results are shown in Figures 11A-11D APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13 vg / kg rAAV. APOE3ch L and APOE3-L refer to KO mice given a low dose of 3e12 vg / kg rAAV. Figure 11A Shown are GFAP / total protein in the cortex as determined by JESS. Figure 11B Shown are GFAP / total protein in the hippocampus as determined by JESS. Figure 11C Shown are the % GFAP area in the whole brain determined by immunofluorescence quantification. Figure 11D Shown are the % GFAP area in the hippocampus determined by immunofluorescence quantification.

[0351] Immunofluorescence staining of GFAP, a marker of encephalitogenic astrocytes, was performed using anti-GFAP antibody (AB5541, Millipore; 1:500), and the cells were stained with Image analysis software (IndicaLabs) quantified the percentage of GFAP area, demonstrating increased GFAP immunoreactivity in the whole brain and hippocampus of APOE KO mice. In mice treated with APOE3(ch) and APOE3 gene therapy, elevated GFAP immunoreactivity was reduced in a dose-dependent manner. Similar results were observed by quantification of GFAP protein and protein in the cortex and hippocampus.

[0352] Example 7: Effects of ApoE3 and ApoE3ch transgenic expression on presynaptic and postsynaptic proteins

[0353] The mice treated in Example 1 were further evaluated to determine the effects of rAAV containing APOE3ch or APOE3 transgene on presynaptic and postsynaptic proteins. Brain lysates were prepared from mice 40 weeks after APOE3(ch) gene therapy or vector treatment. To determine whether APOE3(ch) and APOE transgene expression have an effect on the number of neuronal connections in the brain, the presynaptic and postsynaptic proteins synaptobrevin and PSD-95 were quantified in the cortex and hippocampus by capillary electrophoresis (JESS, Protein Simple).

[0354] The results are shown in Figures 12A-12D APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12 vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13 vg / kg rAAV. Figure 12A Shown are synaptic vesicle protein / total protein in the hippocampus. Figure 12B PSD-95 / total protein in hippocampus is shown. Figure 12C Shown are synaptic vesicle protein / total protein in the cortex. Figure 12D PSD-95 / total protein in the cortex is shown.

[0355] Example 7: Atherosclerotic plaque lesions (9 weeks)

[0356] The effects of rAAV containing APOE3ch or APOE3 transgene on atherosclerosis were evaluated by quantitative atherosclerotic plaque lesions in rAAV-treated ApoE knockout mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052). Aortas were collected from 1-year-old APOE KO mice with severe pre-existing atherosclerosis. The recombinant AAV nucleic acid contained the ApoE / hAAT promoter / enhancer functionally coupled to the nucleic acid encoding ApoE3 or ApoE3ch, along with other expression vector components and ITRs, such as Figure 1 Table 4 summarizes the sequences encoding ApoE.

[0357] Table 4 SEQ ID NO: APOE3ch-N 1 APOE3-N 2 APOE3-3 20 APOE3ch-9 11

[0358] The aorta from the baseline group was used as a measure of atherosclerosis before treatment. The remaining mice were treated with rAAV containing native ApoE3 (ApoE3-N) and native ApoE3ch (ApoE3ch-N) transgenic sequences and CpG-0 codon optimized variants APOE3-3 and APOE3ch-9. rAAV was administered at two doses (2e11 vg / mouse or 2e12 total vg / mouse) for 9 weeks, after which the aorta was collected, stained for atherosclerotic pathology with Oil Red O, and quantified for lesion volume % / aorta.

[0359] Figures 13A-13D Quantification of the percentage of lesion area is shown, where each point on the graph represents one mouse. Figure 13A Shown are the % aortic lesion area in KO mice and KO mice administered with different transgenic sequences encoding the following ApoE: E3N (native ApoE3) 2e11 vg / kg、E3-3(ApoE3-3)2e 11 vg / kg and E3-3 (ApoE3-3)2e 12 vg / kg. Figure 13B Shown from Figure 13A KO baseline and from Figure 13A E3N (native ApoE3)2e 11 vg / kg and E3-3 (ApoE3-3)2e 11 vg / kg group combination. Figure 13C Shown are the % aortic lesion areas in KO mice and KO mice administered with different transgenic sequences encoding: E3chN (native ApoE3ch) 2e 11 vg / kg、E3ch-9(ApoE3ch-9)2e 11 vg / kg and E3ch-9 (ApoE3ch-9) 2e 12 vg / kg. Figure 13D Shown from Figure 13C KO baseline, and E3chN (natural ApoE3ch) 2e 11 vg / kg and E3ch-9 (ApoE3ch-9) 2e 11 vg / kg combination.

[0360] In pre-existing atherosclerotic pathology, APOE3(ch) and APOE(ch) gene therapy demonstrated a greater than 17% reduction in all constructs compared to the baseline group, supporting the potential for reversal of atherosclerosis with APOE3(ch) gene therapy.

[0361] Example 8: Cognitive Research

[0362] It has been reported that APOE KO mice have learning and memory cognitive defects when they are ≥1 year old. One-year-old APOE KO mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #: 002052) were tested on the novel object recognition (NOR) memory test (Antunes and Biala G, Cogn Process. 2012 May; 13 (2): 93-110), and found that there was a significant decrease in recognition memory before APOE3 and APOE3ch gene therapy treatment. The test measures the memory of familiar objects, as measured by the preference for new objects. The time of preference for new objects (> 50%) is a normal cognitive behavior, indicating that the long-term recognition memory of mice is intact.

[0363] Native ApoE3 (E3native), native ApoE3ch (E3chnative), ApoE3-3 (E3-3), and ApoE3ch-9 (E3ch-9) were administered intravenously via the tail vein at a low dose of 2e11 vg / mouse or a high dose of 2e12 total vg / mouse. The constructs are described in Table 4. Figure 14A NOR results for C57BL / 6 and ApoE knockout mice are shown (*p<0.05, unpaired two-tailed t-test). Figure 14B The NOR results for administration of native ApoE and ApoE(ch) sequences are shown. Figure 14C Shown are NOR results with low doses of rAAV encoding CpG-0 ApoE3 (E3-3) and ApoE (Ech-9) rAAV. Figure 14D Shown are NOR results with high doses of rAAV encoding CpG-0 ApoE (E3-3) and ApoE3ch (Ech-9) rAAVs. Five weeks after a single intravenous administration of native APOE3 and APOE3ch sequences and CpG-0 codon-optimized variants E3-3 and E3ch-9 via the tail vein at two doses (2e11 vg / mouse or 2e12 total vg / mouse), NOR memory performance was fully restored to that of age-matched wild-type (WT) mice.

[0364] Example 9: Additional ApoE3 Constructs

[0365] The ability of different codon-optimized, CpG-reduced transgenes encoding ApoE was evaluated. The transgene was inserted into a plasmid, and the plasmid was transfected into AML-12 cells in triplicate. ApoE3 and antigen levels were measured in cell culture supernatants 72 hours after transfection. ApoE3 levels were determined by ELISA and plotted as mean + / - standard deviation.

[0366] Figure 15A A bar graph showing the performance of codon-optimized CpG-reduced cDNA encoding ApoE3 and the CpG-free construct ApoE3-3 is provided. Table 5 provides the different constructs and the sequence identity to some of the full CpG constructs also described in this application.

[0367] Table 5 SEQ ID NO: Percent identity wtApoE3 1 H30 130 97.0% identical to ApoE3ch-46 (SEQ ID NO: 101) H24 129 97.5% identical to ApoE3ch-45 (SEQ ID NO: 100) H6 125 98.2% identical to ApoE3ch-41 (SEQ ID NO: 96) H12 127 97.8% identical to ApoE3ch-43 (SEQ ID NO: 98) H7 126 98.4% identical to ApoE3ch-42 (SEQ ID NO: 97) H15 128 97.6% identical to ApoE3ch-44 (SEQ ID NO: 99) H5 124 98.8% identical to ApoE3ch-40 (SEQ ID NO: 95) E3-3 20

[0368] Figure 15BA bar graph is provided showing the performance of codon-optimized ApoE3 cDNA functionalized by the addition of intron sequences. Introns were inserted into the signal peptide at codons 15G / GC (site 1) and / or at codons 79AG / G (site 2). In those cases where an intron is indicated and no site is provided, the intron was inserted into site 1. References to introns RBP4i, VCLi, and FIXi without indicating no CpG indicate the wild-type sequence. VCLi-no CpG refers to the intron of SEQ ID NO: 121. RBP4i-no CpG refers to the intron of SEQ ID NO: 120. For comparison purposes, codon-optimized ApoE3-3 and H30 variants containing no introns are included as a benchmark. ApoE3 levels were determined by ELISA and plotted as mean + / - standard deviation.

[0369] While the present invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will appreciate that various adjustments, changes, modifications, substitutions, deletions, or additions may be made to the procedures and arrangements without departing from the spirit and scope of the invention.

Claims

1. A polynucleotide comprising a nucleotide sequence encoding ApoE having at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 63-67 or nucleotides 55-951 of SEQ ID NOs: 95-105 and 124-130, wherein the polynucleotide encodes an ApoE3-related protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 32, wherein the protein comprises a cysteine ​​at a position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at a position corresponding to amino acid 158 of SEQ ID NO: 32, wherein the nucleotide sequence encoding ApoE optionally comprises one or more introns.

2. The polynucleotide according to claim 1, wherein the nucleotide sequence encoding ApoE comprises at least one intron.

3. The polynucleotide according to claim 1, wherein the nucleotide sequence encoding ApoE does not contain any introns.

4. The polynucleotide of any one of claims 1-3, wherein the nucleotide sequence encoding ApoE has at least 95% sequence identity to nucleotides 55-951 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130.

5. The polynucleotide of any one of claims 1-3, wherein the nucleotide sequence encoding ApoE has at least 95% sequence identity to any one of SEQ ID NOs: 63-67.

6. The polynucleotide of any one of claims 1-5, wherein the ApoE3-related protein further comprises a serine at the position corresponding to amino acid 136 of SEQ ID NO:

32.

7. The polynucleotide according to any one of claims 1-5, wherein the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO:

32.

8. The polynucleotide of any one of claims 1-5, wherein the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO:

33.

9. The polynucleotide of any one of claims 1-3, wherein the nucleotide sequence encoding ApoE has at least 95% sequence identity with SEQ ID NO: 63, and the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO:

33.

10. The polynucleotide according to any one of claims 1 to 9, wherein the ApoE3-related protein further comprises a 5' signal peptide, and the nucleic acid sequence encoding ApoE3 further comprises a nucleotide sequence encoding the signal peptide.

11. The polynucleotide of claim 10, wherein the signal peptide is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71.

12. The polynucleotide of claim 11, wherein the nucleotide sequence encoding the signal peptide comprises at least 90% identity to the sequence of any one of SEQ ID NOs: 37-41, 43, 45, 47, 49, 51, 53, 55, 57, and 72-75.

13. The polynucleotide of any one of claims 1-12, wherein the nucleotide sequence encoding ApoE comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 20, 26, 31, 11, 15, and 95-105.

14. The polynucleotide of any one of claims 1-13, wherein the ApoE3-related protein comprises the sequence of SEQ ID NO: 34 or SEQ ID NO:

35.

15. The polynucleotide of claim 1 , wherein the nucleotide sequence encoding ApoE comprises the following sequence: (i) nucleotides 55 to 951 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (ii) nucleotides 55 to 954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (iii) nucleotides 1 to 897 of any one of SEQ ID NOs: 63-67; (iv) any one of SEQ ID NOs: 63-67; (v) nucleotides 1 to 951 of any one of SEQ ID NOs: 3-31, 95-105, and 124-140; or (vi) SEQ IDNO: any one of 3-31, 95-105 and 124-130.

16. The polynucleotide of claim 1 , wherein the nucleotide sequence encoding ApoE comprises a sequence having at least 95% identity to: (i) nucleotides 55 to 951 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (ii) nucleotides 55 to 954 of any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (iii) nucleotides 1 to 897 of any one of SEQ ID NOs: 63-67; (iv) any one of SEQ ID NOs: 63-67; (v) nucleotides 1 to 951 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130; or (vi) SEQ ID NOs: Any one of NO: 3-31, 95-105 and 124-130; and the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO: 34 or 35.

17. The polynucleotide according to claim 2, wherein the nucleotide sequence encoding ApoE comprises from 5' to 3': (a) a first exon corresponding to nucleotides 1-43 of SEQ ID NO: 1, wherein the first exon has at least 85% sequence identity with nucleotides 1-43 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the terminal 3' nucleotide of the first exon is G; (b) the first intron at a position corresponding to between nucleotides 43 and 44 of SEQ ID NO: 1; (c) a second exon corresponding to nucleotides 44-236 of SEQ ID NO: 1; wherein the second exon has at least 85% sequence identity with nucleotides 44-236 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the terminal 5' nucleotide of the second exon is G and the terminal 3' nucleotide of the second exon is AG; (d) a second intron at a position corresponding to between nucleotides 236 and 237 of SEQ ID NO: 1; and (e) a third exon corresponding to nucleotides 237-951 of SEQ ID NO: 1, wherein the third exon has at least 85% sequence identity with nucleotides 237-951 of any one of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the terminal 5' nucleotide of the third exon is G; The first exon, the second exon and the third exon together encode the ApoE3-related protein, wherein the ApoE-related protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

35.

18. The polynucleotide of claim 17, wherein the first intron comprises the sequence of SEQ ID NO: The second intron independently comprises a sequence having at least 50% sequence identity to any one of SEQ ID NOs: 119-122, and the second intron independently comprises a sequence having at least 50% sequence identity to any one of SEQ ID NOs: 119-121.

19. The polynucleotide according to claim 18, wherein the first intron consists of the sequence of any one of SEQ ID NOs: 119-121 or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides, and the second intron independently consists of the sequence of any one of SEQ ID NOs: 119-121 or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides.

20. The polynucleotide according to any one of claims 17 to 19, wherein the first exon having at least 95% sequence identity to nucleotides 1-43 of any one of SEQ ID NOs: 11, 20, or 95-105; The second exon has at least 95% sequence identity to nucleotides 44-236 of any one of SEQ ID NOs: 11, 20, or 95-105; and the third exon having at least 95% sequence identity to nucleotides 237-951 of any one of SEQ ID NOs: 11, 20, or 95-105; The ApoE-related protein has at least 98% sequence identity with SEQ ID NO:

35.

21. The polynucleotide of claim 20, wherein the nucleotide sequence encoding ApoE comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 106-115.

22. The polynucleotide of claim 21, wherein the ApoE-related protein comprises the sequence of SEQ ID NO: 34 or 35.

23. The polynucleotide according to claim 22, wherein the nucleotide sequence encoding ApoE comprises the sequence of any one of SEQ ID NOs: 106-115.

24. The polynucleotide of any one of claims 17-21, wherein the ApoE3-related protein comprises the sequence of SEQ ID NO: 34 or 35.

25. The polynucleotide according to any one of claims 1-24, wherein the nucleotide sequence encoding ApoE3 contains 0 to 10 CpGs.

26. The polynucleotide of any one of claims 1-25, wherein the polynucleotide is an expression cassette comprising one or more expression control elements operably linked to the nucleotide sequence encoding ApoE.

27. The polynucleotide of claim 26, wherein the expression cassette comprises a 5' promoter operably linked to the nucleotide sequence encoding ApoE3 and a 3' polyadenylation site operably linked to the nucleotide sequence encoding ApoE3.

28. The polynucleotide of claim 27, wherein the expression cassette comprises, from 5' to 3', a promoter or promoter / enhancer operably linked to the ApoE3-related nucleotide sequence, an intron, a Kozak sequence, the ApoE3-encoding nucleic acid sequence, and a polyadenylation signal.

29. The polynucleotide of claim 27 or 28, wherein the promoter is a liver-specific promoter.

30. The polynucleotide of any one of claims 27-29, comprising a promoter / enhancer, wherein the enhancer has at least 95% sequence identity to SEQ ID NO:

58.

31. The polynucleotide of any one of claims 27-30, wherein the promoter is the hAAT promoter.

32. The polynucleotide of claim 27 or 28, wherein the promoter provides for expression in CNS cells.

33. The polynucleotide of claim 32, wherein the expression cassette further comprises one or more miRNA target sequences to inhibit expression in dorsal root ganglia, liver, or immune cells.

34. The polynucleotide of any one of claims 28-33, wherein the intron comprises the sequence of SEQ ID NO: 60 and the polyadenylation signal comprises the sequence of SEQ ID NO: 61 or SEQ ID NO:

62.

35. The polynucleotide of any one of claims 26-34, wherein the expression cassette further comprises one or more inhibitory nucleic acids that selectively target nucleic acid encoding ApoE2, ApoE3, or ApoE4.

36. The polynucleotide of claim 35, wherein the inhibitory nucleic acid selectively targets a nucleic acid encoding ApoE4.

37. The polynucleotide of any one of claims 26-36, wherein the expression cassette contains up to about 10% CpG.

38. The polynucleotide of any one of claims 26-37, wherein the expression cassette comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 76-80.

39. The polynucleotide of any one of claims 1-38, wherein the polynucleotide is DNA.

40. The polynucleotide of any one of claims 26-39, wherein the expression cassette comprises a nucleotide sequence having at least 95% sequence identity to a modified SEQ ID NO: 76, wherein SEQ ID NO: 76 is modified by replacing nucleotides 1295-2248 with any one of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-115, and 124-130.

41. A recombinant viral vector nucleic acid comprising the polynucleotide of any one of claims 26-40 and 5' and / or 3' viral elements that provide for viral packaging and / or replication.

42. The recombinant viral vector nucleic acid of claim 41, wherein the recombinant viral vector nucleic acid is DNA and comprises adeno-associated virus (AAV) inverted repeats (ITRs) flanking the 5′ end of the polynucleotide and / or AAV ITRs flanking the 3′ end of the polynucleotide.

43. The recombinant viral vector nucleic acid of claim 42, wherein the 5′ and / or 3′ viral elements are each selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B.

44. The recombinant viral vector nucleic acid of claim 43, wherein the 5' ITR comprises a sequence having at least 95% sequence identity to SEQ ID NO: 81, and the 3' ITR comprises a sequence having at least 95% sequence identity to SEQ ID NO:

82.

45. The recombinant viral vector nucleic acid of any one of claims 41-44, wherein the recombinant viral vector nucleic acid contains up to about 10% CpG.

46. ​​A gene delivery vehicle, wherein the gene delivery vehicle is a viral or non-viral vector comprising the polynucleotide of any one of claims 1-40 or the recombinant viral vector nucleic acid of any one of claims 41-45.

47. The gene delivery vehicle of claim 46, wherein the gene delivery vehicle is a viral vector.

48. The gene delivery vehicle of claim 47, wherein the viral vector is a recombinant AAV, a recombinant lentiviral vector, or a recombinant adenoviral vector.

49. The gene delivery vehicle of claim 48, wherein the viral vector is the recombinant AAV, and the recombinant AAV vector comprises a capsid comprising a VP1, VP2, or VP3 having at least 90% sequence identity to VP1, VP2, or VP3 of any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 83, and SEQ ID NO:

84.

50. The gene delivery vector of claim 49, wherein the capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-Bl, AAV-AS, AAV1 / rh.10 capsid; or the capsid comprises the VP1 of SEQ ID NO: 83 or SEQ ID NO:

84.

51. The gene delivery vehicle of claim 46, wherein the gene delivery vehicle is the non-viral vector.

52. The gene delivery vehicle of claim 51 , wherein the non-viral vector is a nanoparticle selected from the group consisting of lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein or peptide-based nanoparticles, DNA dendrimers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes.

53. The gene delivery vehicle of claim 52, wherein the non-viral vector is LNP or LPNP.

54. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-40, the recombinant viral vector nucleic acid of any one of claims 41-45, or the gene delivery vehicle of any one of claims 46-53 and a pharmaceutically acceptable carrier.

55. The pharmaceutical composition of claim 54, wherein the composition comprises recombinant AAV and empty AAV capsids, wherein the ratio of the empty AAV capsids to the recombinant AAV is 100:1 to 1:

100.

56. A method of treating a subject to lower cholesterol, lower LDL / VLDL, increase HDL, or lower the total cholesterol / HDL ratio; or to treat hypercholesterolemia, type III familial hyperlipoproteinemia, familial hypercholesterolemia, dementia, post-stent restenosis, atherosclerosis, coronary heart disease, or Alzheimer's disease or to reduce the likelihood of these diseases; the method comprising administering to the subject an effective amount of a polynucleotide according to any one of claims 1-40, a recombinant viral vector nucleic acid according to any one of claims 41-45, a gene delivery vehicle according to any one of claims 46-53, or a pharmaceutical composition according to claim 54 or 55.

57. The method of claim 56, wherein the method lowers cholesterol, lowers LDL / VLDL, increases HDL, or lowers the total cholesterol / HDL ratio in a subject in need thereof.

58. The method of claim 57, wherein the subject has hypercholesterolemia.

59. The method of claim 56, wherein the method treats or reduces the likelihood of hypercholesterolemia, familial hyperlipoproteinemia type III, vascular dementia, frontotemporal dementia, cerebral amyloid angiopathy, post-stent restenosis, atherosclerosis, coronary artery disease, or Alzheimer's disease in a subject.

60. The method of any one of claims 56-59, wherein the subject is a statin low responder or is statin intolerant.

61. The method of claim 56, wherein the method treats or reduces the likelihood of Alzheimer's disease in a subject.

62. The method of claim 61, wherein the administration comprises intraparenchymal, intracisternal, or intraventricular administration.

63. The method of claim 61, wherein the polynucleotide, the recombinant viral vector nucleic acid, the gene delivery vehicle, or the pharmaceutical composition is administered intravenously.

64. The method of any one of claims 56-63, wherein the subject has at least one ApoE4 allele, is homozygous for EpoE4, or is a PSEN1 mutation carrier.

65. The method of claim 64, further comprising administering an effective amount of an inhibitory nucleic acid to reduce native ApoE4 expression in the subject.

66. The method of any one of claims 56-65, wherein the subject is a human.

67. An AAV vector genome plasmid comprising the recombinant viral nucleic acid of any one of claims 41-45.

68. The AAV genome plasmid of claim 67, wherein the plasmid lacks the rep gene and the cap gene.

69. A method for producing an rAAV vector, the method comprising the step of culturing an rAAV production cell line comprising rAAV helper virus activity, wherein the genome of the production cell comprises the recombinant viral vector nucleic acid, rep gene and cap gene according to any one of claims 41-45, wherein the rAAV vector is produced.

70. A method for producing an rAAV vector, the method comprising the step of culturing an rAAV-permissive cell comprising an AAV genome plasmid according to claim 67 or 68, wherein the rAAV-permissive cell further comprises (a) a rep gene and a cap gene provided as part of the cell genome and / or provided by one or more separate plasmids, and (b) helper virus activity provided by the cell genome and / or provided by one or more separate plasmids.

71. The method of claim 70, wherein the rAAV-permissive cell is a packaging cell, wherein the packaged genome comprises a cap gene and a rep gene.

72. The method of claim 70, wherein (a) the rep gene, the cap gene, and the auxiliary activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided by a rep / cap plasmid and the auxiliary activity is provided by a helper plasmid.

73. A method for obtaining an rAAV vector, the method comprising the following steps: (a) producing the rAAV using the method of any one of claims 70-72 and (b) purifying the rAAV.

74. A polynucleotide comprising a sequence having at least 50% sequence identity to the sequence of any one of SEQ ID NOs: 119-121, wherein the polynucleotide has 0-5 CpGs.

75. The polynucleotide according to claim 74, wherein the polynucleotide comprises the sequence of any one of SEQ ID NOs: 119-121 or a sequence that differs from any one of SEQ ID NOs: 119-121 by 1 to 10 nucleotides, wherein the polynucleotide does not have CpG.

76. The polynucleotide of claim 75, consisting of the sequence of any one of SEQ ID NOs: 119-121.

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