ApoE gene therapy

Abstract

The present invention relates to polynucleotide constructs encoding ApoE3-related 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 related to cholesterol levels, atherosclerosis, coronary heart disease, dementia, cerebral amyloid angiopathy, or Alzheimer's disease.

Description

[Background technology]

[0001]

[0003] ApoE is synthesized as a 317 amino acid precursor protein containing an 18 amino acid leader sequence. Cleavage of the leader sequence yields 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).

[0002]

[0004] ApoE is primarily involved in lipid metabolism, providing a variety of activities, including mediating hepatic and extrahepatic uptake of plasma lipoproteins and cholesterol efflux from lipid-laden macrophages. Processes in which ApoE has been shown to participate 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).

[0003]

[0005] Three major ApoE isoforms exist in humans: ApoE2, ApoE3, and ApoE4. The different isoforms are distinguished by amino acids at two positions. With respect to 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).

[0004]

[0006] Various ApoE isoforms, including minor forms, are associated with increased risk for various diseases or disorders (Zhou et al., (2021) Current Opinion in Neurobiology 69:58-67). A roughly linear relationship between LDL-C and coronary disease risk has been identified for ApoE genotypes (ordered as ε2 / ε2, ε2 / ε3, ε2 / ε4, ε3 / ε3, ε3 / ε4, and ε4 / ε4) (Bennet et al., JAMA. (2007) 298(11):1300-1311). Additional correlations include ApoE2 homozygosity, which often results in type III hyperlipoproteinemia, and ApoE4, which 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).

[0005]

[0007] The substitution identified as the Christchurch substitution results in a substitution of arginine at 136 in ApoE with serine. 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)") have been shown 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).

[0006]

[0008] WO 2022 / 115535, WO 2021 / 108809, and WO 2020 / 243346 refer to the treatment of Alzheimer's disease. Summary of the Invention

[0007]

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

[0008]

[0010] Accordingly, a first aspect of the present invention describes a polynucleotide comprising an ApoE-encoding nucleotide sequence having at least 85% sequence identity to any 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 the position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at the position corresponding to amino acid 158 of SEQ ID NO: 32, and wherein the ApoE-encoding nucleotide sequence optionally comprises one or more introns. The at least 85% identity to any 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.

[0009]

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

[0010]

[0012] References to stated percent identities for two or more reference sequences, and similar phrases throughout this specification providing stated percent identities for two or more reference sequences, provide an independently stated percent identity or percent identity range for each of the referenced sequences. In determining percent identity for polynucleotides, RNA and corresponding DNA are considered the same in the absence of reference to a polynucleotide that is RNA or DNA. Corresponding RNA and DNA include substitutions of uracil for thymine and ribose for deoxyribose backbones.

[0011]

[0013] Various polynucleotide constructs are provided herein, 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, and polynucleotides comprising recombinant viral vector nucleic acids, wherein the 5' and / or 3' ends of the polynucleotide have elements that provide for viral vector and / or viral replication and packaging into a vector genome plasmid.

[0012]

[0014] Reference to one or more expression control elements "operably linked" or "operably coupled" to an ApoE-encoding nucleic acid indicates that the expression control element(s) affect ApoE3-related protein expression, which 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.

[0013]

[0015] Another aspect of the present invention is directed to administering a polynucleotide construct as described herein to achieve one or more of the following: reduce cholesterol, reduce LDL / VLDL, increase HDL, or reduce the total cholesterol / HDL ratio; or treat or reduce 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.

[0014]

[0016] Additional aspects include the polynucleotide constructs described herein for use in medicine or to achieve one or more of the following: reducing cholesterol, reducing LDL / VLDL, increasing HDL, or reducing 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 for the preparation of a medicament for use in medicine or to achieve one or more of the following: reducing cholesterol, reducing LDL / VLDL, increasing HDL, or reducing 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.

[0015]

[0017] Additional embodiments include AAV vector genomes; methods for producing rAAV vectors; methods for obtaining rAAV vectors; and polynucleotides related to any of SEQ ID NOs: 119-121, which have 0 to 5 CpG positions.

[0016]

[0018] Other features and advantages of the present invention will be apparent from the additional description provided herein, including the various examples. The examples provided illustrate different components and methodologies useful in practicing the invention. Such examples do not limit the claimed invention. Based on this disclosure, one of ordinary skill in the art will be able to identify and utilize other components and methodologies useful for practicing the invention. [Brief explanation of the drawings]

[0017] [Figure 1]

[0023] Figure 1 provides a schematic example of a recombinant adeno-associated virus (rAAV) polynucleotide cassette. The example provided provides the locations of the 5' ITR, ApoE / hAAT promoter / enhancer, HBB2 intron, Kozak sequence, ApoE3(ch)-encoding nucleic acid, polyA sequence, and 3' ITR. [Figure 2A] Figure 2A illustrates ApoE3 and ApoE3ch transgene expression and the effect of transgene expression on cholesterol in ApoE knockout mice over a 36-week course. Recombinant AAV containing the ApoE3(ch) transgene was given at a low dose of 3e12vg / kg or a high dose of 1e13vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12vg / kg rAAV or a high dose of 6e12vg / kg rAAV. Figure 2B illustrates hAPOE plasma levels. Week 0 time point represents cholesterol levels before AAV administration. [Figure 2B] Figure 2B illustrates ApoE3 and ApoE3ch transgene expression and the effect of transgene expression on cholesterol in ApoE knockout mice over a 36-week course. Recombinant AAV containing the ApoE3(ch) transgene was given at a low dose of 3e12vg / kg or a high dose of 1e13vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12vg / kg rAAV or a high dose of 6e12vg / kg rAAV. Figure 2B illustrates total cholesterol. Week 0 time point indicates cholesterol levels before AAV administration. [Figure 2C]Figure 2A illustrates ApoE3 and ApoE3ch transgene expression and the effect of transgene expression on cholesterol in ApoE knockout mice over a 36-week course. Recombinant AAV containing the ApoE3(ch) transgene was given at a low dose of 3e12vg / kg or a high dose of 1e13vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12vg / kg rAAV or a high dose of 6e12vg / kg rAAV. Figure 2C illustrates LDL / VLDL. Week 0 indicates cholesterol levels before AAV administration. [Figure 2D] Figure 2A illustrates ApoE3 and ApoE3ch transgene expression and the effect of transgene expression on cholesterol in ApoE knockout mice over a 36-week course. Recombinant AAV containing the ApoE3(ch) transgene was given at a low dose of 3e12vg / kg or a high dose of 1e13vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12vg / kg rAAV or a high dose of 6e12vg / kg rAAV. Figure 2D illustrates HDL. Week 0 time point indicates cholesterol levels before AAV administration. [Figure 2E] Figure 2A illustrates ApoE3 and ApoE3ch transgene expression and the effect of transgene expression on cholesterol in ApoE knockout mice over a 36-week course. Recombinant AAV containing the ApoE3(ch) transgene was given at a low dose of 3e12vg / kg or a high dose of 1e13vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12vg / kg rAAV or a high dose of 6e12vg / kg rAAV. Figure 2E illustrates the total cholesterol / HDL ratio. Week 0 indicates cholesterol levels before AAV administration. [Figure 3A] Figures 3A and 3B illustrate ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol 3 weeks after treatment. Figure 3A illustrates hAPOE levels (**p<0.01, ***p<0.001, ****p<0.0001 vs. vehicle; +, equal to APOE3ch native expression). [Figure 3B] Figures 3A and 3B illustrate ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 3 weeks post-treatment. Figure 3B illustrates total cholesterol (#p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 vs. WT vehicle; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 3C] Figure 3A illustrates ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol 3 weeks after treatment. Figure 3B illustrates LDL / VLDL (##p<0.01, ###p<0.001, ####p<0.0001 vs. WT vehicle; *p<0.05, ****p<0.0001 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 3D] Figure 3A illustrates ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol 3 weeks after treatment. Figure 3B illustrates HDL cholesterol (#p<0.05, ##p<0.01, ###p<0.001 vs. WT vehicle). [Figure 3E] Figure 3A illustrates ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol 3 weeks after treatment. Figure 3B illustrates the total cholesterol / HDL ratio (##p<0.01 vs. WT vehicle; *p<0.05 vs. KO vehicle; percent increase from KO vehicle animals is depicted above the selection bar). [Figure 4A] Figures 4A and 4B illustrate ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol 6 weeks after treatment. Figure 4A illustrates hAPOE levels (**p<0.01, ***p<0.001, ****p<0.0001 vs. vehicle; +, equal to APOE3ch native expression). [Figure 4B]Figure 4A illustrates ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 4B illustrates total cholesterol (##p<0.01, ####p<0.0001 vs. WT vehicle; *p<0.05, ***p<0.001, ****p<0.0001 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 4C] Figure 4A illustrates ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 4B illustrates LDL / VLDL (###p<0.001, ####p<0.0001 vs. WT vehicle; *p<0.01, ****p<0.0001 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 4D] Figure 4A depicts ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 4D depicts HDL cholesterol (#p<0.05, ####p<0.0001 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle; percent increase from KO vehicle animals depicted above selection bar). [Figure 4E] Figure 4D illustrates ApoE3ch transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 4E illustrates the total cholesterol / HDL ratio (##p<0.01 vs. WT vehicle; *p<0.05 vs. KO vehicle). [Figure 5A] Figures 5A and 5B illustrate ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol 3 weeks after treatment. Figure 5A illustrates hAPOE levels (***p<0.001, ****p<0.0001 vs. vehicle; unless indicated by brackets). [Figure 5B]Figures 5A and 5B illustrate ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol 3 weeks after treatment. Figure 5B illustrates total cholesterol (#p<0.05, ###p<0.01, ####p<0.0001 vs. WT vehicle; *p<0.05, ****p<0.0001 vs. vehicle; or comparisons indicated by brackets; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 5C] Figures 5A and 5B depict ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol at 3 weeks post-treatment. Figure 5C depicts LDL / VLDL (#p<0.05, ###p<0.01, ####p<0.0001 vs. WT vehicle; *p<0.05, ****p<0.0001 vs. KO vehicle; or comparisons indicated by brackets; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 5D] Figures 5A and 5B depict ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol at 3 weeks post-treatment. Figure 5D depicts HDL cholesterol (#p<0.05, ###p<0.01, ####p<0.0001 vs. WT vehicle; *p<0.05, ****p<0.0001 vs. KO vehicle; or comparisons indicated by brackets; percent increase from KO vehicle animals is depicted above the selection bar). [Figure 5E] Figure 5B illustrates ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol at 3 weeks post-treatment. Figure 5C illustrates the total cholesterol / HDL ratio (##p<0.01 vs. WT vehicle; *p<0.05 vs. KO vehicle). [Figure 6A] Figures 6A and 6B illustrate ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol 6 weeks after treatment. Figure 6A illustrates hAPOE levels (***p<0.001, ****p<0.0001 vs. vehicle, unless indicated by brackets). [Figure 6B]Figure 6A illustrates ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol 6 weeks after treatment. Figure 6B illustrates total cholesterol (#p<0.05, ####p<0.0001 vs. WT vehicle; ****p<0.0001 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 6C] Figures 6A and 6B depict ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 6C depicts LDL / VLDL cholesterol (#p<0.05, ###p<0.001, ####p<0.0001 vs. WT vehicle; ****p<0.0001 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 6D] Figures 6A and 6B depict ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 6D depicts HDL cholesterol (#p<0.05, ##p<0.01, ####p<0.0001 vs. WT vehicle; **p<0.01 vs. KO vehicle; percent reduction from KO vehicle animals is depicted above the selection bar). [Figure 6E] Figure 6A illustrates ApoE3 transgene expression from different constructs and the effect of transgene expression on cholesterol at 6 weeks post-treatment. Figure 6B illustrates the total cholesterol / HDL ratio (##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle). [Figure 7A] FIG. 7A illustrates hAPOE / total protein resulting from different constructs (FIG. 7A; p<0.05 vs. E3(ch) native construct). [Figure 7B]Figure 7B illustrates liver tissue levels of vector genome copy number / μg gDNA resulting from different constructs. The reference to "ns vs. native" in Figure 7B indicates that the difference between E3ch-native and the different CpG-reduced constructs was not significant (*p<0.05 for constructs E3-8, E3-9, E3-11, E3-12, and E3-15 vs. the E3-native construct). [Figure 8] Figure 1 provides black and white photographs of atheromatous lipid inclusions stained with Oil Red O. 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 3e12vg / kg rAAV. APOE3ch(high) refers to KO mice administered a high dose of 1e13vg / kg rAAV. APOE3(low) refers to KO mice administered a low dose of 3e12vg / kg rAAV. APOE3(high) refers to KO mice administered a high dose of 6e12vg / kg rAAV. [Figure 9] Figure 1 illustrates percent aortic lesion area. 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. 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 vs. KO Vehicle; or comparisons indicated by parentheses. [Figure 10A]Figure 10A is a bar graph illustrating the anti-inflammatory effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Anti-inflammatory effects were measured using the MesoScale Diagnostics mouse cytokine panel. Figure 10B illustrates IL-5 levels. One-way ANOVA, Dunnett's post-hoc test. #p<0.05, ##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle. [Figure 10B] Figure 10B is a bar graph illustrating the anti-inflammatory effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Anti-inflammatory effects were measured using the MesoScale Diagnostics mouse cytokine panel. Figure 10B illustrates IL-6 levels. One-way ANOVA, Dunnett's post-hoc test. #p<0.05, ##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle. [Figure 10C]Figure 10C is a bar graph illustrating the anti-inflammatory effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. The anti-inflammatory effect was measured using the MesoScale Diagnostics mouse cytokine panel. Figure 10C illustrates TNF-α levels. One-way ANOVA, Dunnett's post-hoc test. #p<0.05, ##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle. [Figure 10D] Figure 10B is a bar graph illustrating the anti-inflammatory effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Anti-inflammatory effects were measured using the MesoScale Diagnostics mouse cytokine panel. Figure 10C illustrates IL-17A / F levels. One-way ANOVA, Dunnett's post-hoc test. #p<0.05, ##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle. [Figure 10E]Figure 10E is a bar graph illustrating the anti-inflammatory effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Anti-inflammatory effects were measured using the MesoScale Diagnostics mouse cytokine panel. Figure 10E illustrates CCL2 levels. One-way ANOVA, Dunnett's post-hoc test. #p<0.05, ##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle. [Figure 10F] Figure 10F is a bar graph illustrating the anti-inflammatory effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052). APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Anti-inflammatory effects were measured using the MesoScale Diagnostics mouse cytokine panel. Figure 10F illustrates CXCL2. One-way ANOVA, Dunnett's post-hoc test. #p<0.05, ##p<0.01 vs. WT vehicle; *p<0.05, **p<0.01 vs. KO vehicle. [Figure 11A]Figure 11A is a bar graph illustrating the effect of rAAV containing APOEch or APOE transgenes in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on glial fibrillary acidic protein (GFAP) levels. APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 11B illustrates GFAP / total protein in the cortex as determined by JESS capillary electrophoresis (ProteinSimple). One-way ANOVA followed by Dunn's or Fisher's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 11B] Figure 11B is a bar graph illustrating the effect of rAAV containing APOEch or APOE transgenes in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on glial fibrillary acidic protein (GFAP) levels. APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 11B illustrates GFAP / total protein in the hippocampus as determined by JESS capillary electrophoresis (ProteinSimple). One-way ANOVA followed by Dunn's or Fisher's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 11C]Figure 11C is a bar graph illustrating the effect of rAAV containing APOEch or APOE transgenes in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on glial fibrillary acidic protein (GFAP) levels. APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 11D illustrates the percent area of ​​GFAP in the whole brain determined by immunofluorescence staining using an anti-GFAP antibody (AB5541, Millipore; 1:500) and quantified using HALO® image analysis software (Indica Labs). One-way analysis of variance followed by Dunn's or Fisher's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 11D] Figure 11D is a bar graph illustrating the effect of rAAV containing APOEch or APOE transgenes in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on glial fibrillary acidic protein (GFAP) levels. APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 11D illustrates GFAP % area in the hippocampus as determined by immunofluorescence quantification using Halo® image analysis software (IndicaLabs). One-way ANOVA followed by Dunn's or Fisher's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 12A]Figure 12A is a bar graph illustrating the effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on pre- and postsynaptic proteins in the cortex and hippocampus. APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 12B illustrates synaptophysin / total protein in the hippocampus. Synaptic proteins were determined by JESS capillary electrophoresis (ProteinSimple). Kruskal-Wallis one-way ANOVA followed by Dunn's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 12B] Figure 12B is a bar graph illustrating the effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on pre- and postsynaptic proteins in the cortex and hippocampus. APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 12B illustrates PSD-95 / total protein in the hippocampus. Synaptic proteins were determined by JESS capillary electrophoresis (ProteinSimple). Kruskal-Wallis one-way ANOVA followed by Dunn's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 12C]Figure 12C is a bar graph illustrating the effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on pre- and postsynaptic proteins in the cortex and hippocampus. APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 12D illustrates synaptophysin / total protein in the cortex. Synaptic proteins were determined by JESS capillary electrophoresis (ProteinSimple). Kruskal-Wallis one-way ANOVA followed by Dunn's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 12D] Figure 12D is a bar graph illustrating the effect of rAAV containing the APOE3ch or APOE3 transgene in ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, line #:002052) on pre- and postsynaptic proteins in the cortex and hippocampus. APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 12D illustrates PSD-95 / total protein in the cortex. Synaptic proteins were determined by JESS capillary electrophoresis (ProteinSimple). Kruskal-Wallis one-way ANOVA followed by Dunn's post-hoc test. #p<0.05 vs. WT, *p<0.05, **p<0.01 vs. KO vehicle. [Figure 13A]Figure 13A is a bar graph illustrating the effect of rAAV containing APOE3ch or APOE3 transgenes in 1-year-old ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #:002052) on atherosclerosis reversal of existing lesions. Figure 13B illustrates the % aortic lesion area in KO mice and mice administered different transgene sequences encoding ApoE: E3N (native ApoE3) 2e11 vg / kg, E3-3 (ApoE3-3) 2e11 vg / kg, and E3-3 (ApoE3-3) 2e12 vg / kg. [Figure 13B] Figure 13B is a bar graph illustrating the effect of rAAV containing APOE3ch or APOE3 transgenes in 1-year-old ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #:002052) on atherosclerotic reversal of existing lesions. Figure 13B illustrates the % change in atherosclerotic lesions in the KO baseline from Figure 13A and the combined E3N (native ApoE3) 2e11vg / kg and E3-3 (ApoE3-3) 2e11vg / kg groups from Figure 13A. [Figure 13C] Figure 13C is a bar graph illustrating the effect of rAAV containing APOE3ch or APOE3 transgenes in 1-year-old ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #:002052) on atherosclerosis reversal of existing lesions. Figure 13D illustrates the % aortic lesion area in KO untreated mice and KO mice administered different transgene sequences encoding ApoE3ch: E3N (native ApoE3ch) 2e11 vg / kg, E3ch-9 (ApoE3ch-9) 2e11 vg / kg, and E3ch-9 (ApoE3ch-9) 2e12 vg / kg. [Figure 13D]Figure 13D is a bar graph illustrating the effect of rAAV containing APOE3ch or APOE3 transgenes in 1-year-old ApoE knockout (KO) mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #:002052) on atherosclerotic reversal of existing lesions. Figure 13D illustrates the % change in atherosclerotic lesions in the KO baseline from Figure 13C and the combined E3N (native ApoE3ch) 2e11vg / kg and E3ch-9 (ApoE3ch-9) 2e11vg / kg groups from Figure 13C. [Figure 14A] Figure 14A illustrates the results of cognitive impairment testing in mice administered rAAV containing the APOE3(ch) transgene in a novel object recognition (NOR) memory test. rAAV was administered at a low dose of 2e11 vg / mouse or a high dose of 2e12 total vg / mouse. Figure 14B illustrates the NOR results for 1-year-old C57BL / 6 mice and ApoE knockout mice, showing cognitive impairment in 1-year-old ApoE knockout mice compared to age-matched C57BL / 6 mice (*p<0.05, unpaired two-tailed t-test). [Figure 14B] Figure 14B illustrates the results of cognitive impairment tests in mice administered rAAV containing the APOE3(ch) transgene in a novel object recognition (NOR) memory test. rAAV was administered at a low dose of 2e11 vg / mouse or a high dose of 2e12 total vg / mouse. Figure 14B illustrates the NOR results prior to gene therapy and 5 weeks after gene therapy treatment using native ApoE3 and ApoE3(ch) sequences in the same mice before and after gene therapy treatment. [Figure 14C] Figure 14A illustrates the results of cognitive impairment testing in mice administered rAAV containing the APOE3(ch) transgene in a novel object recognition (NOR) memory test. rAAV was administered at a low dose of 2e11 vg / mouse or a high dose of 2e12 total vg / mouse. Figure 14B illustrates the NOR results with low doses of rAAV encoding CpG-0 ApoE3 (E3-3) and ApoE3ch (E3ch-9) rAAV. [Figure 14D]Figure 14D illustrates the results of cognitive impairment testing in mice administered rAAV containing the APOE3(ch) transgene in a novel object recognition (NOR) memory test. rAAV was administered at a low dose of 2e11 vg / mouse or a high dose of 2e12 total vg / mouse. Figure 14D illustrates the NOR results with high doses of rAAV encoding CpG-0 ApoE3 (E3-3) and ApoE3ch (E3ch-9) rAAV. [Figure 15A] Figure 15A illustrates ApoE expression from different transgenes. Figure 15A provides a bar graph showing the performance of codon-optimized, CpG-reduced cDNAs encoding ApoE3. Plasmids encoding either wild-type (wt) ApoE3 or codon-optimized ApoE3 cDNAs were transfected into AML-12 cells in triplicate, and antigen levels were measured in cell culture supernatants 72 hours post-transfection. ApoE3 levels were assayed by ELISA and plotted as mean ± standard deviation. For comparison purposes, non-intron-containing, codon-optimized ApoE3-3 and H30 variants were included as standards. ApoE3 levels were assayed by ELISA and plotted as mean ± standard deviation. [Figure 15B] Figure 15A illustrates ApoE expression from different transgenes. Figure 15B provides a bar graph showing the performance of codon-optimized ApoE3 cDNAs made functional by the addition of intron sequences. Plasmids encoding either wild-type (wt) ApoE3 or intron-containing cDNA variants of ApoE3 were transfected into AML-12 cells in triplicate, and antigen levels were measured in cell culture supernatants 72 hours after transfection. For comparison purposes, non-intron-containing codon-optimized ApoE3-3 and H30 variants were included as standards. ApoE3 levels were assayed by ELISA and plotted as the mean ± standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0018]

[0034] The invention features a polynucleotide construct comprising an ApoE-encoding nucleotide sequence having at least 85% sequence identity to any 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, the protein comprising 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 the ApoE-encoding nucleotide sequence 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.

[0019]

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

[0020]

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

[0021]

[0037] The polynucleotide encoding ApoE3-related protein can be delivered by non-viral or viral delivery.The viral vector that can be used in gene therapy includes retroviral vector, adenoviral vector, AAV vector and herpes simplex virus vector.Non-viral delivery includes naked DNA and nanoparticles.

[0022]

[0038] References to percent "identical," "identity," and similar terms refer to two sequences with maximal alignment in a particular region. The region provided is with respect to the reference sequence indicated. For example, a sequence "identical" or "identity" to 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. Percent "identical" or "identity" for nucleic acid sequences can be determined in a similar manner, aligning the nucleotides to the reference sequence to achieve maximal alignment, dividing by the total number of nucleotides in the reference sequence, and multiplying by 100. Percent "identical" or "identity" for ApoE coding sequences determined independently of any introns indicates that the intron(s) are removed prior to alignment for purposes of calculation.

[0023]

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

[0024]

[0040] In certain embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA, (poly- and oligonucleotides), chromosomal DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA inhibitory DNA or RNA (RNAi, e.g., 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- and double-stranded oligonucleotide DNA (ODN), immunostimulatory sequences (ISS), riboswitches, and ribozymes.

[0025]

[0041] In certain embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded, triplexed, quadruplexed, linear or circular, and of any length.

[0026]

[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, nanoplasmid, open circular linear double-stranded DNA, or closed linear double-stranded DNA (CELiD / ceDNA / doggyboneDNA). According to certain embodiments, the ssDNA molecule is closed circular or open circular linear DNA.

[0027]

[0043] "Transgene" means a nucleic acid that is intended to be or has been introduced into a cell and operably linked to a promoter. Transgenes include heterologous polynucleotide sequences, such as nucleic acids encoding ApoE3-related proteins and heterologous promoters.

[0028]

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

[0029]

[0045] CpG motifs may be suitably reduced or removed in the nucleotide sequence encoding the ApoE3-related protein and in other sequences present in certain constructs (e.g., expression cassettes and viral vectors), including non-coding sequences such as 5' and 3' untranslated regions (UTRs), stuffer sequences, promoters, enhancers, polyadenylation signals, ITRs, and intron(s).

[0030]

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

[0031]

[0047] The conjunction "and / or" between multiple specified elements encompasses both individual and combined alternatives. For example, if two elements are joined by "and / or," the first alternative means the applicability of the first alternative without the second alternative, the second alternative means the applicability of the second alternative without the first alternative, and the third alternative means the applicability of the first and second alternatives together. Any one of the alternatives is understood to fall within the meaning and thus meets the requirements of the term "and / or." The simultaneous applicability of two or more of the alternatives is also understood to fall within the meaning of the term "and / or."

[0032]

[0048] Unless the context clearly dictates otherwise when used, the terms "or" and "and" have the same meaning as "and / or."

[0033]

[0049] References to terms such as "including," "for example," "eg," "etc.", etc. followed by different elements or examples are open-ended descriptions, and the listed elements or examples are exemplary, and other elements or examples can be provided or used.

[0034]

[0050] The terms "polypeptide," "protein," and "peptide" can be used interchangeably to refer to amino acid sequences, regardless of function. Polypeptides and peptides contain at least two amino acids, and proteins contain at least about 10 amino acids. Provided amino acids include naturally occurring amino acids and amino acids provided by cellular modification.

[0035]

[0051] References to "comprise," and variations such as "comprises" and "comprising," when used in reference to an element or group of elements, are open-ended and do not exclude additional, unspecified elements or method steps. Terms such as "including," "containing," and "characterized by" are synonymous with comprising. In different aspects and embodiments described herein, references to open-ended terms such as "comprising" can be replaced with "consisting of" or "consisting essentially of."

[0036]

[0052] A reference to "consisting of" excludes any element, step, or ingredient not specified in the recited claim element, where such element, step, or ingredient is relevant to the claimed invention.

[0037]

[0053] References to "consisting essentially of" limit the scope of the claim to the materials or steps specified and those that do not materially affect the basic and novel feature(s) of the claimed invention.

[0038]

[0054] The term "about" means a value within 10% of the underlying parameter (i.e., plus or minus 10%). 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 of the values ​​by up to 10%.

[0039]

[0055] All numerical values ​​or numerical ranges include integers within such ranges and fractions of values ​​or integers within ranges unless the context clearly dictates otherwise. Thus, by way of example, a reference to a reduction of 95% or more 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%, etc., and a reference to a numerical range such as "1 to 4" includes 1, 2, 3, 4, as well as 1.1, 1.2, 1.3, 1.4, etc. By way of further example, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0040]

[0056] Additionally, references to numerical ranges such as "0.01 to 10" include 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dosage of about "0.01 mg / kg to about 10 mg / kg" of subject 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.

[0041]

[0057] References to integers with more than or less than include numbers greater than or less than the referenced number, respectively. Thus, for example, a reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and "two or more" administrations includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0042]

[0058] Various references, including articles and patent publications, are cited or described in the background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. None of the references is admitted to be prior art with respect to any invention disclosed or claimed. In some instances, a particular reference is indicated as being incorporated herein by reference to highlight the incorporation.

[0043]

[0059] The definitions provided herein, including in this section and other sections of this application, apply throughout this application.

[0044]

[0060] Unless otherwise defined, 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.

[0045]

[0061] The description is separated into various sections and paragraphs and provides examples of various embodiments. These separations should not be considered as separating the content of a paragraph or section or embodiment from the content of another paragraph or section or embodiment. The description provided has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that may be considered. The discussion of any embodiment is meant to be exemplary only and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples (unless otherwise provided in the claims).

[0046]

[0062] In order to describe numerous embodiments of the present invention, the present invention is generally disclosed herein using categorical language. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is completely or partially excluded. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Thus, even if the present invention is not generally expressed herein as something that the present invention does not include, embodiments that are not expressly excluded in the present invention are nevertheless disclosed herein.

[0047]

[0063] I. ApoE Protein-Encoding Polynucleotides

[0064] The ApoE-encoding nucleotide sequence encodes an ApoE3-related protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 32, where the protein comprises a cysteine ​​at the position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at the position corresponding to amino acid 158 of SEQ ID NO: 32. If intron(s) are present, the percent identity is determined independently of the intron(s). In certain embodiments, no introns are present in the ApoE-encoding nucleic acid.

[0048]

[0065] In different 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.

[0049]

[0066] In different 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 a position corresponding to amino acid 136 of SEQ ID NO: 32 provides a Christchurch substitution.

[0050]

[0067] Additional ApoE3-related proteins can be obtained using the ApoE3 sequences of SEQ ID NO: 32 and SEQ ID NO: 33 as starting constructs based on existing knowledge of various ApoE domains and various ApoE sequences. Exemplary references describing various 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.

[0051]

[0068] In certain embodiments, the ApoE coding sequence comprises a sequence having at least 85% sequence identity to any of SEQ ID NOs:63-67, and nucleotides 55-954 of any 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 provided sequence identity is to any of SEQ ID NOs:63, 64, 65, 66, 67, or nucleotides 1-897 of any of SEQ ID NOs:63, 64, 65, 66, 67; or to any of SEQ ID NOs:3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 25, 27, 28, 29, 30, 95, 106, 110, 112, 114, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 149, 150, 151, 152, 153, 154, 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 NO:130 are 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% of nucleotides 55 to 951 or 55 to 954 of any of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, and SEQ ID NO:130.

[0052]

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

[0053]

[0070] Reference to a sequence provided in this application that includes a stop codon includes embodiments in which no stop codon is present, multiple stop codons are present, and different stop codons are present.

[0054]

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

[0055]

[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 secretory signal of a secreted polypeptide and / or can be in whole or in part synthetic. Generally, known signal peptides are from about 10-15 to 50-60 amino acids in length. Furthermore, known secretory signals from secreted polypeptides can be altered or modified (e.g., by amino acid substitution, deletion, truncation, or insertion) so long as the resulting secretory signal sequence functions to enhance polypeptide secretion.

[0056]

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

[0057]

[0074] In certain embodiments, the signal peptide is selected from ApoE3 signal peptide, human chymotrypsinogen B2 signal peptide ("sp7"; the 18 amino acid signal peptide of NCBI Reference Sequence NP_001020371), alpha 2-HS-glycoprotein (AHSG) signal peptide, CD300 signal peptide, lysosome-associated membrane glycoprotein 1 (LAMP1) signal peptide, Notch2 signal peptide, orosomucoid 1 (ORM1) signal peptide, transferrin (TF) signal peptide, secretecon (the 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 secreted alkaline phosphatase (SEAP).

[0058]

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

[0059]

[0076] In different 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 including the ApoE3 signal peptide, and SEQ ID NO: 35 provides ApoE3(ch) including the ApoE3 signal peptide.

[0060]

[0077] In certain embodiments, the ApoE-encoding nucleotide sequence contains one or more introns. Introns are characterized by 5' and 3' splicing consensus sequences that, together with an adenosine at the branch point, provide the intron boundary. The adenosine is responsible for breaking the phosphodiester bond at the upstream exon-intron boundary. In certain embodiments, the 5' and 3' splicing consensus sequences comprise 5'GU...AG3'. In certain embodiments, the 5' and 3' splicing consensus sequences comprise 5'AU...AC3'. Consensus splicing sites are described, for example, in Jurica and Roybal, "RNA Splicing," in Lennarz and Lane, eds., Encyclopedia of Biological Chemistry (2nd ed.), Academic Press, 2013, pp. 185-190; and Qu et al., Front Genet. April 11, 2017; p. 8:38, each of which is incorporated herein by reference in its entirety.

[0061]

[0078] Branch points are also typically located within consensus sequences (e.g., heptamer sequences) approximately 18-40 nucleotides upstream of the 3' acceptor site, with a polypyrimidine tract generally separating the branch point from the 3' splice site. In certain embodiments, the adenosine-containing branch point is approximately 18-40 nucleotides upstream of the 3' acceptor site, with a polypyrimidine tract containing primarily C and U residues. RNA splicing is described, for example, by Jurica and Roybal, "RNA Splicing," in Lennarz and Lane, eds., Encyclopedia of Biological Chemistry (2nd ed.), Academic Press, 2013, pp. 185-190; and Berger et al., Wiley Interdiscip Rev RNA. July 2016;7(4):487-98, each of which is incorporated herein by reference in its entirety.

[0062]

[0079] In further embodiments, the ApoE-encoding nucleotide sequence has one or two introns and / or the introns are located in naturally occurring positions: natural intron splicing 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.

[0063]

[0080] In certain embodiments, the ApoE-encoding nucleotide sequence (ApoE transgene) is arranged 5' to 3' as follows: (a) a first exon corresponding to nucleotides 1 to 43 of SEQ ID NO: 1, which has at least 85% sequence identity to nucleotides 1 to 43 of any of SEQ ID NOs: 3 to 31, 95 to 105 or 124 to 130, provided that the 3' terminal nucleotide of the first exon is G, and in further embodiments related 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 to 43 of any of SEQ ID NOs: 3 to 31, 95 to 105, and 124 to 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 to 236 of SEQ ID NO: 1, which has at least 85% sequence identity to nucleotides 44 to 236 of any of SEQ ID NOs: 3 to 31, 95 to 105 or 124 to 130, provided that the 5'-terminal nucleotide of the second exon is G (in a further embodiment, GC) and the 3'-terminal nucleotide of the second exon is AG, and in further embodiments related 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 to 236 of any of SEQ ID NOs: 3 to 31, 95 to 105 or 124 to 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, which has at least 85% sequence identity to nucleotides 237-951 of any of SEQ ID NOs: 3-31, 95-105, or 124-130, provided that the 5'-terminal nucleotide of the third exon is G; in further embodiments related to 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 of SEQ ID NOs: 3-31, 95-105, or 124-130. the first, second and third exons 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 comprises at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 35, or 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.

[0064]

[0081] Each of the possibilities within (a), (b), (c), (d), and (e) can be independently combined with respect to each provided sequence and sequence identity. For example, (1) the first exon, independently of the second and third exons, 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 1-43 of any of SEQ ID NOs: 3-31, 95-105, or 124-130; (2) the second exon, independently of the first and third exons, 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 of SEQ ID NOs: 3-31, 95-105, or 124-130. and (3) the third exon, independently of the first and second exons, 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 to 236 of any of SEQ ID NOs: 3-31, 95-105, or 124-130.

[0065]

[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 the position or region that coincides with the indicated position or region of the reference sequence when maximum alignment is performed. Maximum alignment takes into account any additions, deletions, and / or substitutions. Preferably, only substitutions are present.

[0066]

[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 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% identity to any of SEQ ID NOs: 119-122.

[0067]

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

[0068]

[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 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 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 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, or 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.

[0069]

[0086] In certain embodiments, the ApoE-encoding nucleotide sequence 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 of SEQ ID NOs: 106-115.

[0070]

[0087] In certain embodiments, the one or more introns are selected from the group consisting of a rabbit β-globin intron containing a splicing donor / splice acceptor, an SV40 intron containing a splicing donor / splice acceptor, a human β-globin intron, intron 2 of the human hemoglobin β gene, hFIX int1 (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 with intron 2 of 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 portions of the human coagulation factor IX gene and expressed in a pLIVE vector, Mirus synthetic introns present in Bio, Madison, WI); human hemoglobin subunit beta (HBB2) synthetic intron, and optimized HBB2; and chimeric introns such as an intron composed of the 5' splicing donor of the first human β-globin intron and a branch site and 3' acceptor site from an intron between the leader and body of an 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) July 20;3:16049; and the HBB-IGG intron provided by the pCMVNT™ vector).

[0071]

[0088] In certain embodiments, the intron is between 50 and 1,500 bases.

[0072]

[0089] In certain embodiments, any of the ApoE-encoding nucleotide sequences and introns provided herein contain 0 to 5, 0 to 10, or 0 to 15 CpGs; 2, 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 up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, or up to about 5.0% CpGs; preferably 0 CpGs.

[0073]

[0090] II. Expression Cassettes

[0091] A polynucleotide expression cassette comprises an ApoE-encoding nucleic acid operably linked to one or more expression control elements. An "expression control element" is a nucleic acid sequence that affects expression of an ApoE-encoding nucleic acid. Expression control can be affected, for example, at the level 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 adjacent to the transcribed sequence or at a distance from the transcribed sequence. There can be one or more expression control elements. Examples of expression control elements include promoters, enhancers, introns, polyadenylation signals, Kozak sequences, post-transcriptional regulatory elements, and termination sequences.

[0074]

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

[0075]

[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 typically active in specific cells, tissues, or organs because they are recognized by transcriptional activator proteins or other regulators of transcription that are specific to that specific cell, tissue, or organ type (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th ed., Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al. (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0076]

[0094] Incorporation of tissue-specific regulatory elements in an expression construct confers at least partial tissue tropism to the expression of an ApoE3-related protein. Reference to a promoter or enhancer that is specific for a particular cell type of a tissue indicates that the promoter or enhancer provides a relatively high level of expression and / or secretion in the indicated cell or tissue type. Examples of liver-specific promoters are the transthyretin (TTR) gene promoter; the human alpha-1 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 (AFP), 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 alpha-antitrypsin promoter; LSP (845 nt) (requires an intronless scAAV); and the LSP1 promoter. Examples of enhancers active in the liver are apolipoprotein E (ApoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0077]

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

[0078]

[0096] Examples of CNS-specific promoters include: 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 2neuron-specific promoters such as calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or 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 the proopiomelanocortin (POMC) promoter; and neuron / spinal cord-specific promoters (see, e.g., U.S. Patent Application Publication No. 2021 / 214749 and Adeno-Associated Virus (AdV) Inhibitors, both of which are incorporated herein by reference in their entireties). See Vectors (2019), Castle, ed., 1st ed., Springer New York, New York, NY.

[0079]

[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-α promoter, synthetic promoters, hybrid promoters, and promoters with multiple tissue specificities.

[0080]

[0098] Expression control elements can also affect expression in a regulatable manner, with a signal or stimulus increasing or decreasing expression. Regulatable elements that increase expression of the nucleic acid they are transcribing in response to a signal or stimulus are also referred to as "inducible elements" (i.e., induced by the signal). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 1998 / 10088); the tetracycline-repressible 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 regulated by specific physiological conditions such as temperature, acute phase, or development.

[0081]

[0099] In certain embodiments, the expression cassette further comprises one or more introns independent of the ApoE-encoding nucleotide acids. A variety of different introns can be used to enhance gene expression. Examples of introns that can be used include a rabbit β-globin intron containing a splice donor / splice acceptor, an SV40 intron containing a splice donor / splice acceptor, a human β-globin intron, intron 2 of the human hemoglobin β gene, hFIX int1 (intron 1 of the human clotting factor IX gene), CBA-rHHB (a synthetic intron derived from a fusion of intron 1 of the chicken β-actin gene with intron 2 of 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 portions of the human clotting factor IX gene and expressed in pLIVE vectors, Mirus synthetic introns present in Bio, Madison, WI); human hemoglobin subunit beta (HBB2) synthetic intron, and optimized HBB2; and chimeric introns such as an intron composed of the 5' splicing donor of the first human β-globin intron and a branch site and 3' acceptor site from an intron between the leader and body of an 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) July 20; 3:16049; and the HBB-IGG intron provided by the pCMVNT™ vector).

[0082]

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

[0083]

[0101] Polyadenylation signal sequences confer the formation of poly(A) tails that promote nuclear export, translation, and / or mRNA stability, and may also be involved in transcription termination. Examples of polyadenylation signal sequences include the SV40 late polyadenylation signal, the bovine growth hormone poly(A) (bGHpA) signal sequence, synthetic poly(A), mouse β-globin pA, rabbit β-globin pA, and pA based on H4 (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).

[0084]

[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.

[0085]

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

[0086]

[0104] In certain embodiments, the expression cassette further comprises an miRNA target sequence, which in further embodiments 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. Based on the presence of certain miRNAs in specific cells, incorporating an miRNA target sequence can be used to reduce expression in specific cell or tissue types. 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).

[0087]

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

[0088]

[0106] In certain embodiments, the nucleotide sequence encoding the expression cassette has 0 to 5, 0 to 10, 0 to 15, 0 to 50, or 0 to 100 CpGs; 6, 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, CpGs at positions 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82; 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% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.

[0089]

[0107] III. Recombinant Viral Vector Nucleic Acid

[0108] Polynucleotide recombinant viral nucleic acids contain 5' and / or 3' viral elements that confer viral packaging and can confer additional activities such as self-priming, DNA replication, promoter activity, genome integration, or episomal concatemerization. The 5' and 3' elements are generally located at or near the 5' and 3' ends of the recombinant viral nucleic acid and can be naturally occurring or modified versions of naturally occurring sequences. Examples of 5' and 3' elements include adenovirus ITRs, adeno-associated virus ITRs and packaging sequences; retrovirus 5' and 3' long terminal repeats (LTRs) and packaging sequences (Naso et al. (2017) BioDrugs, 31(4), pp. 317-334; Bulcha et al. (2021) Sig. Transduct. Target Ther. 6: pp. 53 (2021); and Liu and Seol (2020) BMB Reports; 53(11): pp. 565-575).

[0090]

[0109] The term "recombinant" as a modifier of nucleic acid or vector refers to a combination of elements that do not occur in nature. For example, a recombinant viral vector nucleic acid provides 5' and / or 3' viral elements along with an expression cassette that includes 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 include a naturally occurring or modified capsid that encapsidates the recombinant viral vector nucleic acid.

[0091]

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

[0092]

[0111] In certain embodiments, the viral vector nucleic acid has 0 to 5, 0 to 10, 0 to 15, 0 to 50, 0 to 100, or 0 to 150 CpG positions; 7, 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, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, CpGs at positions 2, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82; 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% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.

[0093]

[0112] IV. Viral Vectors

[0113] In certain embodiments, the gene delivery vehicle is a viral vector. A viral vector comprises a protein capsid that encapsidates a recombinant viral nucleic acid and can deliver the nucleic acid to cells or tissues. Depending on the particular vector, the viral vector can 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.

[0094]

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

[0095]

[0115] As more naturally occurring virus isolates are discovered or capsid variants are generated, there may or may not be serological differences from any of the currently existing serotypes. Thus, if the novel virus does not have serological differences, it will likely be a subgroup or variant of the corresponding serotype.

[0096]

[0116] IV.A. Adenoviral Vectors

[0117] Adenoviruses are non-enveloped, double-stranded DNA viruses. Recombinant adenoviral vectors contain recombinant adenoviral nucleic acid that lacks 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 generated. The Ad genome is flanked at its ends by hairpin-like inverted terminal repeats (ITRs) that vary in length from 30 to 371 bp. ITRs function as self-priming structures that promote primase-independent DNA replication. 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).

[0097]

[0118] In certain embodiments, the recombinant adenoviral vector is a third-generation vector, also referred to as "gutless" or "helper-independent." Gutless vectors can be generated from recombinant adenoviral nucleic acid that is free of all or substantially all viral sequences except for the ITRs and packaging signal. Gutless adenoviral vectors are high-capacity vectors that can accommodate DNA inserts of up to approximately 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 integration. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombinant 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 incorporated herein by reference in its entirety).

[0098]

[0119] In certain embodiments, recombinant adenoviral vectors can be generated based on rare human or chimpanzee serotypes. The use of chimpanzee and rare human serotypes can be useful in reducing host immune responses 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).

[0099]

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

[0100]

[0121] IV.B. AAV Vectors

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

[0101]

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

[0102]

[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 AAV3B ITRs. In further embodiments, the 5' and 3' ITRs are present and both ITRs are from the same serotype genome.

[0103]

[0125] In further 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 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 of SEQ ID NOs: 82, 89, 91 and 93.

[0104]

[0126] In certain embodiments, the 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 acid that can be incorporated into AAV capsids. scAAV and shAAV contain inverted dimer repeats that provide intramolecular double-stranded DNA. scAAV can be generated by mutating the ITR terminal resolution site so that rep cannot nick the terminal resolution site. shAAV can utilize a short hairpin to generate double-stranded AAV nucleic acid. Being double-stranded DNA, scAAV and shAAV offer the advantage of avoiding the DNA synthesis step required for single-stranded rAAV nucleic acid upon cell entry. A potential drawback of scAAV and shAAV is the size of the DNA insert that can be incorporated, which is reduced by about half compared to single-stranded rAAV nucleic acids (U.S. Pat. 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 incorporated by reference herein in its entirety).

[0105]

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

[0106]

[0128] In certain embodiments, the AAV capsid comprises at least one amino acid sequence similar to that of VP1, VP2, or VP3 of any 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; and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions). 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 Application Publication 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.

[0107]

[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., ITRs) as the encapsidating capsid proteins.

[0108]

[0130] In different embodiments, the rAAV capsid comprises at least 80%, at least 85%, or both of VP1, VP2, or VP3 of any 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 VP1 of SEQ ID NO:83 or SEQ ID NO:84. , 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.

[0109]

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

[0110]

[0132] In certain embodiments, including treatment of CNS diseases or disorders, the capsid confers CNS expression. Examples of such rAAV capsids and designs of rAAV capsids capable of conferring CNS expression are provided in Chen et al., (2021) Journal of Controlled Release 333, pp. 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, pp. 106-115 (2022), and Ittner et al., Br. J. Pharmacol. (2019) 176:3649-3655, each of which is incorporated herein by reference in its entirety.

[0111]

[0133] Recombinant AAV can be produced, for example, by using appropriate helper viruses or plasmids and cell lines to supply the viral proteins necessary for vector production in trans. In certain embodiments, rAAV is produced using an rAAV vector genome plasmid. The plasmid contains a portion of the rAAV nucleic acid that is ultimately packaged or encapsidated to form viral (e.g., rAAV) particles. The "plasmid backbone" contains elements important for propagation and recombinant virus production. With the exception of possible 3' ITR and / or 5' ITR cloning residues, the plasmid backbone is not itself packaged or encapsidated into viral (e.g., AAV) particles.

[0112]

[0134] Recombinant AAV can be produced from various types of cell lines. In a specific embodiment, human HEK293 cells are used (American Type Culture Collection accession number ATCC CRL1573). Other host cell lines suitable for rAAV production are described, for example, in Robert et al. (2017) Biotechnol. J., 12:1600193; and International Application PCT / US2017 / 024951, the disclosures of which are incorporated herein in their entirety.

[0113]

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

[0114]

[0136] AAV requires helper virus functions to complete its replication cycle. Helper virus functions can be provided by various viruses in permissive cell lines. Permissive cell lines are cell lines that can support viral replication. Examples of helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HBoV1, which can be used with permissive primate cells; and baculovirus, which can be used with 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).

[0115]

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

[0116]

[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.

[0117]

[0139] Recombinant AAV can be produced from various 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 PCT / US2017 / 024951, the disclosures of which are incorporated herein in their entireties.

[0118]

[0140] Recombinant AAV can be cultured under a variety of different conditions suitable for cell growth and gene expression. References describing rAAV production include Clement 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 ed., Springer New York, New York, NY, each of which is incorporated herein by reference in its entirety.

[0119]

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

[0120]

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

[0121]

[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.

[0122]

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

[0123]

[0145] In certain embodiments, the rAAV vector is obtained by generating and purifying rAAV using the methods described herein. Purification of rAAV can be accomplished using techniques such as gradient-based purification, column-based methods, and multiplexing methods (see, e.g., Ayuso et al. (2010), Curr Gene Ther. (2010) 10(6):423-36, which is incorporated herein by reference in its entirety).

[0124]

[0146] In certain embodiments, AAV helper functions are introduced into host cells by transfecting them with an AAV helper construct, either prior to or simultaneously with transfection of the AAV expression vector. Host cells with AAV helper functions can 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 complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and are unable to replicate and package themselves. These constructs can be in the form of, for example, a plasmid, phage, transposon, cosmid, virus, or virion. Numerous AAV helper constructs have been described, such as the plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Numerous other vectors encoding Rep and / or Cap expression products are known. Recombinant AAV can be produced as described, for example, in U.S. Pat. No. 9,408,904; and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein in their entireties.

[0125]

[0147] IV.C. Retroviral Vectors

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

[0126] 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. A preferred non-viral vector is a nanoparticle. A variety of different nanoparticles can be utilized, including lipid nanoparticles (LNPs), polymeric 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) NanoImpact 20, 100261; Neshat et al. (2020) Current See 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).

[0127]

[0150] If desired, the nanoparticles can be targeted to a cell type, for example, using 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).

[0128]

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

[0129]

[0152] The preparation of different nanoparticles and the incorporation of nucleic acids and other compounds into them are known in the art. Examples of publications illustrating 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. October; 68:1-11 (each of which is incorporated herein by reference in its entirety). Factors that can influence small molecule incorporation into nanoparticles include hydrophobicity and the presence of ionizable moieties (see, e.g., Nii and Ishii, International Journal of Pharmaceutics (2005) 298, 198; and Chen et al., Journal of Controlled Release (2018) 286, 46).

[0130] VA lipid-based delivery system

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

[0131]

[0154] "Lipid nanoparticle" or "LNP" refers to a lipid-based vesicle useful for delivery of nucleic acid molecules and having nanoscale dimensions. In different 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.

[0132]

[0155] DNA is negatively charged.Therefore, it can be beneficial for LNP to contain cationic lipid, such as amino lipid.Exemplary amino lipids are listed 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, 8,466,127, and the like, all of which are incorporated herein in their entirety. 2, and 7,745,651, and U.S. Patent Application Publication Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125. In certain embodiments, the LNP comprises an amino lipid described in U.S. Patent No. 9,512,073, which is incorporated herein in its entirety.

[0133]

[0156] The terms "cationic lipid" and "amino lipid" are used interchangeably herein to include lipids and their salts 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 pHs below the pKa of the cationic lipid and substantially neutral at pHs above the pKa. Cationic lipids can also be titratable cationic lipids. In certain embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) group; a C18 alkyl chain, each alkyl chain of which may independently have one or more double bonds, one or more triple bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0134]

[0157] Cationic lipids include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DM, also known as DLin-C2K-DMA, XTC2, and C2K). A), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA, also known as MC3), salts thereof, and mixtures thereof. Other cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-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).

[0135]

[0158] Still further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleylcarbamoyloxy-3-dimethylamino ... Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3- 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-hydroxyethylammonium chloride (DC-Chol), nium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxopentane Examples of suitable amines include N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (DSS), or mixtures thereof.

[0136]

[0159] Many commercially available preparations of cationic lipids can be used, such as LIPOFECTIN® (containing DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE® (containing DOSPA and DOPE, available from GIBCO / BRL).

[0137]

[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. U.S. Patent Application Publication No. 2017 / 0367988 describes Merck-32.

[0138]

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

[0139]

[0162] LNPs can include neutral lipids. Neutral lipids can include lipid molecular species that exist in either uncharged or neutral zwitterionic forms at physiological pH. Such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by considerations such as particle size and stability. In certain embodiments, the neutral lipid component can be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0140]

[0163] Lipids with a variety of acyl chain groups of varying chain lengths and degrees of saturation are available or can be isolated or synthesized. In certain embodiments, lipids containing saturated fatty acids with carbon chain lengths in the C14-C22 range can be used. In certain embodiments, lipids containing mono- or di-unsaturated fatty acids with carbon chain lengths in the C14-C22 range can be used. Additionally, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups, such as serine and inositol.

[0141]

[0164] In further embodiments providing a neutral lipid, the neutral lipid may 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, e.g., 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%.

[0142]

[0165] LNPs can contain additional components such as sterols and polyethylene glycol. Sterols can impart fluidity to the LNP. As used herein, "sterol" refers to naturally occurring sterols of plant (phytosterols) or animal (zoosterol) origin, as well as non-naturally occurring synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Suitable sterols include those conventionally used in the field of liposome, lipid vesicle, or lipid particle preparations, most commonly cholesterol. Plant sterols include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156. In different embodiments providing a sterol, the sterol is present in an amount from about 1% by weight of the LNP to about 80% by weight of the LNP, or from about 10% by weight of the LNP to about 25% by weight of the LNP.

[0143]

[0166] Polyethylene glycol (PEG) is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 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-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0144]

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

[0145]

[0168] PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "anchor" lipid moieties for fixing PEG moieties to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) described in U.S. Patent No. 5,820,873, PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. In certain embodiments, PEG-modified lipids can be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, PEG can be in an amount from about 0.1% by weight of the LNP to about 50% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.

[0146]

[0169] In further embodiments relating to LNP size, prior to the step of encapsulating nucleic acid, the LNPs have 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.

[0147]

[0170] In certain embodiments related to LNPs, the LNPs are described in Billingsley et al., Nano Lett. 2020, 20, p. 1578 or Billingsley et al., WO 2021 / 077066 (both of which are incorporated herein by reference in their entireties). Billingsley et al. and WO 2021 / 077066 describe LNPs containing lipid-anchored PEG, cholesterol, phospholipids, and ionizable lipids. In certain embodiments, the LNPs contain a C14-4 polyamine core and / or have a particle size of about 70 nm. The C14-4 has the following structure: [ka]

[0148]

[0171] In certain embodiments, the LNPs are composed of cationic lipids or lipopeptides as described by U.S. Patent Nos. 10,493,031, 10,682,374, or WO 2021 / 077066 (each of which is incorporated herein by reference in its entirety). In certain embodiments, the LNPs contain cationic lipids, cholesterol-based lipids, and / or one or more PEG-modified lipids. In certain embodiments, the LNPs contain cKK-E12 (Dong et al., PNAS (2014) 111(11), 3955): [ka]

[0149]

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

[0150]

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

[0151]

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

[0152]

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

[0153]

[0176] Additional lipids that can be utilized include those described in 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, 21, 8, 1570-1578; and Liu et al., Adv. Mate., each of which is incorporated herein by reference in its entirety. r. 2019, 31, 1902575, e.g., BAMEA-O16B; Cheng et al., Adv. Mater., 2018, 30, 1805308, e.g., 5A2-SC8; Hajj and Ball, Small, 2019, 15, 1805097, e.g., 306Oi10; Du et al., U.S. Patent Application Publication No. 20160376224; and Tanaka et al., Adv. Funct. Mater., 2020, 30, 1910575.

[0154]

[0177] In further embodiments, the nanoparticles are LNPs. In further embodiments, the LNPs, in mole percent, comprise, consist essentially of, or consist of the following components: (1) about 20% to about 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipids, 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 phospholipids, 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 phospholipids are neutral lipids; and the phospholipids are DOPE or DSPC.

[0155]

[0178] In further embodiments, the LNPs, by mole percent, comprise, consist essentially of, or consist of the following components: (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) is GenVoy-ILM™ LNP (Precision NanoSystems).

[0156] Nanoparticles based on VB polymers

[0179] Polymer-based delivery systems can be fabricated from a variety of different natural and synthetic materials. DNA and other compounds can be entrapped in the polymer matrix of polymeric nanoparticles or adsorbed or conjugated to the surface of the 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, polymethacrylates, poly-L-lysine, pullulan, dextran, and hyaluronic acid, poly-β-aminoesters (Thomas et al., (2019) Molecules 24, 3744).

[0157]

[0180] Polymer-based nanoparticles can have a variety of sizes ranging from about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, from about 50 nm to about 200 nm, from about 100 nm to about 150 nm, and even smaller than about 150 nm.

[0158] VC lipid polymer nanoparticles

[0181] Lipid-polymer nanoparticles are hybrid nanoparticles that provide both lipid and polymer components and can be considered LNPs or LPNPs. LPNP configurations can provide an outer polymer layer and an inner lipid layer, or an outer lipid layer and an inner polymer layer. The presence of two different types of materials facilitates the design of nanoparticles to provide delayed release of components. Different lipid and polymer components can be selected based on 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. October; 68:1-11).

[0159] VD Protein and Peptide-Based Nanoparticles

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

[0160]

[0183] CPPs are short peptides (6-30 amino acid residues) that potentially allow intracellular penetration for the delivery of therapeutic molecules. Most CPPs consist primarily of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphipathic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., HIV-1 Tat protein) or obtained synthetically (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) such as 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) such as transportan, VT5, bactenecin-7 (Bac7), proline-rich peptides (PPRs), SAP (VRLPPP)3, TP10, pep-1, and MPG; membrane-directed CPPs (which simultaneously exhibit hydrophobic and amphipathic properties and contain both large aromatic and small residues) such as H625, SPIONs-PEG-CPP, and NP; and hydrophobic CPPs (containing only nonpolar motifs or residues) such as SG3, PFVYLI, pep-7, and fibroblast growth factor.

[0161]

[0184] Protein and peptide nanoparticles can be provided in a variety of sizes ranging from, for example, 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 less than about 150 nm.

[0162] VE peptide cage nanoparticles

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

[0163]

[0186] Various types of protein "shells" can assemble and be loaded with various types of materials. Protein cages can be generated using viral coat protein(s) (e.g., from cowpea chlorotic mottle virus protein coat) as well as non-viral proteins (e.g., U.S. Pat. Nos. 6,180,389 and 6,984,386; U.S. Patent Application Publication Nos. 20040028694; and 20090035389, each of which is incorporated by reference in its entirety).

[0164]

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

[0165]

[0188] The protein cages can have a variety of core sizes, such as 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 even less than about 150 nm.

[0166] VF exosomes

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

[0167]

[0190] Various modifications can be made to exosomes to facilitate cargo delivery and cellular targeting. Modifications to facilitate cargo delivery include structures for associating with cargo, such as protein scaffolds and polymers. Modifications for cellular targeting include targeting ligands and altered surface charge. Publications describing the production, modification, and use of exosomes for the delivery of various cargoes include Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) NanoImpact 20, 100261; and Dooley et al., (2021) Molecular Therapy 29(5), 1729 (each of which is incorporated herein by reference).

[0168] VI. Pharmaceutical Compositions

[0191] Pharmaceutical compositions include pharmaceutically acceptable carriers that facilitate administration and / or storage of the polynucleotide constructs, viral vectors, and non-viral vectors described herein. References to "pharmaceutically acceptable" indicate that the component does not cause substantial undesirable biological effects in the amounts employed. Pharmaceutically acceptable carriers can contain a variety of components, such as one or more pharmaceutically acceptable excipients, such as salts, sugars, buffers, solvents, preservatives, proteins, and surfactants. Certain excipients can have more than one function. Pharmaceutically acceptable excipients and carriers that can be used for viral vectors are provided, for example, in WO 2021 / 071835.

[0169]

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

[0170]

[0193] In embodiments, the pharmaceutical composition contains a formulation that can be injected into a subject. Examples of components of an injection formulation include isotonic sterile saline solution, salts (e.g., monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, and mixtures of their salts), buffered saline, sugars (e.g., dextrose), and water for injection. Pharmaceutical compositions include dried, e.g., lyophilized, compositions that allow the formation of a solution suitable for administration upon addition of sterile water or saline.

[0171]

[0194] Additionally, suspensions can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, suspensions can also contain suitable stabilizers or agents that increase the solubility of the compounds, facilitating the preparation of concentrated solutions.

[0172]

[0195] "Effective amount" or "sufficient amount" means an amount that provides an 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 or immunosuppressive agents), treatments, protocols, or treatment regimens; and can provide a long-term or short-term response.

[0173]

[0196] A pharmaceutical composition containing 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 contains sufficient genetic material to allow the recipient to produce a therapeutically effective amount of the protein in the subject.

[0174]

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

[0175]

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

[0176]

[0199] Additional guidance and examples of pharmaceutical compositions and delivery systems are provided, for example, in 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, edited by 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.

[0177]

[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 to reduce the likelihood or severity of a disease or order and / or to treat a diagnosed disease or disorder. In certain embodiments, the polynucleotide component, vector choice, route of administration, and / or particular pharmaceutical composition are selected in view of the particular disease or disorder to be treated.

[0178]

[0202] Subjects with a particular disease or disorder or at increased risk of a particular disease or disorder can be identified, for example, based on symptoms, biomarkers, and genetic markers. Exemplary treatments include one or more of the following: reducing cholesterol, reducing LDL / VLDL, increasing HDL, or reducing 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.

[0179]

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

[0180]

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

[0181]

[0205] In certain embodiments, treatment is achieved by providing peripheral ApoE3-associated expression, e.g., hepatic expression. High hepatic expression can be achieved, for example, using a polynucleotide expression cassette containing a promoter or promoter / enhancer that confers high hepatic expression; a viral vector that confers liver tropism; and liver-targeting nanoparticles. In different embodiments, the promoter is the human alpha-1-antitrypsin (hAAT) promoter, apolipoprotein AI promoter, albumin promoter, hepatitis B virus core promoter, alpha-fetoprotein (AFP), human factor IX promoter, thyroxine-binding globulin promoter, TTR minimal enhancer / promoter, alpha-antitrypsin promoter, or LSP1 promoter; and / or enhancers are provided for apolipoprotein E (ApoE) HCR-1 and HCR-2. In further embodiments, the rAAV has a serotype based on AAV2 or AAV3B, or a VP1 based on SEQ ID NO: 83 or SEQ ID NO: 84.

[0182]

[0206] In certain embodiments, Alzheimer's disease is treated using nucleic acid expression components, vector components, and / or techniques that provide for CNS expression of an ApoE3-related protein; subjects are selected based on biomarkers or genetic markers associated with Alzheimer's disease; and / or subjects are diagnosed with Alzheimer's disease. In further embodiments, the ApoE3-related protein comprises a Christchurch substitution.

[0183]

[0207] In certain embodiments, Alzheimer's disease is treated using nucleic acid expression components, vector components, and / or technologies that confer peripheral ApoE3-related protein expression. Peripheral ApoE isoforms, separated from those in the brain by the blood-brain barrier, have been shown to differentially affect Alzheimer's pathogenesis and cognition (Liu et al., (2022), Nature Neuroscience 25:1020-1033). In further embodiments, the ApoE3-related protein comprises a Christchurch substitution.

[0184]

[0208] Alzheimer's disease subjects can be identified based on abnormal decline in cognitive performance, which can be performed in conjunction with amyloid plaque measurements (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 different embodiments, the subject is a human subject with one or two ApoE4 alleles or is a PSEN1 (presenilin 1) mutation carrier.

[0185]

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

[0186]

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

[0187]

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

[0188]

[0212] In certain embodiments, treatment of CNS diseases or disorders, such as Alzheimer's disease, is carried out using techniques that facilitate passage of gene delivery vehicles across the blood-brain barrier. In further embodiments, passage of the blood-brain barrier is facilitated using focused ultrasound in combination with microbubbles (Cammalleri et al., J Clin Neurophysiol. (2020) March;37(2):104-117, which is incorporated herein by reference in its entirety).

[0189]

[0213] In certain embodiments, the components conferring CNS expression include the PGK promoter, the CBh promoter, or the EF1α promoter.

[0190]

[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, pp. 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 Application Publication No. 202 / 1214749, each of which is incorporated herein by reference in its entirety.

[0191]

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

[0192]

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

[0193]

[0217] CNS delivery devices, systems, and techniques also include those described in, for example, U.S. Patent No. 8,128,600, U.S. Patent Application Publication No. 2020 / 0324089, U.S. Patent Nos. 1,1129,643, 11,154,377, U.S. Patent Application Publication Nos. 2021 / 0343397, 2021 / 0282866, U.S. Patent Nos. 9,572,928, 8,337,458, 10,722,265, and U.S. Patent Application Publication No. 2021 / 214749, each of which is incorporated herein by reference in its entirety.

[0194]

[0218] The optimal dose can vary depending on various factors, such as the particular therapeutic agent and the desired endpoint. The dose, amount, number, frequency, or duration can be proportionally increased or decreased, taking into account any adverse side effects, complications, or other risk factors of the treatment or therapy, and the condition of the subject.

[0195]

[0219] "Unit dosage form" means 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 that can contain a pharmaceutically acceptable carrier, or in a composition in a freeze-dried or lyophilized state. In the case of a freeze-dried state, a sterile liquid carrier can be added prior to administration. Individual unit dosage forms can be included in a multi-dose kit or container.

[0196]

[0220] An "effective amount" achieves a 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 disease or disorder progression. A preferred therapeutically effective amount is effective to reduce multiple or all adverse symptoms.

[0197]

[0221] In certain embodiments, a pharmaceutical composition comprising a viral or non-viral vector is administered to a subject at a suitable dose to reduce cholesterol, reduce LDL / VLDL, increase HDL, or reduce the total cholesterol / HDL ratio; or to treat or reduce the likelihood of 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. In different embodiments, a suitable dosage is about 0.01 mg / kg to about 10 mg / kg of vector per kg of the subject's body weight, about 0.01 mg / kg to about 0.1 mg / kg of vector per kg of the subject's body weight, about 0.1 mg / kg to about 1.0 mg / kg of vector per kg of the subject's body weight, or about 1.0 mg / kg to about 10 mg / kg of vector per kg of the subject's body weight.

[0198]

[0222] Generally, the rAAV dose is at least 1 x 10 per kilogram of subject body weight to achieve a therapeutic effect. 8 vector genomes (vg / kg) or more, e.g., 1 x 10 per kilogram of subject body weight 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1 x 10 14 In a different embodiment, the rAAV dose ranges from about 5×10 vector genomes (vg / kg) or more. 11 rAAV vg / kg or approximately 5 × 10 11 rAAV over vg / kg; approx. 1×1012 rAAV vg / kg or approximately 1 × 10 12 rAAV vg / kg; approx. 2×10 12 rAAV vg / kg or approximately 2 × 10 12 rAAV vg / kg > approx. 3×10 12 rAAV vg / kg or approximately 3 x 10 12 rAAV vg / kg; approx. 4×10 12 rAAV vg / kg or approximately 4 × 10 12 rAAV over vg / kg; approx. 5×10 12 rAAV vg / kg or approximately 5 × 10 12 rAAV over vg / kg; approx. 1×10 13 rAAV vg / kg or approximately 1 × 10 13 rAAV vg / kg; approx. 2×10 13 rAAV vg / kg or approximately 2 × 10 13 rAAV vg / kg > approx. 3×10 13 rAAV vg / kg or approximately 3 x 10 13 rAAV vg / kg; approx. 4×10 13 rAAV vg / kg or approximately 4 × 10 13 rAAV over vg / kg; approx. 5×10 13 rAAV vg / kg or approximately 5 × 10 13 rAAV vg / kg; approx. 6×10 13 rAAV vg / kg or approximately 6 x 10 13 rAAV > vg / kg.

[0199]

[0223] An example dose range for rAAV vg / kg is approximately 5 x 10 11 ~Approx. 6×10 13 rAAV vg / kg dose range: approximately 5 × 10 11 ~Approx. 5.5×10 11 rAAV vg / kg dose range: approximately 5.5 × 10 11 ~Approx. 6×10 11 rAAV vg / kg dose range: approximately 6 × 10 11 ~Approx. 6.5×10 11 Dose range of rAAV vg / kg: approximately 6.5 × 10 11 ~Approx. 7×10 11 rAAV vg / kg dose range: approximately 7 × 1011 ~Approx. 7.5×10 11 rAAV vg / kg dose range: approximately 7.5 × 10 11 ~Approx. 8×10 11 rAAV vg / kg dose range: approximately 8 × 10 11 ~Approx. 8.5×10 11 rAAV vg / kg dose range: approximately 8.5 × 10 11 ~Approx. 9×10 11 rAAV vg / kg dose range: approximately 9 × 10 11 ~Approx. 9.5×10 11 rAAV vg / kg dose range: approximately 9.5 × 10 11 ~Approx. 1×10 12 rAAV vg / kg dose range: approximately 1 × 10 12 ~Approx. 1.5×10 12 Dose range of rAAV vg / kg: approximately 1.5 x 10 12 ~about 2×10 12 Dose range of rAAV vg / kg: approximately 2 × 10 12 ~Approx. 2.5×10 12 rAAV vg / kg dose range: approximately 2.5 × 10 12 ~Approx. 3×10 12 rAAV vg / kg dose range: approximately 3 × 10 12 ~Approx. 3.5×10 12 rAAV vg / kg dose range: approximately 3.5 × 10 12 ~Approx. 4×10 12 rAAV vg / kg dose range: approximately 4 × 10 12 ~Approx. 4.5×10 12 rAAV vg / kg dose range: approximately 4.5 × 10 12 ~Approx. 5×10 12 rAAV vg / kg dose range: approximately 5 × 10 12 ~Approx. 5.5×10 12 rAAV vg / kg dose range: approximately 5.5 × 10 12 ~Approx. 6×10 12 rAAV vg / kg dose range: approximately 6 × 10 12 ~Approx. 6.5×10 12 Dose range of rAAV vg / kg: approximately 6.5 × 10 12 ~Approx. 7×10 12 rAAV vg / kg dose range: approximately 7 × 10 12~Approx. 7.5×10 12 rAAV vg / kg dose range: approximately 7.5 × 10 12 ~Approx. 8×10 12 rAAV vg / kg dose range: approximately 8 × 10 12 ~Approx. 8.5×10 12 rAAV vg / kg dose range: approximately 8.5 × 10 12 ~Approx. 9×10 12 rAAV vg / kg dose range: approximately 9 × 10 12 ~Approx. 9.5×10 12 rAAV vg / kg dose range: approximately 9.5 × 10 12 ~Approx. 1×10 13 rAAV vg / kg dose range: approximately 1 × 10 13 ~Approx. 1.5×10 13 Dose range of rAAV vg / kg: approximately 1.5 x 10 13 ~about 2×10 13 Dose range of rAAV vg / kg: approximately 2 × 10 13 ~Approx. 2.5×10 13 rAAV vg / kg dose range: approximately 2.5 × 10 13 ~Approx. 3×10 13 rAAV vg / kg dose range: approximately 3 × 10 13 ~Approx. 3.5×10 13 rAAV vg / kg dose range: approximately 3.5 × 10 13 ~Approx. 4×10 13 rAAV vg / kg dose range: approximately 4 × 10 13 ~Approx. 4.5×10 13 rAAV vg / kg dose range: approximately 4.5 × 10 13 ~Approx. 5×10 13 rAAV vg / kg dose range: approximately 5 × 10 13 ~Approx. 5.5×10 13 rAAV vg / kg dose range: approximately 5.5 × 10 13 ~about 6×10 13 rAAV vg / kg dose range: approximately 6 × 10 13 ~Approx. 1×10 14 Dose ranges of rAAV vg / kg are included.

[0200]

[0224] In certain embodiments, the rAAV vg / kg is about 5×1011 vg / kg, approximately 6×10 11 vg / kg, approximately 7×10 11 vg / kg, approx. 8×10 11 vg / kg, approx. 9×10 11 vg / kg, approximately 1×10 12 vg / kg, approx. 2×10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 4×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 6×10 12 vg / kg, approximately 7×10 12 vg / kg, approx. 8×10 12 vg / kg, approx. 9×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 2×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 4×10 13 vg / kg, approx. 5×10 13 vg / kg, or approximately 6 × 10 13 It is administered at a dose of 1000 mg / kg.

[0201]

[0225] In certain embodiments, the doses and dose ranges for other viral vectors are as provided herein for rAAV. For example, in certain embodiments, the doses and dose ranges for recombinant adenoviral vectors, recombinant retroviral vectors (e.g., lentivirus), and recombinant herpes simplex viral vectors are the same as those exemplified above for rAAV.

[0202]

[0226] In certain embodiments, the polynucleotide constructs, viral vectors, and non-viral vectors described herein are administered in combination with an additional compound or treatment for a particular disease of the disorder; and / or in combination with a compound that reduces the immune response generated against the polynucleotide, delivery vehicle, and / or produced protein. The additional compound or treatment can be administered separately; and can be provided in different ways, such as administered prior to, substantially simultaneously with, or subsequent to the administration of the polynucleotide constructs, viral vectors, and non-viral vectors described herein.

[0203]

[0227] In certain embodiments, the administration of the polynucleotide constructs, viral vectors, and non-viral vectors described herein is in combination with an immunosuppressant agent or regimen. Such agents and regimens can be used, if necessary, to achieve immune tolerance or mitigate an immune response to the produced ApoE3-related protein, the provided polynucleotide, or the provided delivery vehicle. Examples of immunosuppressant agents and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (Synthetic Vaccine Particle (SVP)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles)), ImmTOR-IL™ (ImmTOR with a Treg-selective IL-2 agonist), B cell depletion, immunoadsorption, and plasmapheresis.

[0204]

[0228] In certain embodiments, a viral or non-viral vector is administered in combination with one or more immunosuppressive agents, wherein the one or more immunosuppressive agents are administered prior to, substantially simultaneously with, or after administration of the vector or non-viral vector. In certain embodiments, the one or more immunosuppressive agents are administered contemporaneously with the vector or non-viral vector. In certain embodiments, the one or more immunosuppressive agents are administered 1 to 12, 12 to 24, or 24 to 48 hours; or 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 14, 14 to 20, 20 to 25, 25 to 30, 30 to 50 days, or more than 50 days prior to administration of the viral or non-viral vector. In certain embodiments, the one or more immunosuppressive agents are administered 1 to 12, 12 to 24, or 24 to 48 hours after administration of the viral or non-viral vector; or 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 14, 14 to 20, 20 to 25, 25 to 30, 30 to 50 days, or more than 50 days. Administration of the immunosuppressive agent after a period following vector or non-viral vector administration can occur, for example, if there is a decrease in the encoded protein after initial expression levels for a period following vector or non-viral vector administration, e.g., 20 to 25, 25 to 30, 30 to 50, 50 to 75, 75 to 100, 100 to 150, 150 to 200, or more than 200 days.

[0205]

[0229] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid, e.g., a corticosteroid. In certain embodiments, the immunosuppressant is prednisone, prednisolone, a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g., CellCept®, Myfortic®), a CD52 inhibitor (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, belatacept), an anti-CD3 mAb, an anti-LFA-1 mAb (e.g., efalizumab), an anti-CD40 mAb (e.g., ASKP1240), an anti-CD22 mAb (e.g., epratuzumab), an anti-CD20 mAb (e.g., rituximab, orelizumab, ofatumumab, veltuzumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., atacicept), anti-C5 mAb (e.g., eculizumab), mycophenolate ester, 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 mAbs (e.g., anti-IL-6 antibody sirukumab, anti-IL-6 receptor antibody tocilizumab (Actemra®)), IL-10 inhibitors, TGF-β inhibitors, B cell-targeting antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particle (SVP™)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g., ibrutinib), cyclophosphamide, fingolimod, inhibitors of B cell-activating factor (BAFF) (e.g., anti-BAFF mAb, e.g., belimumab), inhibitors of proliferation-inducing ligand (APRIL), anti-IL-1b mAb (e.g., canakinumab (Haris®)), C3a inhibitor, tregitope (see, e.g., U.S. Pat. No. 10,213,496), or combinations and / or derivatives thereof.

[0206]

[0230] Immunosuppressive protocols, including the use of rapamycin alone or in combination with IL-10, can be used to decrease, reduce, 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 through the induction of regulatory T cells (Tregs).

[0207]

[0231] Strategies for reducing (overcoming) or avoiding humoral immunity to viral vectors such as rAAV in systemic gene transfer include administering high vector doses; using AAV empty capsids as decoys to adsorb anti-AAV antibodies; administering immunosuppressants to reduce, reduce, inhibit, prevent, or eradicate humoral immune responses to rAAV; changing the rAAV capsid serotype or genetically engineering the rAAV capsid to make it less susceptible 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 the use of AAV-specific plasmapheresis columns to selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from 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.

[0208]

[0232] Empty capsids used as decoy probes can be provided at different ratios relative to the viral vector. The amount of empty capsid administered can be adjusted 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 to 50:1, about 50:1 to 25:1, 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 empty AAV capsids to rAAV vectors administered 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 capsids is the same as the rAAV serotype.

[0209]

[0233] In certain embodiments, the viral vehicle is delivered using a method that avoids the bloodstream and viral antibodies.Examples of such techniques include delivery to the liver via the hepatic artery; and endoscopic retrograde cholangiopancreatography (ERCP) delivery to the liver.Delivery to the CNS via the carotid artery.Other ductal systems, such as the ducts of the submandibular gland, can also be used as an entry point to deliver viral vectors to subjects who have developed or have existing anti-antibodies to viral vectors.

[0210]

[0234] Additional strategies for reducing humoral immunity to rAAV (which can be applied to other viral vectors) include methods for removing, depleting, capturing, and / or inactivating AAV antibodies, commonly referred to as apheresis, and more specifically, plasmapheresis when blood products are involved. Apheresis or plasmapheresis is a process in which a human subject's plasma is circulated ex vivo through a device that alters the plasma through the addition, removal, and / or replacement of components before returning it to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure can be used to deplete, capture, inactivate, reduce, or remove immunoglobulins (antibodies) that bind to AAV, which may contribute to rAAV neutralization, thereby reducing the titer of AAV antibodies in the treated subject. An example is using a device composed of an AAV capsid affinity matrix column and passing a blood product (e.g., plasma) through the AAV capsid affinity matrix, which provides binding for AAV antibodies of various isotypes (see, e.g., Bertin et al., 2020, Sci. Rep. 10, p. 864, which is incorporated herein by reference in its entirety).

[0211]

[0235] In certain embodiments, the 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 neonatal Fc receptors (FcRn), such as anti-FcRn antibodies, and / or agents that bind to the recombinant viral vector, or to a nucleic acid or polypeptide, protein, or peptide encoded by a polynucleotide encapsidated by the recombinant viral vector, or that reduce circulating antibodies that bind to the polynucleotide, to reduce IgG recirculation and enhance IgG clearance in vivo. In certain embodiments, antibody binding to the viral vector is reduced or inhibited by an agent that reduces the interaction of IgG with FcRn, proteases, or glycosidases.

[0212]

[0236] In certain embodiments, the polynucleotide constructs, viral vectors, and non-viral vectors described herein can be used in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or an endoglycosidase (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. Such treatment can be performed, for example, to reduce or eliminate neutralizing antibodies against the gene delivery vehicle (e.g., viral vector capsid) and enable treatment of patients previously considered ineligible for gene therapy or who develop antibodies resulting from gene therapy. Such strategies are described, for example, in Leborgne et al. (2020) Nat. Med., 26:1096-1101.

[0213]

[0237] In certain embodiments, the subject treatment method is performed in combination with a compound that reduces native ApoE expression in the subject. Native ApoE expression can be inhibited, for example, using an inhibitory nucleic acid that selectively targets the native ApoE coding sequence. Reference to "selectively targeting" the native ApoE sequence indicates that the expression of the ApoE3-related protein encoded by the polynucleotide is not significantly affected. The inhibitory nucleic acid can be provided on the same polynucleotide and / or vector that encodes the ApoE3-related protein, or 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.

[0214]

[0238] In certain embodiments, the expression cassette further comprises an inhibitory nucleic acid that selectively targets one or two of naturally occurring ApoE2-, ApoE3-, and ApoE4-encoding nucleic acids. In further 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, naturally occurring ApoE3 is targeted in conjunction with the use of an ApoE3-related protein comprising the Christchurch mutation.

[0215]

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

[0216]

[0240] VIII. Kit

[0241] The present invention includes kits comprising packaging materials and one or more components therein. The kits typically include a label or package insert, including descriptions or instructions for the components therein for in vitro, in vivo, or ex vivo use. The kits can include a collection of such components, e.g., a viral or non-viral vector, and optionally a second active ingredient, such as another compound, agent, drug, or composition.

[0217]

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

[0218]

[0243] The label or package insert may include the identity of one or more components therein, the clinical pharmacology of the active ingredient(s), including dosage, mechanism of action, pharmacokinetics, and pharmacodynamics. The label or package insert may include information identifying the manufacturer, lot number, location and date of manufacture, and expiration date. The label or package insert may include information regarding the disease for which the kit components can be used. The label or package insert may include instructions to a clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or regimen. The instructions may include dosage, frequency, or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0219]

[0244] The label or package insert can include information about one or more benefits the component may provide, such as a prophylactic or therapeutic benefit. The label or package insert can include information about possible adverse side effects, complications, or reactions, such as a note to the subject or clinician regarding situations in which it is not appropriate to use a particular composition. Adverse side effects or complications could also occur if the subject has taken, will take, or is currently taking one or more other drugs that may be incompatible with the composition, or if the subject has undergone, will be undergoing, or is currently undergoing another treatment protocol or regimen that may be incompatible with the composition; therefore, the instructions could include information about such incompatibilities.

[0220]

[0245] Labels or package inserts include "printed matter," e.g., paper or cardboard, that is separate from or affixed to a component, kit, or packaging material (e.g., a box), or that is attached to the ampoule, test tube, or vial containing the kit component. Labels or package inserts can additionally include computer-readable media, such as bar-coded printed labels, disks, CD or DVD-ROM / RAM, DVDs, optical disks such as MP3s, magnetic tape, or electronic storage media such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, flash media, or memory-type cards.

[0221]

[0246] IX. mRNA therapeutics

[0247] In certain embodiments, RNA versions of the nucleic acids encoding the ApoE-related proteins described herein are provided as mRNA constructs capable of expressing the encoded proteins inside cells. The mRNA constructs include a 5' cap, a 5' UTR, a coding RNA, a 3' UTR, and a poly(A) tail. The UTR and poly(A) tail can serve various functions, such as being involved in mRNA subcellular localization and regulating translation efficiency and mRNA stability. The design and generation of mRNA constructs, including various modifications, are exemplified in various publications, such as Ouranidis et al. (2022) Biomedicines, 10, 50; Qin et al., Signal Transduct Target Ther. (2022) May 21;7(1):166; and U.S. Patent Application Publication No. 2013 / 0259924, each of which is incorporated herein by reference in its entirety.

[0222]

[0248] In certain embodiments, the mRNA construct is delivered to a cell or subject using nanoparticles, examples of which include those provided in Sections VA-VE, supra, Ouranidis et al., (2022) Biomedicines, 10, p. 50, and U.S. Patent Application Publication No. 2013 / 0259924.

[0223]

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

[0224]

[0250] X. Additional Aspects and Embodiments

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

[0225]

[0252] A first aspect is directed to a polynucleotide comprising an ApoE-encoding nucleotide sequence having at least 85% sequence identity to any of SEQ ID NOs: 63-67, or nucleotides 55-951 of any 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 the position corresponding to amino acid 112 of SEQ ID NO: 32 and an arginine at the position corresponding to amino acid 158 of SEQ ID NO: 32; the ApoE-encoding nucleotide sequence optionally comprises one or more introns. The percent identity is determined independently of any introns present in the ApoE-encoding nucleotide sequence.

[0226]

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

[0227]

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

[0228]

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

[0229]

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

[0230]

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

[0231]

[0258] Embodiment E4 further describes the first aspect, embodiments E1a, E1b, E2a, E2b, and E3, wherein the ApoE3-related protein comprises a serine at a position corresponding to amino acid 136 of SEQ ID NO:32.

[0232]

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

[0233]

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

[0234]

[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 ApoE3-encoding nucleic acid sequence further comprises a nucleotide sequence encoding the signal peptide.

[0235]

[0262] Embodiment E8 further describes embodiment E7, wherein the signal peptide has at least 90% identity 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 has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71; or differs from any of SEQ ID NOs: 36, 42, 44, 46, 48, 50, 52, 54, 56, and 68-71 by either 1, 2, or 3 amino acids.

[0236]

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

[0237]

[0264] Embodiment E10a further describes the first aspect, embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, and E9, wherein the ApoE-encoding nucleic acid sequence has at least 90% sequence identity to any of SEQ ID NOs: 3-31. In further embodiments, the ApoE-encoding nucleic acid sequence 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 of SEQ ID NOs: 3-31; or differs from any 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 ID NOs: 11 and 20.

[0238]

[0265] Embodiment E10b further describes the first aspect, embodiments E1a, E1b, E2a, E2b, E3, E4, E5, E6, E7, E8, and E9, in which the ApoE-encoding nucleic acid sequence has at least 90% sequence identity to any of SEQ ID NOs: 95-105 and 124-130. In further embodiments, the ApoE-encoding nucleic acid sequence 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 of SEQ ID NOs: 95-105 and 124-130; or differs from any 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.

[0239]

[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.

[0240]

[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 ApoE-encoding nucleotide sequence comprises: (i) nucleotides 55-951 of any of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, and 27-30; (ii) nucleotides 55-954 of any of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, and 27-30; (iii) nucleotides 1-897 of any of SEQ ID NOs: 63-67; (iv) any of SEQ ID NOs: 63-67; (v) nucleotides 1-951 of any of SEQ ID NOs: 3-31; or (vi) the sequence of any of SEQ ID NOs: 3-31.

[0241]

[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 ApoE-encoding nucleotide sequence comprises: (i) nucleotides 55 to 951 of any of SEQ ID NOs: 95-105 and 124-130; (ii) nucleotides 55 to 954 of any of SEQ ID NOs: 95-105 and 124-130; (iii) nucleotides 1 to 951 of any of SEQ ID NOs: 95-105 and 124-130; or (iv) the sequence of any of SEQ ID NOs: 95-105 and 124-130.

[0242]

[0269] Embodiment 12c is a variant of the present invention, wherein the ApoE-encoding nucleotide sequence is selected from the group consisting of: (i) nucleotides 55-951 of any of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (ii) nucleotides 55-954 of any of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (iii) nucleotides 1-897 of any of SEQ ID NOs: 63-67; (iv) any of SEQ ID NOs: 63-67; (v) SEQ ID NOs: 3-31, 95-105 and 124-130; or (vi) a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any of SEQ ID NOs: 3-31, 95-105, and 124-130; and wherein the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO: 34 or 35.

[0243]

[0270] Embodiment 12d is an embodiment in which the ApoE-encoding nucleotide sequence is, from 5' to 3': (a) a first exon corresponding to nucleotides 1-43 of SEQ ID NO: 1, having at least 85% sequence identity to nucleotides 1-43 of any of SEQ ID NOs: 3-31, 95-105, or 124-130, provided that the 3'-terminal nucleotide of the first exon is G. In further embodiments related 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 of SEQ ID NOs: 3-31, 95-105, and 124-130; in further embodiments, the sequence identity is with respect to nucleotides 1-43 of any 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, having at least 85% sequence identity to nucleotides 44-236 of any of SEQ ID NOs: 3-31, 95-105 and 124-130, provided that the 5'-terminal nucleotide of the second exon is G (in a further embodiment, GC) and the 3'-terminal nucleotide of the second exon is AG. In further embodiments related to 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 to nucleotides 44-236 of any 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 of SEQ ID NOs: 11, 20, 95-105 and 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, having at least 85% sequence identity to nucleotides 237-951 of any of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the 5'-terminal nucleotide of the third exon is G, and the first, second, and third exons together encode an ApoE3-related protein. In further embodiments related to 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 of SEQ ID NOs: 3-31, 95-105, and 124-130; in further embodiments, the sequence identity is with nucleotides 237-951 of any 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 further embodiments, the ApoE-related protein comprises an amino acid sequence at least 96%, at least 97%, at least 98%, 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.

[0244]

[0271] Each of the possibilities involving (a), (b), (c), (d), and (e) can be independently combined for each sequence. For example, (1) the first exon, independently of the second and third exons, 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 1-43 of any of SEQ ID NOs: 3-31, 95-105, and 124-130; (2) the second exon, independently of the first and third exons, can have at least 100% sequence identity to nucleotides 44-236 of any of SEQ ID NOs: 3-31, 95-105, and 124-130. and (3) the third exon, independently of the first and second exons, 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 to 236 of any of SEQ ID NOs: 3 to 31, 95 to 105, and 124 to 130.

[0245]

[0272] Embodiment 12e further describes embodiment 12d, in which 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 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% identity to any of SEQ ID NOs: 119-122.

[0246]

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

[0247]

[0274] Embodiment 12g is 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 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 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 of SEQ ID NOs: 11, 20, and 95-105, and the ApoE-related protein has at least 98% sequence identity to SEQ ID NO: 35; Embodiments 12d, 12e, and 12f are further described.

[0248]

[0275] Embodiment 12h further describes embodiment 12g, in which the ApoE-encoding nucleotide sequence 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 of SEQ ID NOs:106-115.

[0249]

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

[0250]

[0277] Embodiment E13 is an embodiment in which the ApoE-encoding nucleic acid sequence comprises 0-5, 0-10, or 0-15 CpGs; 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, Further described are 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, comprising any of: CpGs at positions 76, 77, 78, 79, 80, 81, and 82; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, or up to about 5.0% CpGs. Preferably, 0 to 10, 0 to 5, or 0 CpG sites.

[0251]

[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, in which the polynucleotide is an expression cassette further comprising one or more expression control elements operably linked to the ApoE-encoding nucleic acid sequence. In further embodiments, one or more expression control elements selected from a promoter, a promoter / enhancer, an intron, a polyadenylation signal, and a Kozak sequence are present. In further embodiments, the expression cassette comprises a promoter operably linked 5' to the ApoE-encoding nucleic acid sequence and a polyadenylation site operably linked 3' to the ApoE-encoding nucleic acid; or the expression cassette comprises a promoter, an intron, a Kozak sequence, the ApoE-encoding nucleic acid sequence and a polyadenylation signal operably linked 5' to 3' to the ApoE-encoding nucleic acid sequence.

[0252]

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

[0253]

[0280] Embodiment E16 further describes embodiments E14 and E15, in which 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.

[0254]

[0281] Embodiment E17 further describes embodiment E14, wherein the promoter confers expression in CNS cells. In a further embodiment, the promoter is any of the CAG promoter, the CBh promoter, the EF1α promoter, or the hSynapsin promoter. In a further embodiment, the expression cassette further comprises an miRNA target sequence for inhibiting dorsal root ganglion cell, liver cell, and / or immune cell expression.

[0255]

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

[0256]

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

[0257]

[0284] Embodiment E20 is an embodiment of the present invention, wherein the expression cassette has 0 to 5, 0 to 10, 0 to 15, 0 to 50, or 0 to 100 CpG positions; 3, 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%, about 11%, about 12%, about 13%, about 14%, or about 15% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.

[0258]

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

[0259]

[0286]

[0023] 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 for viral packaging and / or replication. In further embodiments, the recombinant viral vector nucleic acid is DNA; and / or based on the AAV genome and comprises AAV 5' and 3' ITRs; the recombinant viral vector nucleic acid is DNA and based on the adenovirus genome and comprises 5' and 3' ITRs and a packaging signal; and the recombinant viral vector nucleic acid is RNA and based on a retrovirus genome (e.g., a lentivirus) and comprises 5' and 3' ITRs and a packaging signal. Reference to "based on" a viral genome indicates the ability to replicate and be packaged into the capsid of the referenced virus.

[0260]

[0287] Embodiment E23 further describes E22, wherein the polynucleotide is an rAAV nucleic acid comprising ITRs adjacent to the 5' end of the polynucleotide and / or the 3' end of the polynucleotide. In a further embodiment, the ITRs are adjacent to the 5' and 3' ends of the polynucleotide.

[0261]

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

[0262]

[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.

[0263]

[0290] Embodiment E26 is a variant of the embodiment in which the recombinant viral vector nucleic acid has 0 to 5, 0 to 10, 0 to 15, 0 to 50, 0 to 100, or 0 to 150 CpG positions; , 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, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, CpGs at positions 6, 77, 78, 79, 80, 81, and 82; 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% of CpGs; and / or up to about 0.5%, up to about 1.0%, Further described are E22, E23, E24, and E25, which contain any of up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.

[0264]

[0291] Embodiment E27 is directed to a gene delivery vehicle that is a viral or non-viral vector comprising the polynucleotide of any of the first aspects, E1-E21, or a recombinant viral vector of any of E22-E26.

[0265]

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

[0266]

[0293] Embodiment E29 further describes E28, wherein the viral vector is a rAAV, and wherein the rAAV vector comprises a capsid comprising a VP1, VP2, or VP3 with at least 90% sequence identity to any 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 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 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.

[0267]

[0294] Embodiment E30 further describes E27, wherein the gene delivery vehicle is a non-viral vector. In a further embodiment, the non-viral vector is a nanoparticle; selected from the group consisting of a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipid-polymer nanoparticle (LPNP), a protein- or peptide-based nanoparticle, a DNA dendrimer or DNA-based nanocarrier, a carbon nanotube, a microparticle, a microcapsule, an inorganic nanoparticle, a peptide-cage nanoparticle, and an exosome; an LNP; or an LPNP.

[0268]

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

[0269]

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

[0270]

[0297] A second aspect of the present invention is directed to a method of reducing cholesterol, reducing LDL / VLDL, increasing HDL, or reducing 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, comprising the step of administering to a subject an effective amount of any of the first aspects, any of the polynucleotides E1 to E21, any of the recombinant viral vectors E22 to E26, or any of the gene delivery vehicles E27 to E30, or the pharmaceutical composition of E31 or E32.

[0271]

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

[0272]

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

[0273]

[0300] Embodiment 35 further describes the second aspect, embodiments E33 and E34, in which the method treats or reduces 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 in a subject.

[0274]

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

[0275]

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

[0276]

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

[0277]

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

[0278]

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

[0279]

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

[0280]

[0307] Embodiment 41 further describes a second aspect, embodiments E33, E34, E35, E36, E37, E38, E39a, E39b, and E40, in which 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 a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNAi, a ribozyme, and an antisense RNA.

[0281]

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

[0282]

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

[0283]

[0310] A fourth aspect is directed to the use of any of the first aspect, a polypeptide of any of embodiments E1 to E21, a recombinant viral vector of any of embodiments E22 to E26, a gene delivery vehicle of any of embodiments E27 to E30, or a pharmaceutical composition of embodiment E31 or E32, for the preparation of a medicament for use in medicine or as provided in the second aspect and any of embodiments E33 to E42.

[0284]

[0311] A fifth aspect is directed to an AAV vector genome plasmid comprising the recombinant viral nucleic acid of any of embodiments E22-26.

[0285]

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

[0286]

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

[0287]

[0314] Embodiment 44 is directed to a method of producing an rAAV vector, comprising culturing rAAV-permissive cells comprising the AAV genome plasmid of the sixth aspect or embodiment 43, wherein the rAAV-permissive cells further comprise (a) rep and cap genes provided either as part of the cellular genome and / or by one or more separate plasmids; and (b) helper virus activity provided by the cellular genome and / or by one or more separate plasmids.

[0288]

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

[0289]

[0316] Embodiment 46 further describes embodiment 44, wherein either (a) the rep gene, the cap gene, and the helper 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 helper activity is provided by a helper plasmid.

[0290]

[0317] A seventh aspect is directed to a method of obtaining an rAAV vector, the method comprising: (a) producing rAAV using the method of the sixth aspect or any of embodiments 44-46; and (b) purifying the rAAV.

[0291]

[0318] An eighth aspect is directed 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 of SEQ ID NOs: 119-121, wherein the polynucleotide has 0 to 5 CpGs. Preferably, the polynucleotide is an intron capable of spliceosome-mediated excision from a pre-mRNA transcript.

[0292]

[0319] Embodiment 47 further describes the eighth aspect, wherein the polynucleotide comprises the sequence of any of SEQ ID NOs: 119-121, or a sequence that differs from any of SEQ ID NOs: 119-121 by 1-10 nucleotides, and the polynucleotide has no CpGs. In a further embodiment, the polynucleotide consists of the sequence of any of SEQ ID NOs: 119-121.

[0293]

[0320] XI. Sequence

[0321] Table 1 provides various nucleic acid and amino acid sequences. The sequences shown in bold provide the codons.

[0294]

[0322] In different 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.

[0295]

[0323] [Table 1] JPEG2025533927000008.jpg179149 JPEG2025533927000009.jpg198149 JPEG2025533927000010.jpg198149 JPEG2025533927000011.jpg190149 JPEG2025533927000012.jpg198149 JPEG2025533927000013.jpg198149 JPEG2025533927000014.jpg190149 JPEG2025533927000015.jpg198149 JPEG2025533927000016.jpg212149 JPEG2025533927000017.jpg190149 JPEG2025533927000018.jpg189149 <h2 style=";text-align:left;direction:ltr">JPEG2025533927000019.jpg192149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000020.jpg181149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000021.jpg188149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000022.jpg198149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000023.jpg196149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000024.jpg197149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000025.jpg197149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000026.jpg197149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000027.jpg197149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000028.jpg196149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000029.jpg196149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000030.jpg198149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000031.jpg205149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000032.jpg198149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000033.jpg202149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000034.jpg195149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000035.jpg200149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000036.jpg200149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000037.jpg201149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000038.jpg200149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000039.jpg200149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000040.jpg201149<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533927000041.jpg200149<h2 style=";text-align:left;direction:ltr"> JPEG2025533927000042.jpg200149 JPEG2025533927000043.jpg199149 JPEG2025533927000044.jpg205149 JPEG2025533927000045.jpg205149

[0296] [CROSS-REFERENCE TO RELATED APPLICATIONS]

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

[0297] [Reference to electronically submitted sequence listing]

[0002] The contents of the electronic sequence listing (JP25-5288-XY_SEQL.xml; size: 210,677 bytes; and creation date: June 5, 2025) are incorporated herein by reference in their entirety. [Example]

[0298]

[0324] Examples are provided below that further illustrate various features of the invention and methodologies for practicing the invention. The examples provided do not limit the claimed invention.

[0299]

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

[0326] The effect of ApoE3(ch) and ApoE3 transgene expression on cholesterol was assessed using wild-type C57BL / 6 mice and ApoE knockout (KO) mice and rAAV containing transgenes expressing ApoE3(ch) or ApoE3. The recombinant AAV nucleic acid is composed of a rAAV polynucleotide containing the ApoE / hAAT promoter / enhancer operably coupled to either the ApoE3- or ApoE(ch)-encoding nucleic acid, along with other expression vector components and ITRs, as shown in Figure 1.

[0300]

[0327] Female wild-type C57BL / 6 mice 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 by 6 months of age. Baseline plasma was collected at 8 weeks of age. At 9 weeks of age, mice were intravenously administered 200 mL of vehicle diluent (PBS180 / 0.001% Pluronic F66, pH 7.3) as a control, or 200 mL of diluent containing low or high doses of rAAV (transgene and capsid) via the tail vein. Recombinant AAV containing the ApoE3(ch) transgene was given at a low dose of 3e12vg / kg or a high dose of 1e13vg / kg. Recombinant AAV containing the ApoE3 transgene was given at a low dose of 3e12vg / kg rAAV or a high dose of 6e12vg / 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.

[0301]

[0328] [Table 2]

[0302]

[0329] Plasma was collected weekly for the first 8 weeks after AAV infusion and monthly thereafter for measurement of total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein / very-low-density lipoprotein (LDL / VLDL) cholesterol using the EnzyChrom™ AF HDL and LDL / VLDL Assay Kit (Bioassay Systems, Hayward, CA, USA) according to the manufacturer's instructions, and ApoE3 protein by ELISA (Mabtech Ab, Sweden).

[0303]

[0330] Figures 2A-2E illustrate ApoE3 and ApoE3(ch) transgene expression and the effect of transgene expression on cholesterol in ApoE knockout mice over a 36-week course. Recombinant AAV containing the ApoE(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 rAAV or a high dose of 6e12 vg / kg rAAV. Figure 2A illustrates hAPOE production, Figure 2B illustrates total cholesterol, Figure 2C illustrates HDL, Figure 2D illustrates the LDL / VLDL cholesterol ratio, and Figure 2E illustrates the total / HDL cholesterol ratio. 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 total cholesterol / HDL ratio (p<0.0001; Spearman r=-0.6792), but significantly positively correlated with HDL cholesterol (p<0.0001, Spearman r=0.4584).

[0304]

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

[0332] The effects of various CpG-depleted ApoE(ch) and ApoE constructs on cholesterol were evaluated using wild-type C57BL / 6 mice and ApoE knockout (KO) mice, and rAAV containing various CpG-depleted ApoE3(ch) and ApoE3 transgenes. The recombinant AAV nucleic acid contained the ApoE / hAAT promoter / enhancer operably coupled to either the ApoE3- or ApoE(ch)-encoding nucleic acid, along with other expression vector components and ITRs, as diagrammed in Figure 1.

[0305]

[0333] Female wild-type C57BL / 6 mice 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, mice were intravenously administered via the tail vein with 200 mL of vehicle diluent (PBS180 / 0.001% Pluronic F66, pH 7.3) as a control, or 200 mL of diluent containing 1 e13 vg / kg (2 e11 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.

[0306]

[0334] Plasma was collected at weeks 3 and 6 after rAAV infusion for measurement of total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein / very-low-density lipoprotein (LDL / VLDL) cholesterol using the Enzychrome™ AF HDL and LDL / VLDL Assay Kit (Bioassay Systems, Hayward, CA, USA) and ApoE3 protein by ELISA (Mabtech Ab, Sweden) according to the manufacturer's instructions. Results are shown in Figures 3A-E, 4A-E, 5A-E, and 6A-E. SEQ ID NOs for the various constructs are provided in Table 3.

[0307]

[0335] [Table 3]

[0308]

[0336] Figures 3A-3E illustrate ApoE3(ch) transgene expression from various constructs and the effect of transgene expression on cholesterol at 3 weeks: Figure 3A illustrates hAPOE levels, Figure 3B illustrates total cholesterol, Figure 3C illustrates the LDL / VLDL cholesterol ratio, Figure 3D illustrates HDL cholesterol, and Figure 3E illustrates the total cholesterol / HDL ratio.

[0309]

[0337] Figures 4A-4E illustrate ApoE3(ch) transgene expression from various constructs and the effect of transgene expression on cholesterol at 6 weeks: Figure 4A illustrates hAPOE levels, Figure 4B illustrates total cholesterol, Figure 4C illustrates the LDL / VLDL cholesterol ratio, Figure 4D illustrates HDL cholesterol, and Figure 4E illustrates the total cholesterol / HDL ratio.

[0310]

[0338] Figures 5A-5E illustrate ApoE3 transgene expression from various constructs and the effect of transgene expression on cholesterol at 3 weeks: Figure 5A illustrates hAPOE levels, Figure 5B illustrates total cholesterol, Figure 5C illustrates the LDL / VLDL cholesterol ratio, Figure 5D illustrates HDL cholesterol, and Figure 5E illustrates the total cholesterol / HDL ratio.

[0311]

[0339] Figures 6A-6E illustrate ApoE3 transgene expression from various constructs and the effect of transgene expression on cholesterol at 6 weeks: Figure 6A illustrates hAPOE levels, Figure 6B illustrates total cholesterol, Figure 6C illustrates the LDL / VLDL cholesterol ratio, Figure 6D illustrates HDL cholesterol, and Figure 6E illustrates the total cholesterol / HDL ratio.

[0312]

[0340] Example 3: Liver expression

[0341] Liver tissue samples were obtained from mice treated in Example 2 at 6 weeks, and hAPOE / total protein (FIG. 7A) and vector genome copy number (VCGN) / μg gDNA (FIG. 7B) were determined. hAPOE levels were determined by JESS. VCGN was assessed by qPCR per microgram of gDNA. No immune infiltration was observed in liver tissue by H&E pathology evaluation (data not shown).

[0313]

[0342] Example 4: Atherosclerotic lesions

[0343] The effects of ApoE3(ch) and ApoE3 transgenes on atheromatous lesions were evaluated using wild-type and ApoE knockout mice and rAAV containing transgenes expressing ApoE3(ch) or ApoE3. Aortas from the mice used in Example 1 were evaluated for atheromatous lesions 40 weeks after AAV injection by visualizing and quantifying atheromatous lipid inclusions.

[0314]

[0344] Aortas were harvested 40 weeks after AAV infusion by immersion fixation in paraformaldehyde (4%) for 24 hours and then transferred to a sucrose gradient. Aortas were washed in dPBS equilibrated in 60% isopropanol, and atheromatous lipid inclusions were stained with Oil Red O. Aortas were cut whole-mount in en face preparations for imaging, and representative black-and-white images from different groups are provided in Figure 8. Figure 9 illustrates the quantification of percent lesion area; each point on the graph represents data from a mouse.

[0315]

[0345] Example 5: Anti-inflammatory effects of ApoE3 and ApoE3ch transgene expression

[0346] Mice treated in Example 1 were further evaluated to determine the anti-inflammatory effects of rAAV containing APOE3ch or an 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 obtain a sufficient volume for measuring inflammatory markers using a mouse cytokine panel manufactured by MesoScale Diagnostics (V-PLEX Mouse Cytokine 19-Plex Kit). Six inflammatory plasma proteins were assessed in vehicle-treated APOE KO and WT mice and normalized by APOE3(ch) gene therapy.

[0316]

[0347] The results are shown in Figures 10A-10F. APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 10A illustrates IL-5 levels, Figure 10B illustrates IL-6 levels, Figure 10C illustrates TNF-α levels, Figure 10D illustrates IL-17A / F levels, Figure 10E illustrates CCL2 levels, and Figure 10F illustrates CXCL2 levels. One-way ANOVA, Dunnett's post-hoc test. #p<0.05 vs. WT vehicle, ##p<0.01 vs. KO vehicle. * p<0.05, ** p<0.01.

[0317]

[0348] Example 6: Effect of ApoE3 and ApoE3ch transgene expression on GFAP

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

[0318]

[0350] The results are shown in Figures 11A-11D. APOE3ch-H and APOE3-H refer to KO mice given a high dose of 1e13vg / kg rAAV. APOE3chL and APOE3-L refer to KO mice given a low dose of 3e12vg / kg rAAV. Figure 11A illustrates GFAP / total protein in the cortex as determined by JESS. Figure 11B illustrates GFAP / total protein in the hippocampus as determined by JESS. Figure 11C illustrates the % area of ​​GFAP in the whole brain as determined by immunofluorescence quantification. Figure 11D illustrates the % area of ​​GFAP in the hippocampus as determined by immunofluorescence quantification.

[0319]

[0351] Immunofluorescence staining for the brain inflammatory astroglial marker GFAP was performed using an anti-GFAP antibody (AB5541, Millipore; 1:500), and percent GFAP area quantified using Halo® image analysis software (Indica Labs) demonstrated increased GFAP immunoreactivity throughout the brain and in the hippocampus of APOE KO mice. Elevated GFAP immunoreactivity was reduced in a dose-dependent manner in mice administered APOE3(ch) and APOE3 gene therapy. Similar results were observed with GFAP protein and protein quantification in the cortex and hippocampus.

[0320]

[0352] Example 7: Effects of ApoE3 and ApoE3ch transgene expression on pre- and postsynaptic proteins

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

[0321]

[0354] The results are shown in Figures 12A-12D. APOE3ch(low) and APOE3(low) refer to KO mice given a low dose of 3e12vg / kg rAAV. APOE3ch(high) and APOE3(high) refer to KO mice given a high dose of 1e13vg / kg rAAV. Figure 12A illustrates synaptophysin / total protein in the hippocampus. Figure 12B illustrates PSD-95 / total protein in the hippocampus. Figure 12C illustrates synaptophysin / total protein in the cortex. Figure 12D illustrates PSD-95 / total protein in the cortex.

[0322]

[0355] Example 7: Atheromatous Lesions (9 weeks)

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

[0323]

[0357] [Table 4]

[0324]

[0358] Aortas from the baseline group served as a measure of atherosclerosis before treatment. The remaining mice were treated with rAAV containing native ApoE3 (ApoE3-N) and native ApoE3ch (ApoE3ch-N) transgene sequences and the 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 aortas were harvested, atherosclerotic lesions were stained with Oil Red O, and the % lesion volume / aorta was quantified.

[0325]

[0359] Figures 13A-13D illustrate the quantification of percent lesion area, with each point on the graph representing one mouse. Figure 13A shows the KO mice and KO mice administered different transgene sequences encoding ApoE as follows: E3N (native ApoE3)2e 11 vg / kg, E3-3 (ApoE3-3) 2e 11 vg / kg, and E3-3 (ApoE3-3) 2e 12Figure 13B illustrates the % aortic lesion area in vg / kg of the KO baseline and E3N (native ApoE3)2e mice from Figure 13A. 11 vg / kg group and E3-3 (ApoE3-3) 2e 11 Figure 13C illustrates the KO mice and those administered different transgene sequences encoding the following genes: E3chN (native ApoE3ch)2e 11 vg / kg, E3ch-9 (ApoE3ch-9) 2e 11 vg / kg, and E3ch-9 (ApoE3ch-9) 2e 12 Figure 13D illustrates the % aortic lesion area in vg / kg of KO baseline and E3chN (native ApoE3ch)2e 11 vg / kg and E3ch-9 (ApoE3ch-9) 2e 11 The combination with vg / kg is shown.

[0326]

[0360] All constructs of APOE3(ch) and APOE(ch) gene therapy demonstrated a greater than 17% reduction in existing atherosclerotic lesions compared to the baseline group, supporting the feasibility of reversing atherosclerosis using APOE3(ch) gene therapy.

[0327]

[0361] Example 8: Cognitive Studies

[0362] APOE KO mice have been reported to have cognitive impairments in learning and memory at age 1 or older. One-year-old APOE KO mice (B6.129P2-Apoetm1Unc / J, Jackson Labs, strain #:002052) were tested in the novel object recognition (NOR) memory test (Antunes and Biala G, Cogn Process. 2012 May;13(2):93-110) and found to have significantly impaired recognition memory prior to APOE3 and APOE3ch gene therapy treatment. The test measures memory for a familiar object as measured in preference for a novel object. A time preference for the novel object (>50%) is normal cognitive behavior and indicates intact long-term recognition memory in the mice.

[0328]

[0363] Native ApoE3 (E3 native), native ApoE3ch (E3ch native), 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 listed in Table 4. Figure 14A illustrates the NOR results for C57BL / 6 mice and ApoE knockout mice ( * *p<0.05, unpaired two-tailed t-test). Figure 14B illustrates the NOR results of administering native ApoE and ApoE(ch) sequences. Figure 14C illustrates the NOR results with low-dose rAAV encoding CpG-0 ApoE3 (E3-3) and ApoE(Ech-9) rAAV. Figure 14D illustrates the NOR results with high-dose rAAV encoding CpG-0 ApoE (E3-3) and ApoE3ch (Ech-9) rAAV. NOR memory performance was completely restored to that of age-matched wild-type (WT) mice 5 weeks after a single intravenous administration via the tail vein of two doses (2e11 vg / mouse or 2e12 total vg / mouse) of native APOE3 and APOE3ch sequences and the CpG-0 codon-optimized variants E3-3 and E3ch-9.

[0329]

[0364] Example 9: Additional ApoE3 constructs

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

[0330]

[0366] Figure 15A provides a bar graph showing the performance of codon-optimized CpG-reduced cDNA encoding ApoE3 and the CpG-free construct ApoE3-3. Sequence identities for various constructs and some full CpG constructs also described in this application are provided in Table 5.

[0331]

[0367] [Table 5]

[0332]

[0368] Figure 15B provides a bar graph showing the performance of codon-optimized ApoE3 cDNAs made functional by the addition of intron sequences. Introns were inserted into the signal peptide at codon 15G / GC (site 1) and / or at codon 79AG / G (site 2). Where one intron is shown and no site is given, the intron was inserted at site 1. References to intronic RBP4i, VCLi, and FIXi without a CpG no-notation indicate the wild-type sequence. VCLi-noCpG refers to the intron of SEQ ID NO: 121. RBP4i-noCpG refers to the intron of SEQ ID NO: 120. For comparison purposes, the non-intron-containing codon-optimized ApoE3-3 and H30 variants were included as references. ApoE3 levels were assayed by ELISA and plotted as the mean ± standard deviation.

[0333]

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

Claims

1. 1. A polynucleotide comprising an ApoE-encoding nucleotide sequence having at least 85% sequence identity to any 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, the protein comprising 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 ApoE-encoding nucleotide sequence optionally comprises one or more introns.

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

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

4. 4. The polynucleotide of any one of claims 1 to 3, wherein the ApoE-encoding nucleotide sequence has at least 95% sequence identity to nucleotides 55 to 951 of any 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 to 3, wherein the ApoE-encoding nucleotide sequence has at least 95% sequence identity to any of SEQ ID NOs: 63 to 67.

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

32.

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

33.

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

33.

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

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

12. 12. The polynucleotide of claim 11, wherein the nucleotide sequence encoding the signal peptide has at least 90% identity to any of the sequences 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 to 12, wherein the ApoE-encoding nucleotide sequence comprises a sequence having at least 95% sequence identity to any of SEQ ID NOs: 20, 26, 31, 11, 15, and 95-105.

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

35.

15. 2. The polynucleotide of claim 1, wherein the ApoE-encoding nucleotide sequence comprises: (i) nucleotides 55 to 951 of any 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 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 of SEQ ID NOs: 63-67; (iv) any of SEQ ID NOs: 63-67; (v) nucleotides 1 to 951 of any of SEQ ID NOs: 3-31, 95-105, and 124-140; or (vi) any of SEQ ID NOs: 3-31, 95-105, and 124-130.

16. The ApoE-encoding nucleotide sequence is selected from the group consisting of: (i) nucleotides 55 to 951 of any 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 of SEQ ID NOs: 3-10, 12-14, 16-19, 21-25, 27-30, 95-105, and 124-130; (iii) nucleotides 55 to 954 of any of SEQ ID NOs: 63-67; 2. The polynucleotide of claim 1, wherein the ApoE3-related protein comprises the amino acid sequence of SEQ ID NO: 34 or 35; (iv) any of SEQ ID NOs: 63-67; (v) nucleotides 1-951 of any of SEQ ID NOs: 3-31, 95-105, and 124-130; or (vi) a sequence at least 95% identical to any of SEQ ID NOs: 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 ApoE-encoding nucleotide sequence is, from 5' to 3': (a) a first exon corresponding to nucleotides 1 to 43 of SEQ ID NO: 1, which has at least 85% sequence identity to nucleotides 1 to 43 of any of SEQ ID NOs: 3 to 31, 95 to 105, and 124 to 130, provided that the 3'-terminal nucleotide of the first exon is G; (b) a 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 to 236 of SEQ ID NO: 1, wherein the second exon has at least 85% sequence identity to nucleotides 44 to 236 of any of SEQ ID NOs: 3 to 31, 95 to 105, and 124 to 130, provided that the 5'-terminal nucleotide of the second exon is G and the 3'-terminal 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, having at least 85% sequence identity to nucleotides 237-951 of any of SEQ ID NOs: 3-31, 95-105, and 124-130, provided that the 5'-terminal nucleotide of the third exon is G.

3. The polynucleotide of claim 2, wherein the first, second and third exons together encode the ApoE3-related protein, and the ApoE-related protein comprises an amino acid sequence at least 95% identical to SEQ ID NO:

35.

18. 18. The polynucleotide of claim 17, wherein the first intron comprises a sequence having at least 50% sequence identity to any of SEQ ID NOs: 119-122, and the second intron independently comprises a sequence having at least 50% sequence identity to any of SEQ ID NOs: 119-121.

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

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

35.

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

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

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

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

25. 25. The polynucleotide of any one of claims 1 to 24, wherein the ApoE3-encoding nucleotide sequence comprises 0 to 10 CpG positions.

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

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

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

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

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

58.

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

32. 29. The polynucleotide of claim 27 or 28, wherein the promoter directs expression in a CNS cell.

33. 33. The polynucleotide of claim 32, wherein the expression cassette further comprises one or more miRNA target sequences for inhibiting dorsal root ganglion cell, liver cell, or immune cell expression.

34. 34. The polynucleotide of any one of claims 28 to 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. 35. The polynucleotide of any one of claims 26 to 34, wherein the expression cassette further comprises one or more inhibitory nucleic acids that selectively target ApoE2, ApoE3, or ApoE4 encoding nucleic acids.

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

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

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

39. The polynucleotide of any one of claims 1 to 38, which is DNA.

40. 40. The polynucleotide of any one of claims 26 to 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 has been modified by replacing nucleotides 1295 to 2248 with any 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 a polynucleotide according to any one of claims 26 to 40 and 5' and / or 3' viral elements which provide for viral packaging and / or replication.

42. 42. The recombinant viral vector nucleic acid of claim 41, which is DNA and comprises an adeno-associated virus (AAV) inverted repeat (ITR) adjacent to the 5' end of the polynucleotide and / or an AAV ITR adjacent to the 3' end of the polynucleotide.

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

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

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

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

48. 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. 49. The gene delivery vehicle of claim 48, wherein the viral vector is a recombinant AAV, and the recombinant AAV vector comprises a capsid comprising a VP1, VP2, or VP3 having at least 90% sequence identity to the VP1, VP2, or VP3 of any 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. 50. The gene delivery vehicle 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-B1, AAV-AS, AAV1 / rh.10 capsid; or wherein the capsid comprises VP1 of SEQ ID NO:83 or SEQ ID NO:

84.

51. The gene delivery vehicle of claim 46, which is a non-viral vector.

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

55. 55. The pharmaceutical composition of claim 54, comprising a recombinant AAV and an empty AAV capsid, wherein the ratio of the empty AAV capsid to the recombinant AAV is 100:1 to 1:

100.

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

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

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

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

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

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

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

63. 62. 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. 64. The method of any one of claims 56 to 63, wherein the subject has at least one ApoE4 allele, is an EpoE4 homozygote, or is a PSEN1 mutation carrier.

65. 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. 66. The method of any one of claims 56 to 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 to 45.

68. 68. The AAV genome plasmid of claim 67, which is deficient in the rep and cap genes.

69. 46. ​​A method for producing an rAAV vector, comprising culturing an rAAV producer cell line containing rAAV helper virus activity, wherein the genome of the producer cell contains the recombinant viral vector nucleic acid of any one of claims 41 to 45, a rep gene, and a cap gene, and wherein the rAAV vector is produced.

70. 69. A method for producing an rAAV vector, comprising culturing rAAV-permissive cells containing the AAV genome plasmid of claim 67 or 68, wherein the rAAV-permissive cells further comprise (a) rep and cap genes provided either as part of the cellular genome and / or by one or more separate plasmids, and (b) helper virus activity provided by the cellular genome and / or by one or more separate plasmids.

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

72. 71. The method of claim 70, wherein either (a) the rep gene, the cap gene, and the helper 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 helper activity is provided by a helper plasmid.

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

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

75. 75. The polynucleotide of claim 74, comprising the sequence of any of SEQ ID NOs: 119-121, or a sequence which differs from any of SEQ ID NOs: 119-121 by 1-10 nucleotides, and which has no CpGs.

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

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