Methods for the treatment and prevention of Alzheimer's disease and APOE pharmaceutical compositions

Abstract

This disclosure provides methods and compositions for the treatment of Alzheimer's disease. The methods and compositions of this disclosure include AAV vectors and AAV viral vectors, which include a transgene nucleic acid molecule containing a nucleic acid sequence encoding an APOE2 polypeptide.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of the filing dates of Application No. 63 / 496,910 filed on 18 April 2023, Application No. 63 / 592,123 filed on 20 October 2023, and Application No. 63 / 592,469 filed on 23 October 2023, the disclosures thereof being incorporated herein by reference.

[0002] Submission of sequence listings in XML format The sequence listing XML associated with this application is provided electronically in XML file format and is incorporated herein by reference. The XML file containing the sequence listing is named 1676199WO1.xml. The XML file is 47,801 KB in size, was created on April 17, 2024, and is filed electronically through the USPTO Patent Centre.

[0003] Technical field This disclosure relates to a method for preventing or treating Alzheimer's disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an AAV vector containing a nucleic acid encoding an apolipoprotein (APOE). Methods for generating an AAV viral vector encoding an APOE polypeptide are also provided. [Background technology]

[0004] background There is a long-recognized but unaddressed need for effective therapeutic agents for the treatment or prevention of Alzheimer's disease (AD). An AAV vector encoding an apolipoprotein (APOE) variant having at least one mutation that provides a protective role against the development of Alzheimer's disease is disclosed herein. The AAV vector can be formulated for administration to individuals with Alzheimer's disease or at risk of developing it. Methods for generating APOE AAV vectors are also provided. Common APOE alleles (APOE4, APOE3, APOE2) are by far the primary genetic risk modifiers for AD, with the APOE4 allele increasing risk and lowering age of onset, and the APOE2 allele decreasing risk and significantly delaying age of onset. Extensive human genetic data have shown that APOE4 and APOE2 are codominant. APOE2 / APOE4 heterozygotes do not have a four times higher risk of Alzheimer's disease (AD) than APOE3 / APOE4 heterozygotes; they have the same AD risk as APOE3 / APOE3 homozygotes. APOE4 homozygotes have a significantly increased risk of developing AD (14.5 times higher than APOE3 homozygotes), and also an earlier age of onset (approximately 5 years earlier per APOE4 allele compared to APOE3 homozygotes). 45% to 50% of AD patients carry at least one APOE4 allele, compared to only 15% of healthy controls of the same age. In contrast, APOE2 is a protective allele, reducing AD risk by approximately 50% (1.8 times lower risk) and significantly delaying the age of onset, even in the presence of the APOE4 allele. Therefore, nearly equivalent expression of APOE2 offsets the adverse effects of the E4 allele in humans.

[0005] Amyloid loading is associated with the neuropathology of Alzheimer's disease (AD). Both animal and clinical studies have shown that APOE genotype also predicts the timing and amount of brain Aβ peptide deposition and amyloid loading (APOE4 > APOE3 > APOE2). Amyloid loading is one mechanism by which APOE isoforms exert their effects. Another mechanism is the tau pathway.

[0006] Further gene variants increase the risk of developing Alzheimer's disease (AD). Individuals carrying the dominant PSEN1-E280A gene have been shown to have early onset of amyloid-beta (Aβ) and amyloid accumulation in the central nervous system (CNS).

[0007] Notably, subjects carrying the autosomal dominant PSEN E280A allele had good cognitive function at age 70, well past the age when relatives typically develop cognitive decline. This individual was found to be homozygous for APOE3 but carried the R136S mutation, known as the Christchurch mutation, within APOE3. In vitro experiments showed that APOE3ch behaved like the APOE2 allele; both isoforms did not adequately bind to heparin, the extracellular matrix molecule involved in the transmission and uptake of toxic forms of tau. Therefore, protection from the onset of AD may be associated with the loss of binding to heparan sulfate proteoglycans (HSPGs), which are thought to transmit tau. Thus, the APOE Christchurch mutation suggests a protective role against Alzheimer's disease.

[0008] The methods and compositions disclosed herein can be used to provide an APOE polypeptide having the R136S Christchurch mutation, such as APOE2, for delaying and / or preventing the onset of AD in individuals at risk of developing AD. [Overview of the project]

[0009] overview This disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising, in the 5' to 3' direction, a first AAV ITR sequence including SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:23; an enhancer sequence including SEQ ID NO:3; a promoter sequence including SEQ ID NO:4; a chimeric intron including SEQ ID NO:5; a nucleic acid sequence encoding apolipoprotein 2 (APOE2) polypeptide or apolipoprotein 3 (APOE3) polypeptide containing the Christchurch mutation, including SEQ ID NO:6 or SEQ ID NO:20; a poly(A) sequence including SEQ ID NO:7; and a second ITR sequence including SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:23.

[0010] In some aspects, Christchurch mutations include the R154S mutation related to unprocessed APOE polypeptides; or the R136S mutation related to mature APOE polypeptides lacking a signal peptide.

[0011] In some cases, the rAAV vector contains nucleic acid sequences described in SEQ ID NO:8, SEQ ID NO:21, and SEQ ID NO:22.

[0012] This disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide containing a Christchurch mutation or an apolipoprotein 3 (APOE3) polypeptide containing a Christchurch mutation, wherein the rAAV vector comprises SEQ ID NO:8, SEQ ID NO:21, or SEQ ID NO:22.

[0013] In some cases, rAAV vectors are packaged as rAAV viral vectors containing the AAV capsid protein.

[0014] In some contexts, the AAV capsid protein is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, or AAVrh10. In some contexts, the AAV capsid protein is AAVrh10.

[0015] This disclosure provides a pharmaceutical composition comprising an AAV virus vector in any aspect thereof.

[0016] This disclosure provides a method for treating Alzheimer's disease in a human subject, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to any aspect of this disclosure.

[0017] In some cases, the therapeutically effective dose of the vector is approximately 1 × 10⁻⁶. 11 ~Approx. 1×10 16 It is a genome copy.

[0018] In some cases, the target organisms are APOE2 / APOE4 heterozygotes, APOE4 / APOE4 homozygotes, or APOE3 / APOE4 heterozygotes.

[0019] In some cases, the composition is administered systemically, intracisionally, via the cisterna magna, or via CI-C2 administration.

[0020] In some aspects, the pharmaceutical composition is approximately 5.0 × 10 9 gc / mL CSF ~ approx. 5.0×10 12 It is administered at a dose of gc / mL CSF. In some cases, the pharmaceutical composition is (i) approximately 1.4 × 10⁻⁶ 10 gc / mL CSF, (ii) approximately 4.4×10 10gc / mL CSF, or (iii) approximately 1.4 × 10 11 It is administered at a dose of gc / mL CSF. In some cases, the pharmaceutical composition is approximately 1.4 × 10⁻⁶. 14 It is administered at the GC dose.

[0021] In some cases, the pharmaceutical composition is administered in a total volume of approximately 5 mL, 10 mL, 15 mL, or 20 mL.

[0022] In some phases, subjects experience an increase of at least approximately 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the expression of APOE2 Christchurch or APOE3 Christchurch.

[0023] In some contexts, the expression of APOE2 Christchurch or APOE3 Christchurch occurs in the central nervous system. In other contexts, the expression of APOE2 Christchurch or APOE3 Christchurch is measured in the cerebrospinal fluid (CSF).

[0024] In some aspects, after administration of the pharmaceutical composition, the expression level of at least one of total tau (T-tau) and phosphorylated tau (P-tau) is reduced in the subjects compared to baseline before administration.

[0025] In some phases, T-tau and / or P-tau expression levels are reduced by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100%.

[0026] In some situations, after administration of the pharmaceutical composition, the amyloid beta 42 / amyloid beta 40 (Aβ 42 / 40 ) ratio increases.

[0027] In some situations, the Aβ 42 / 40 ratio increases by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100%.

[0028] In some situations, an immunosuppressant is administered to the subject prior to treatment with the pharmaceutical composition. In some situations, the immunosuppressant is prednisone.

[0029] In some situations, prednisone is administered once daily at 40 mg one week before administration of the AAV viral vector; once daily at 40 mg from week 1 to week 2 after administration of the AAV viral vector; once daily at 30 mg during week 3 after administration of the AAV viral vector; once daily at 20 mg during week 4 after administration of the AAV viral vector; once daily at 10 mg during week 5 after administration of the AAV viral vector; once daily at 5 mg during week 6 after administration of the AAV viral vector; once daily at 2.5 mg during week 7 after administration of the AAV viral vector; and once every other day at 2.5 mg during week 8 after administration of the AAV viral vector.

[0030] [[ID=2Q]]Any of the above situations, or any other situation described herein, can be combined with any other situation.

[0031] It should be noted that there seems to be a typo in the original text where [[ID=2Q]] is likely meant to be . This has been left as is in the translation to maintain consistency with the provided text.Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. Herein, the singular includes the plural unless otherwise specified in the context; for example, the terms “a,” “an,” and “the” are understood to be singular or plural, and the term “or” is understood to be inclusive. For example, “one element” means one or more elements.

[0032] Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. References cited herein are not considered prior art to the claimed invention. In case of any conflict, including definitions, this specification shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit. Other features and advantages of this disclosure are evident from the detailed description and claims below. [Brief explanation of the drawing]

[0033] [Figure 1A] This is a table showing APOE variants. The Alzheimer's disease risk region of APOE (amino acids 112-158) includes APOE3 (average risk), APOE4 (high risk), and APOE2 (low risk). Sequences of the Columbian APOE3Ch variant and the therapeutic gain-of-function APOE2Ch variant are also shown, compared to the APOE2 variant in amyloid and tau mouse models. [Figure 1B]This graph shows the heparin binding of APOE variant, APOE2, APOE3, APOE4, APOE2 Christchurch, and APOE3 Christchurch. The concentration of APOE variant is shown on the left y-axis. The concentration of sodium chloride (NaCl) is shown on the right y-axis. The fraction number is shown on the x-axis, with smaller fraction numbers correlated to weaker heparin binding and larger fraction numbers correlated to stronger heparin binding. [Figure 1C] This document presents an experimental design for comparing AAVrh.10hAPOE2 versus AAVrh.10hAPOE2Ch using AAVrh.10 null and PBS control treatments in APP.PSEN1 / TRE4 ("amyloid") and P301S.PSEN1 / TRE4 ("tau") mice. [Figure 2-1] Figures 2A–2E show a series of graphs illustrating the levels of viral genome copies (Figures 2A and 2B), APOE mRNA (Figures 2C and 2D), and APOE protein (Figures 2E and 2F) after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-null), APOE2 (AAV-Rh10-E2), APOE2 + hemagglutinin (HA) tag (AAV-Rh10-E2HA), APOE2 Christchurch (AAV-Rh10-E2CH), or APOE2 Christchurch + HA tag (AAV-Rh10-E2CHHA) to the hippocampus of amyloid (Figures 2A, 2C, and 2E) and tau (Figures 2B, 2D, and 2F) mice. The y-axis represents viral genome copies per microgram of DNA. Data are expressed as geometric mean ± SD. The number of mice ("n") used in each analysis is shown below each bar. [Figure 2-2] See the explanation in Figure 2-1. [Figure 2-3] See the explanation in Figure 2-1. [Figure 3-1]Figures 3A–3D show a series of graphs illustrating the levels of Aβ42 (Figure 3A), Aβ40 (Figure 3B), total tau (Figure 3C), and total tau (Figure 3D) detected in the hippocampus of amyloid and tau mice treated with AAVrh10 viral capsids encoding null (AAV-Rh10-null), APOE2 (AAV-Rh10-E2), or APOE2 Christchurch (AAV-Rh10-E2CH). The y-axis represents the APOE level, expressed as ng of APOE per microgram of total protein. Data are expressed as geometric mean ± SD. The number of mice "n" for each analysis is shown below each bar. [Figure 3-2] See the explanation in Figure 3-1. [Figure 4-1] Figures 4A–4T show histological parameters of β-amyloid, X-34, Iba-1, and GFAP in amyloid mice treated with AAVrh.10hE2 and AAVrh.10hAPOE2Ch, compared to AAVrh.10 null and PBS control groups. Figures 4A–4D, 4F–4I, 4K–4N, and 4P–4S are representative images. Figures 4E (amyloid, Aβ aggregates), 4J (X-34, amyloid fibrils), 4O (Iba-1, microglia activation), and 4T (GFAP, astroglial activation) are quantitative histochemical analyses. The analyses were performed 3 months after therapy. Data are shown as ± standard error; the number of mice "n" for each analysis is listed. For β-amyloid, Iba-1, and GFAP, specific staining for neurological markers in the CA-1 region of the hippocampus is shown in red. Nuclei stained with DAPI are shown in blue. The dentate gyrus (a high-density cluster of nuclei) is visible in the lower right corner of each panel. For X34 staining, a monochrome image of the CA-1 region of the hippocampus is shown. Red indicates high-intensity staining (staining above the fixed intensity threshold). In the X34 panel, the nuclei of the dentate gyrus are visible as unstained ovals in the lower right corner of the field of view. Bar = 100 μm. [Figure 4-2] See the explanation in Figure 4-1. [Figure 4-3] See the explanation in Figure 4-1. [Figure 4-4] See the explanation in Figure 4-1. [Figure 5-1] Figures 5A–5O show the histological parameters AT8, Iba-1, and GFAP in tau mice treated with AAVrh.10hAPOE2 and AAVrh.10hAPOE2Ch compared to AAVrh.10 null and PBS control treatments. Figures 5A–5D, 5F–5I, and 5K–5N are representative images. Figures 5E (AT8, tau loading, and aggregates), 5J (Iba-1, microglia activation), and 5O (GFAP, astroglial activation) are quantitative histochemical analyses. The analyses were performed 3 months after therapy. Data are shown as ± standard error; the number of mice "n" for each analysis is listed. For AT8, Iba-1, and GFAP, specific staining for neurological markers in the CA-1 region of the hippocampus is shown in red. Nuclei stained with DAPI are shown in blue. The dentate gyrus is visible in the lower right corner of each panel. Bar = 100 μm. [Figure 5-2] See the explanation in Figure 5-1. [Figure 5-3] See the explanation in Figure 5-1. [Figure 6-1] Figures 6A–6H show the assessment of neurological integrity and hippocampus-dependent memory in amyloid mice and tau mice using nesting, Y-maze, novel object recognition, and Burns maze tests. Evaluation of AAVrh.10hAPOE2Ch(E2CH), AAVrh.10hAPOE2(E2), AAVrh.10 null, and PBS hippocampal treatment in amyloid mice and tau mice in their performance on nesting (Figure 6A), nesting (Figure 6B), Y-maze (Figure 6C), Y-maze (Figure 6D), novel object recognition (Figure 6E), novel object recognition (Figure 6F), Burns maze (Figure 6G), and Burns maze (Figure 6H). Analysis of amyloid mice was performed at 5.5–6 months of age (3 months after therapy) for amyloid mice and at 8.5–9 months of age (3 months after therapy) for tau mice. Data are shown as ± standard error; the number of mice (n) for each analysis is listed. [Figure 6-2] See the explanation in Figure 6-1. [Figure 6-3] See the explanation in Figure 6-1. [Figure 6-4] See the explanation in Figure 6-1. [Figure 7-1] Figures 7A–7D show the total number of Y-maze entries and alternations evaluated in all experimental groups in both amyloid and tau mice. For each analysis, treatment with AAVrh.10hAPOE2Ch(E2CH) and AAVrh.10hAPOE2(E2) is compared to AAVrh.10 null (null) and PBS controls. Figure 7A shows the total number of entries in amyloid mice; Figure 7B shows the total number of alternations in amyloid mice; Figure 7C shows the total number of entries in tau mice; Figure 7D shows the total number of alternations in tau mice. All data are expressed as mean ± SEM. The number of mice for each analysis is listed. [Figure 7-2] See the explanation in Figure 7-1. [Figure 8] Figures 8A and 8B show the escape latency during the training period of the Burns maze test in all experimental groups, including AAVrh.10hAPOE2Ch(E2CH) and AAVrh.10hAPOE2(E2), compared to AAVrh.10 null (null) and PBS control. Figure 8A: Burns maze escape latency over a 7-day training period for amyloid mice; and Figure 8B: Burns maze escape latency over a 7-day training period for tau mice. Data are shown mean ± SEM. In the amyloid mice in Figure 8A, improvement in learning ability was observed for both E2 and E2CH, while in the tau mice in Figure B, only the E2CH treatment followed the same pattern. All data are expressed as mean ± SEM. The number of mice is detailed in Figures 6A-6H. [Figure 9A]Figures 9A and 9B show quantitative assessments of neurodegeneration by immunohistochemical examination in the hippocampus of both amyloid and tau mice. Figure 9A is a graph showing that the ratio of NeuN (neuron marker) staining to DAPI (nucleus) represents neuronal viability. Figure 9B is a graph showing the intensity of Olig2 (oligodendrocyte marker) staining, quantified with QuPath software and normalized with DAPI staining. This staining provides a measure of myelin integrity / neuronal health, which has been shown to correlate strongly with neurodegeneration. Each dot represents a different animal within the same treatment group and consists of the mean of three independent stained sections. Columns and error bars represent mean ± SEM. [Figure 9B] See the explanation in Figure 9A. [Figure 10A] Figures 10A and 10B show the assessment of neuronal health using spatial transcriptomics. Differential gene expression (DEG) in the hippocampus and cortex was evaluated using spatial transcriptomics for representative samples from each cohort of amyloid and tau mice. Transcripts associated with neuronal health were represented using two classifications: synaptic / neuronal integrity markers and neurogenesis markers. Each dot represents the mean expression change compared to the PBS group in Figures 10A (amyloid mice) and 10B (tau mice). [Figure 10B] See the explanation in Figure 10A. [Figure 11A-1] Figures 11A and 11B show RTqPCR evaluations of microglial phenotypes. mRNA expression levels of CD68, Clec7a, Trem2, CD163, CD206, and Arg1 were evaluated in the hippocampus for each cohort of amyloid and tau mice. Figure 11A shows amyloid mice, and Figure 11B shows tau mice. Each dot represents a different animal, and the error bars represent the geometric mean ± geometric standard deviation. [Figure 11A-2] Refer to the explanation in Figure 11A-1. [Figure 11B-1] Refer to the explanation in Figure 11A-1. [Figure 11B-2]Refer to the explanation in Figure 11A-1. [Figure 12A] Figures 12A–12D show the phenotypes of microglia in amyloid mice and tau mice as evaluated by spatial transcriptomics. Figures 12A (amyloid mice) and 12B (tau mice) show the relative expression levels of inflammatory and anti-inflammatory genes by each of the three categories of genes: anti-inflammatory, DAM activation, and homeostasis. Each dot represents the mean expression change compared to the PBS group. Figures 12C (amyloid mice) and 12D (tau mice) show individual genes from Figures 12A and 12B, separated into a similar ontology, with horizontal bars indicating whether the mean expression of these groups for each experimental cohort was upregulated or downregulated. [Figure 12B] See the explanation in Figure 12A. [Figure 12C] See the explanation in Figure 12A. [Figure 12D] See the explanation in Figure 12A. [Figure 13A] Figures 13A and 13B show serum total cholesterol and triglyceride levels in amyloid and tau mice. Levels were measured in n=4 mice from each treatment group. Figure 13A (amyloid mice) and Figure 13B (tau mice). The reference range shows the homeostasis level of wild-type C57BL / 6 mice. Error bars represent mean ± SEM values. Post-hoc pairwise comparisons using P-value ANOVA and Tukey correction for all comparisons excluded triglycerides in amyloid mice, which did not follow a normal distribution and were evaluated by the Kruskal-Wallis test with the post-hoc Dunn test. [Figure 13B] See the explanation in Figure 13A. [Figure 14]This graph shows the sensitivity of APOE ELISA for detecting different APOE variants. 293 cells were transfected with pAAV-hAPOE2, pAAV-hAPOE4, and pAAV-hAPOE2Ch, grown in serum-free medium for 72 hours, and then the medium was collected. Triple ELISA measurements were performed with three different dilutions. The horizontal line represents the mean value for each dilution relative to each treatment. [Figure 15-1] Figures 15A-15D show the evaluation of disease pathogenesis in naive amyloid and tau mice at the age of vector administration. Figure 15A shows immunohistochemical staining for amyloid beta in brain sections of amyloid mice, which shows a positive signal in the cortex and hippocampus at 2.5 months of age at administration, indicated by yellow asterisks, and the positive signal increased up to 5.5 months in control mice; Figure 15B shows time-dependent changes in soluble and insoluble Aβ42 and Aβ40 by ELISA quantification in hippocampal lysates of control mice; Figure 15C shows immunofluorescence staining for tau tangle in the hippocampal region of tau mice (red = AT8, blue = DAPI), indicated by yellow asterisks, which was observed at 5.5 months of age at administration and increased up to 8.5 months in control mice; Figure 15D shows the levels of total tau and phosphate tau assessed by ELISA in hippocampal lysates of control tau mice at 5.5 and 8.5 months of age, which show a similar pattern to AT8 staining. Each dot represents a different animal in the associated cohort. [Figure 15-2] See the explanation in Figure 15-1. [Figure 16-1]Figures 16A–16C show the effects of treatment on lipid-related transcripts in amyloid and tau mice. Spatial transcriptomics analysis was performed on n=1 sections from each treatment group in each cohort to evaluate the transcription of lipid-related genes across the entire section. Figure 16A is a heatmap of relative expression levels for all lipid-related transcripts. Each row represents a different transcript matrixed by the experimental group; Figures 16B (amyloid mice) and 16C (tau mice) show gene ontology (GO) analysis of lipid-related transcripts differentially expressed in the E2 and E2Ch groups compared to PBS. Each bar represents the relevant GO term plotted against the enrichment analyzed by Fisher's exact test. [Figure 16-2] See the explanation in Figure 16-1. [Modes for carrying out the invention]

[0034] Detailed explanation This disclosure provides a method for treating or preventing Alzheimer's disease in a subject, comprising the step of administering an adeno-associated virus (AAV) vector containing an apolipoprotein (APOE) polypeptide having a Christchurch mutation. In some aspects, the subject is an APOE2 / APOE4 heterozygote, an APOE4 / APOE4 homozygote, an APOE3 / APOE3 homozygote, or an APOE3 / APOE4 heterozygote. In some aspects, the method comprises the step of administering a therapeutically effective dose of a recombinant adeno-associated virus (rAAV) vector containing a nucleic acid sequence encoding an APOE polypeptide having a Christchurch mutation or a fragment thereof to the subject. In some aspects, the APOE Christchurch mutation is R136S. In some aspects, the APOE polypeptide having a Christchurch mutation is an APOE2 polypeptide or an APOE3 polypeptide. In some aspects, the vector is 5.0 × 10⁻⁶ 9 Genome copy (gc) / mL CSF ~ approximately 5.0 × 10⁻⁶ 12The vector is administered in therapeutically effective doses within the gc / mL CSF range. In some cases, the vector is administered via C1-C2 administration or intracisor macrocentromatic (ICM) administration.

[0035] AAV Vector In some aspects, an isolated nucleic acid sequence containing a nucleic acid sequence encoding an apolipoprotein polypeptide with the Christchurch mutation can be a recombinant AAV vector (rAAV vector).

[0036] As used herein, “rAAV vector” refers to a vector comprising, essentially consisting of, one or more transgene sequences and one or more AAV inverted terminal repeats (ITRs). In some aspects, an rAAV vector comprises one or more enhancers, promoters, at least one nucleic acid capable of encoding at least one protein, intron sequences, and polyA sequences.

[0037] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the apolipoprotein 2 (APOE2) Christchurch mutation for use in the treatment or prevention of Alzheimer's disease in subjects where such treatment is needed.

[0038] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the APOE2 Christchurch mutation for use in the treatment of Alzheimer's disease in subjects where such treatment is necessary.

[0039] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the APOE2 Christchurch mutation for reversing or stabilizing symptoms of Alzheimer's disease in subjects where this is necessary.

[0040] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the APOE2 Christchurch mutation for improving symptoms of Alzheimer's disease in subjects where such improvement is needed.

[0041] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the apolipoprotein 3 (APOE) Christchurch mutation for use in the treatment or prevention of Alzheimer's disease in subjects where such treatment is needed.

[0042] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the APOE3 Christchurch mutation for use in the treatment of Alzheimer's disease in subjects where such treatment is necessary.

[0043] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the APOE3 Christchurch mutation for reversing or stabilizing symptoms of Alzheimer's disease in subjects where such reversal is necessary.

[0044] In some aspects, this disclosure relates to rAAV vectors containing nucleic acids encoding the APOE3 Christchurch mutation for improving symptoms of Alzheimer's disease in subjects where such improvement is needed.

[0045] In some aspects, the rAAV vector used in the methods of the present disclosure comprises, in the 5' to 3' direction, a first AAV ITR sequence; an enhancer sequence; a promoter sequence; a chimeric intron sequence; a nucleic acid sequence encoding an APOE2 polypeptide containing the Christchurch variant (APOE2 Christchurch variant or APOE2 Christchurch); a poly(A) sequence; and a second ITR sequence. In some aspects, the AAV vector of the present disclosure encoding the APOE2 Christchurch variant may contain, be essentially, a nucleic acid sequence, or may consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 8. In some aspects, the AAV vectors of this disclosure encoding the APOE2 Christchurch variant may contain, essentially consist of, or comprise a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:22.

[0046] In some aspects, the rAAV vector used in the methods of the present disclosure comprises, in the 5' to 3' direction, a first AAV ITR sequence; an enhancer sequence; a promoter sequence; a chimeric intron sequence; a nucleic acid sequence encoding an APOE3 polypeptide containing a Christchurch variant (APOE3 Christchurch variant or APOE3 Christchurch); a poly(A) sequence; and a second ITR sequence. In some aspects, the AAV vector of the present disclosure encoding an APOE3 Christchurch variant may contain, essentially consist of, or comprise a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:21.

[0047] In some aspects, the rAAV vector used in the methods of the present disclosure comprises, in the 5' to 3' direction, a first AAV ITR sequence; an enhancer sequence; a promoter sequence; a chimeric intron sequence; a nucleic acid sequence encoding an APOE3 polypeptide containing a Christchurch variant (APOE3 Christchurch variant or APOE3 Christchurch); a poly(A) sequence; and a second ITR sequence. In some aspects, the AAV vector of the present disclosure encoding an APOE3 Christchurch variant may contain, essentially consist of, or comprise a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:24 or SEQ ID NO:25.

[0048] In some aspects, the enhancer sequence may be essentially from or consist of a human cytomegalovirus (CMV) enhancer sequence, which may include such enhancer sequence. The CMV enhancer sequence may be essentially from or consist of a nucleic acid sequence, which may include a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:3.

[0049] In some aspects, the promoter sequence may be essentially derived from or consist of the chicken β-actin promoter sequence, which may include the chicken β-actin promoter sequence. The chicken β-actin promoter sequence may be essentially derived from or consist of the nucleic acid sequence, which may include the nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:4.

[0050] In some aspects, the chimeric intron sequence may be essentially derived from or comprise a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:5.

[0051] In some aspects, the polyA sequence may essentially consist of, or comprise, a polyA sequence that may include, a β-globin polyA sequence. The β-globin polyA sequence may essentially consist of, or comprise, a nucleic acid sequence that may include, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:7.

[0052] Nucleic acid sequence encoding the APOE2 polypeptide Apolipoproteins (APOEs) are proteins involved in fat metabolism in the body. They belong to a family of proteins that bind to fat and interact with low-density lipoprotein receptors (LDLRs) for the normal processing of triglyceride-rich lipoproteins. In peripheral tissues, APOEs are produced in the liver and macrophages, mediating cholesterol metabolism. In the central nervous system, APOEs are produced by astrocytes, transporting cholesterol to neurons via APO receptors, which are members of the LDLR family.

[0053] APOE is synthesized as a 317-amino acid protein, which is processed in cells, cleaving the 18-amino acid signal peptide (SEQ ID NO: 19) at the N-terminus to produce a mature protein of 299 amino acids. There are three main variants of APOE: APOE2, APOE3, and APOE4. The three variants differ from each other at two locations: residue 112 (130 residues including the signal peptide) and residue 158 (176 residues including the signal peptide). The amino acid differences of the three APOE variants are listed in Table 1. Table 1 also shows the prevalence and relative risk of developing AD in the world population.

[0054] APOE2 is characterized by having cysteine ​​at position 112 (residue 130 including the signal peptide) and cysteine ​​at position 158 (residue 176 including the signal peptide). APOE3 is characterized by having cysteine ​​at position 112 (residue 130 including the signal peptide) and arginine at position 158 (residue 176 including the signal peptide). APOE4 is characterized by having arginine at position 112 (residue 130 including the signal peptide) and arginine at position 158 (residue 176 including the signal peptide).

[0055] Previous studies have identified a further variant of APOE3 in which an arginine-to-serine mutation (R136S) at position 136 is present. This mutation is called the Christchurch mutation. In some contexts, the Christchurch mutation may refer to the R154S mutation when referring to an unprocessed APOE polypeptide that still contains the signal peptide. The Christchurch mutation may be inserted into other APOE variants disclosed herein, such as APOE2 or APOE4. In some contexts, the APOE2 polypeptide may be modified herein to also contain the R136S (R154S) Christchurch mutation.

[0056] (Table 1) Overview of APOE variants: APOE2, APOE3, and APOE4 TIFF2026516699000001.tif53147

[0057] In some aspects, the APOE2 polypeptide containing the signal peptide may contain, essentially consist of, or comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:9.

[0058] In some aspects, a mature APOE2 polypeptide lacking a signal peptide can be essentially derived from, or consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:14.

[0059] In some aspects, an APOE3 polypeptide containing a signal peptide may contain, essentially consist of, or comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:10.

[0060] In some aspects, a mature APOE3 polypeptide lacking a signal peptide can be essentially derived from, or consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:15.

[0061] In some aspects, an APOE4 polypeptide containing a signal peptide may contain, essentially consist of, or comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:13.

[0062] In some aspects, a mature APOE4 polypeptide lacking a signal peptide can be essentially derived from, or consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:16.

[0063] In some aspects, the APOE3 Christchurch mutant polypeptide containing the signal peptide may contain, essentially consist of, or be composed of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:12.

[0064] In some aspects, a mature APOE3 Christchurch mutant polypeptide lacking a signal peptide can be essentially derived from, or consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:18.

[0065] In some aspects, the APOE2 Christchurch mutant polypeptide containing the signal peptide may contain, essentially consist of, or be composed of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:11.

[0066] In some aspects, a mature APOE2 Christchurch mutant polypeptide lacking a signal peptide can be essentially derived from, or consist of, an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:17.

[0067] In some aspects, the nucleic acid sequence encoding the apolipoprotein 2 (APOE2) Christchurch mutant polypeptide may include, or may be essentially composed of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:6.

[0068] In some aspects, the nucleic acid sequence encoding the apolipoprotein 3 (APOE3) Christchurch mutant polypeptide may be essentially derived from, or consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:20.

[0069] In some aspects, this disclosure provides nucleic acid sequences comprising SEQ ID NO:8 or SEQ ID NO:21 or variants thereof for treating Alzheimer's disease.

[0070] Inverted terminal repeat sequence In some cases, the first ITR may include, essentially be, or consist of, nucleic acid sequences or their complements that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:1. In some cases, the first ITR may include, essentially be, or consist of nucleic acid sequences or their complements that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:2. In some aspects, the first ITR may include, essentially consist of, or be composed of nucleic acid sequences or their complements that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:23.

[0071] In some aspects, the second ITR may include, essentially be, or consist of, nucleic acid sequences or their complements that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:2.

[0072] In some cases, the second ITR may include, be essentially derived from, or consist of nucleic acid sequences or their complements that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:1. In some cases, the first ITR may include, be essentially derived from, or consist of nucleic acid sequences or their complements that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:23.

[0073] AAV virus vector The AAV vectors described herein may be packaged as AAV viral vectors.

[0074] The term "rAAV viral vector" refers to a viral particle consisting of at least one rAAV capsid protein and a capsidized polynucleotide AAV vector. Therefore, the production of an rAAV viral vector necessarily includes the production of an rAAV vector. The term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to capsidate, protect, transport, and release the viral genome into host cells. The capsid is generally composed of an oligomeric structural subunit of the protein ("capsid protein"). As used herein, the term "capsidized" means enclosed within a viral capsid. The viral capsid of AAV consists of a mixture of three viral capsid proteins: VP1, VP2, and VP3.

[0075] rAAV viral vectors useful for implementing this disclosure can be constructed by utilizing methodologies well known in the field of molecular biology. Typically, an AAV viral vector containing a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements necessary for the production of a viral protein that mediates cell transduction.

[0076] The term "gene transfer" or "gene delivery" refers to a method or system for precisely inserting foreign DNA into a host cell. Such methods can result in transient expression of unintegrated transfer DNA, extrachromosomal replication and expression of transfer replicons (e.g., episomes), or integration of transfer genetic material into the host cell's genomic DNA.

[0077] Examples of viral vectors include, but are not limited to, adenovirus vectors, retrovirus vectors, lentivirus vectors, herpesvirus vectors, and adeno-associated virus (AAV) vectors.

[0078] Such recombinant viruses can be generated by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Representative examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for generating such replication-deficient recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056, and WO94 / 19478.

[0079] In one embodiment, an adeno-associated virus (AAV) vector is used.

[0080] In other embodiments, the rAAV vector is AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or any other serotype of AAV that can infect humans, monkeys, or other species.

[0081] In an exemplary embodiment, the rAAV vector is AAVrh10.

[0082] The term "rAAV vector" non-limitingly refers to a vector derived from an adeno-associated virus serotype, including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc. An AAV vector may have one or more AAV wild-type genes, either entirely or partially deleted, such as the rep and / or cap genes, but retain a functional adjacent ITR sequence. The functional ITR sequence is necessary for the rescue, replication, and packaging of the AAV virion. Therefore, an AAV vector is defined herein as containing at least a sequence desired in cis for viral replication and packaging (e.g., a functional ITR). The ITR does not need to be a wild-type nucleotide sequence and may be altered by nucleotide insertions, deletions, or substitutions, as long as it provides functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to provide at least a regulatory element containing a transcription start region, the DNA of interest (i.e., the APOE2 gene), and a transcription termination region as components functionally linked in the direction of transcription.

[0083] The regulatory elements are selected to be functional in mammalian cells. The resulting construct, containing functionally linked components, is surrounded (at 5' and 3') by functional AAV ITR sequences. “Adeno-associated virus inverted terminal sequences” or “AAV ITR” means a region recognized in the art, found at each end of the AAV genome, that functions together in cis as a starting point for DNA replication and as a packaging signal for the virus. AAV ITRs, along with AAV rep coding regions, efficiently provide excision and rescue of nucleotide sequences inserted between two adjacent ITRs, as well as their incorporation into the mammalian cell genome. In some aspects, the ITR sequences of this disclosure may contain deletions of one or more nucleotides at one or more positions in the ITR sequence. In some aspects, the deleted nucleotides in the ITR sequence may be repaired in vivo or during vector replication. Nucleotide sequences of AAV ITR regions are known. For AAV2 sequences, see, for example, Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (BN Fields and DM Knipe, eds.). As used herein, "AAV ITR" does not necessarily contain the wild-type nucleotide sequence and may be altered by nucleotide insertions, deletions, or substitutions. Furthermore, AAV ITRs may, non-limitingly, originate from any of several AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. Moreover, the 5' and 3' ITRs adjacent to the selected nucleotide sequence in the AAV vector do not necessarily have to be identical or originate from the same AAV serotype or isolate, as long as they function as intended, i.e., to allow for the excision and rescue of the sequence of interest from the host cell genome or vector, and to allow for the incorporation of heterologous sequences into the receptor cell genome when the AAV Rep gene product is present in the cell.Furthermore, AAV ITRs may, without limitation, originate from any of several AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. Moreover, the 5' and 3' ITRs adjacent to the selected nucleotide sequence in the AAV expression vector do not necessarily have to be identical or originate from the same AAV serotype or isolate, as long as they function as intended—that is, to enable the excision and rescue of the sequence of interest from the host cell genome or vector, and to enable the integration of the DNA molecule into the receptor cell genome if the AAV Rep gene product is present in the cell.

[0084] Exemplary vectors are those derived from AAV serotypes that exhibit tropism to and high transduction efficiency into cells of the mammalian central nervous system. An overview and comparison of transduction efficiencies of different serotypes is provided in Cearley CN et al., 2008. In other non-limiting examples, some vectors include those derived from any of the serotypes such as AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, or AAVrh10.

[0085] The selected nucleotide sequence is functionally ligated to a regulatory element that directs its transcription or expression in vivo in the target. Such a regulatory element may typically include a regulatory sequence associated with the selected gene.

[0086] Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, phosphoglycerate kinase (PKG) promoters, CAG, MCK (muscle creatine kinase), SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, e.g., CMV very early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters. Promoters may be of human origin or of other species, e.g., mouse. Furthermore, sequences derived from non-viral genes, e.g., mouse metallothionein genes, may also be used herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA).

[0087] An example of a heterologous promoter is the CMV promoter.

[0088] Examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and aufin.

[0089] AAV expression vectors containing a DNA molecule of interest surrounded by AAV ITRs can be constructed by directly inserting a selected sequence into an AAV genome from which the main AAV open reading frame ("ORF") has been excised. Other parts of the AAV genome can also be deleted, as long as enough portions of the ITR remain to allow replication and packaging functions to be enabled. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patents 5,173,414 and 5,139,941; International Publications WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992; Muzyczka, 1992; Kotin, 1994; Shelling and Smith, 1994; and Zhou et al., 1994. Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing them and fused to the 5' and 3' of a selected nucleic acid construct present in another vector using standard ligation techniques. AAV vectors containing ITRs are described, for example, in U.S. Patent 5,139,941. In particular, several AAV vectors available from the American Cell Culture Lineage Preservation Center ("ATCC") under accession numbers 53222, 53223, 53224, 53225, and 53226 are described therein. Furthermore, chimeric genes can be synthetically generated to contain AAV ITR sequences positioned at the 5' and 3' of one or more selected nucleic acid sequences. Preferred codons can be used for the expression of chimeric gene sequences in mammalian CNS cells. Complete chimeric sequences are assembled from duplicated oligonucleotides prepared by standard methods. See, for example, Edge, 1981; Nambair et al., 1984; Jay et al., 1984.To generate AAV virions, an AAV expression vector is introduced into suitable host cells using known techniques, for example, by transfection. Several transfection techniques are generally known in the art. See, for example, Graham et al., 1973; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al., 1981. Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome-mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Felgner et al., 1987), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., 1987).

[0090] The AAV viral vectors of this disclosure comprise (i) the AAV vectors described herein; and (ii) the AAV capsid protein.

[0091] In some contexts, an AAV capsid protein can be any AAV capsid protein. In some contexts, an AAV capsid protein can be AAV1 capsid protein, modified AAV1 capsid protein, AAV2 capsid protein, modified AAV2 capsid protein, AAV4 capsid protein, modified AAV4 capsid protein, AAV5 capsid protein, modified AAV5 capsid protein, AAV6 capsid protein, modified AAV6 capsid protein, AAV7 capsid protein, modified AAV7 capsid protein, AAV8 capsid protein, modified AAV8 capsid protein, AAV9 capsid protein, modified AAV9 capsid protein, AAV10 capsid protein AAV capsid protein, modified AAV10 capsid protein, AAV11 capsid protein, modified AAV11 capsid protein, AAV12 capsid protein, modified AAV12 capsid protein, AAV13 capsid protein, modified AAV13 capsid protein, AAVPHP.B capsid protein, modified AAVPHP.B capsid protein, AAVrh74 capsid protein, modified AAVrh74 capsid protein, MyoAAV capsid protein, modified MyoAAV capsid protein, AAVrh.10 capsid protein, or modified AAVrh.10 capsid protein. In some contexts, AAV capsid protein is AAVrh.10 capsid protein.

[0092] Treatment method This disclosure provides a method for treating or preventing Alzheimer's disease (AD) in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an rAAV vector comprising a nucleic acid sequence encoding a Christchurch mutation-bearing APOE2 polypeptide or a fragment thereof, or a Christchurch mutation-bearing APOE3 polypeptide or a fragment thereof.

[0093] This disclosure provides a method for treating Alzheimer's disease in a subject, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising an APOE2 Christchurch rAAV virus vector or an APOE3 Christchurch rAAV virus vector.

[0094] This disclosure provides a method for treating Alzheimer's disease in a subject, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising an APOE2 Christchurch rAAV viral vector or an APOE3 Christchurch rAAV viral vector, wherein, after administration, the subject experiences an increase in APOE2 or APOE3 expression. In some aspects, the increased APOE2 or APOE3 expression is APOE2 Christchurch polypeptide or APOE3 Christchurch polypeptide.

[0095] The disclosure covers any combination of APOE variants, including being an APOE2 homozygous, APOE3 homozygous, APOE4 homozygous, APOE2 / APOE4 heterozygous, APOE2 / APOE3 heterozygous, or APOE3 / APOE4 heterozygous. In some aspects, the disclosure covers an APOE4 homozygous.

[0096] The subjects of this disclosure may have a variety of cognitive impairments associated with Alzheimer's disease, including no impairment, mild cognitive impairment (CI), mild to moderate dementia, or severe dementia. In some aspects, the subjects of this disclosure are at least 50 years of age. In some aspects, the subjects of this disclosure may be of any age.

[0097] In some aspects, the subjects of this disclosure have CSF biomarkers consistent with Alzheimer's disease. In some aspects, the subjects of this disclosure are determined to be positive by amyloid-targeted positron emission tomography (PET).

[0098] In some cases, subjects experience an increase in the expression of the APOE2 Christchurch mutant polypeptide compared to baseline before administration. Quantification of APOE2 Christchurch mutant expression can be carried out according to any method known in the art. In some cases, subjects experience an increase in APOE2 expression of at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100% compared to baseline before administration.

[0099] In some cases, subjects experience an increase in the expression of the APOE3 Christchurch mutant polypeptide compared to baseline before administration. Quantification of APOE3 Christchurch mutant expression can be carried out according to any method known in the art. In some cases, subjects experience an increase in APOE3 expression of at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100% compared to baseline before administration.

[0100] The expression of APOE2 Christchurch mutations or APOE3 Christchurch mutations can occur anywhere in the central nervous system, including any region of the brain and the cerebrospinal fluid (CSF).

[0101] The expression of APOE2, APOE3, and APOE4 with or without Christchurch mutations can be quantified according to any method known in the art. Quantification of APOE2, APOE3, and APOE4 can be performed using mass spectrometry (MS), Western blotting, chromatography, or chromatographic-coupled mass spectrometry (i.e., LC-MS).

[0102] In some cases, APOE2 Christchurch mutations or expression are reflected as a percentage of APOE4 expression and / or total APOE expression. In some cases, APOE2 Christchurch mutation expression is calculated as total APOE2 expression divided by APOE4 expression. Multiplying this ratio by 100 gives the percentage. In some cases, APOE2 Christchurch mutation expression is calculated as total APOE2 Christchurch mutation expression divided by total APOE expression (total expression of APOE2 Christchurch, APOE2, APOE3, and / or APOE4). Multiplying this ratio by 100 gives the percentage. APOE expression can be assessed according to any appropriate protein quantification method.

[0103] In some cases, APOE3 Christchurch mutations or expression are reflected as a percentage of APOE4 expression and / or total APOE expression. In some cases, APOE3 Christchurch mutation expression is calculated as total APOE3 expression divided by APOE4 expression. Multiplying this ratio by 100 gives the percentage. In some cases, APOE3 Christchurch mutation expression is calculated as total APOE3 Christchurch mutation expression divided by total APOE expression (total APOE3 Christchurch, APOE2, APOE3, and / or APOE4 expression). Multiplying this ratio by 100 gives the percentage. APOE expression can be assessed according to any appropriate protein quantification method.

[0104] Hippocampal volume decreases over time in individuals with AD from the prodromal stage onward. Hippocampal volume typically correlates with cognitive and functional changes over time and serves as a useful marker of disease progression. In some cases, subjects experience an increase in hippocampal volume after treatment with the APOE2 Christchurch mutation or APOE3 Christchurch mutation pharmaceutically active compositions of this disclosure. In some cases, subjects' hippocampal volume remains unchanged after treatment with the APOE2 Christchurch mutation or APOE3 Christchurch mutation pharmaceutically active compositions of this disclosure. In some cases, subjects experience a reduction in the rate of hippocampal volume loss after treatment with the APOE2 Christchurch mutation or APOE3 Christchurch mutation pharmaceutically active compositions of this disclosure. In some cases, hippocampal volume loss is assessed by brain MRI.

[0105] Cognitive assessment The improvement in cognition after administration of APOE2 Christchurch mutation or APOE3 Christchurch mutation pharmaceutical compositions can be evaluated according to any method known in the art. Such evaluations include, but are not limited to, the Clinical Dementia Scale (CDR), the Alzheimer's Disease Assessment Scale-Cognitive Assessment (ADAS-Cog 13), or the Mini-Mental State Examination.

[0106] The Clinical Dementia Rating Scale (CDR) is a clinician-assessed dementia staging system that tracks the progression of cognitive impairment across six categories: memory, orientation, judgment and problem-solving, community activities, home and recreation, and personal care. Each category is scored on a five-point scale: none=0, suspected=0.5, mild=1, moderate=2, and severe=3. The global CDR score is established by clinical scoring rules and has values ​​of 0 (no dementia), 0.5 (suspected dementia), 1 (mild dementia), 2 (moderate dementia), and 3 (severe dementia). The CDR-SB is obtained by summing the ratings for each of the six categories and ranges from 0 to 18, with higher scores indicating greater cognitive impairment.

[0107] CDR-SB is expected to be performed at the time of screening / baseline visit prior to administration of the APOE2 Christchurch mutation or APOE3 Christchurch mutation pharmacologic compositions of this disclosure. These evaluations are expected to be used as a measure of clinical efficacy.

[0108] The Alzheimer's Disease Assessment Scale-Cognitive Subscale (13 items) (ADAS-Cog 13) is a structured scale that assesses memory, orientation, attention, reasoning, language, and constructive actions. A higher score indicates greater functional impairment.

[0109] The MMSE is a concise 30-point questionnaire used to assess cognitive impairment, with lower scores indicating greater impairment. The MMSE assesses 11 categories of cognition, including orientation to time, memory, attention, concentration, naming, repetition, comprehension, and the ability to construct sentences and copy two intersecting polygons.

[0110] Biomarker testing APOE2 expression can be assessed in any region of the CNS after administration of the APOE2 Christchurch mutagenic composition of this disclosure. In some cases, APOE2 expression is assessed in the brain. In some cases, APOE2 expression is assessed in the CSF. Amyloid beta (Aβ) 42, Aβ42 / 40, T-tau, and P-tau are considered core biomarkers for AD. T-tau and P-tau are thought to increase after amyloid accumulation in the brain (amyloid cascade hypothesis). Both amyloid and tau accumulation are associated with inflammation in the brain at various points in disease progression, with amyloid leading inflammation during early accumulation (MCI cases) and tau leading inflammation after the amyloid load in the brain approaches AD levels (prodromal AD). Aβ42 is reduced in the CSF of AD participants, while T-tau and P-tau are increased. Although not a core CSF biomarker, amyloid-beta 40 is also evaluated.

[0111] APOE3 expression can be assessed in any region of the CNS after administration of the pharmaceutical composition of the APOE3 Christchurch mutation of this disclosure. In some cases, APOE3 expression is assessed in the brain. In some cases, APOE3 expression is assessed in the CSF. Amyloid beta (Aβ) 42, Aβ42 / 40, T-tau, and P-tau are considered core biomarkers for AD. T-tau and P-tau are thought to increase after amyloid accumulation in the brain (amyloid cascade hypothesis). Both amyloid and tau accumulation are associated with inflammation in the brain at various points in disease progression, with amyloid leading inflammation during early accumulation (MCI cases) and tau leading inflammation after the amyloid load in the brain approaches AD levels (prodromal AD). Aβ42 is reduced in the CSF of AD participants, while T-tau and P-tau are increased. Although not a core CSF biomarker, amyloid-beta 40 is also evaluated.

[0112] In some cases, after administration of the pharmaceutical composition, amyloid beta 42 (Aβ 42 ), amyloid beta 40 (Aβ 40 The expression levels of at least one of ), T-tau, and P-tau are reduced in the subjects compared to baseline before administration. In some aspects, after administration of the pharmaceutical composition, amyloid beta 42 (Aβ 42 ), amyloid beta 40 (Aβ 40 The expression levels of at least one of ), T-tau, and P-tau increase in subjects compared to baseline before administration. In some aspects, after administration of the pharmaceutical composition, amyloid beta-42 (Aβ) 42 ), amyloid beta 40 (Aβ 40 The expression levels of ), T-tau, and at least one P-tau remain constant in the subjects compared to baseline before administration.

[0113] In some situations, Aβ 42The expression level is reduced by at least approximately 5%, at least approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 100% compared to baseline before administration.

[0114] In some situations, Aβ 40 The expression level is reduced by at least approximately 5%, at least approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 100% compared to baseline before administration.

[0115] In some phases, T-tau expression levels are reduced by at least approximately 5%, at least approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 100% compared to baseline before administration.

[0116] In some phases, P-tau expression levels are reduced by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100% compared to baseline before administration.

[0117] In some cases, after administration of the pharmaceutical composition, Aβ 42 The expression level changes. In some cases, after administration of the pharmaceutical composition, Aβ 42The expression level of increases. In some cases, the expression level of Aβ42 decreases after administration of the pharmaceutical composition. In some cases, the expression level of Aβ42 decreases after administration of the pharmaceutical composition. 42 Changes in the expression level of [substance name] are target-specific.

[0118] In some cases, after administration of the pharmaceutical composition, amyloid beta 42 / amyloid beta 40 (Aβ 42 / 40 The ratio changes. In some cases, after administration of the pharmaceutical composition, Aβ 42 / 40 The ratio increases. In some cases, after administration of the pharmaceutical composition, Aβ 42 / 40 The ratio decreases. In some cases, Aβ after administration of the pharmaceutical composition 42 / 40 The change in ratio is specific to the target.

[0119] In some situations, Aβ 42 / 40 The ratio increases by at least approximately 5%, at least approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 100% compared to baseline before administration.

[0120] In certain embodiments, the gene encoded by the nucleic acid sequence in the AAV vector is the APOE2 gene with the Christchurch mutation.

[0121] In certain embodiments, the gene encoded by the nucleic acid sequence in the AAV vector is the APOE3 gene with the Christchurch mutation.

[0122] In its broadest sense, as used herein, the term “prevent” or “prevention” means preventing a disease or condition from developing in a person who has not yet been diagnosed with the disease or condition or who does not have any clinical symptoms.

[0123] Where used herein, the terms “to treat” or “to treat” mean to reverse, alleviate, or inhibit the progression of a disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition. “Therapeutic effective dose” is intended to be the minimum amount of the activator necessary to provide a therapeutic benefit to the subject. For example, “therapeutic effective dose” for a patient is the amount that induces, improves, stabilizes, slows the rate of progression, or otherwise improves a physiological condition associated with a pathological symptom, disease progression, or disorder, or a physiological condition that is resistant to succumbing to the disorder.

[0124] As used herein, the term “subject” refers to mammals, such as rodents, felines, canines, and primates. In some contexts, the subject as provided in this disclosure is human. In this disclosure, “subject that needs to be described” refers to a subject, such as a human, and more specifically, a subject having Alzheimer’s disease.

[0125] As used herein, the term “gene” refers to a polynucleotide containing at least one open reading frame that, after transcription and translation, can encode a particular polypeptide or protein.

[0126] As used herein, the terms “coding sequence,” “sequence encoding a specific protein,” or “coding nucleic acid” refer to nucleic acid sequences that, under the control of appropriate regulatory sequences, are transcribed in vitro or in vivo (in the case of DNA) and translated into polypeptides (in the case of mRNA). The boundaries of a coding sequence are determined by the 5' (amino)-terminus start codon and the 3' (carboxy)-terminus translation stop codon. Examples of coding sequences include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.

[0127] In certain embodiments, the Disclosure relates to a method for preventing or treating Alzheimer's disease in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition containing a nucleic acid encoding the APOE2 Christchurch mutation.

[0128] In certain embodiments, the Disclosure relates to a method for treating Alzheimer's disease in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition containing a nucleic acid encoding the APOE2 Christchurch mutation.

[0129] In certain embodiments, the Disclosure relates to a method for reversing or stabilizing the symptoms of Alzheimer's disease in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition containing a nucleic acid encoding the APOE2 Christchurch mutation.

[0130] In certain embodiments, the Disclosure relates to a method for preventing or treating Alzheimer's disease in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition containing a nucleic acid encoding the APOE3 Christchurch mutation.

[0131] In certain embodiments, the Disclosure relates to a method for treating Alzheimer's disease in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition containing a nucleic acid encoding the APOE3 Christchurch mutation.

[0132] In certain embodiments, the Disclosure relates to a method for reversing or stabilizing the symptoms of Alzheimer's disease in a subject in need, comprising the step of administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition containing a nucleic acid encoding the APOE3 Christchurch mutation.

[0133] As used herein, the terms “asymptomatic” or “presymptomatic” refer to individuals who have a disease defined by genetic diagnosis (Alzheimer’s disease) but do not have detectable clinical cardiac symptoms.

[0134] As used herein, the term "symptomatic" refers to individuals who have a disease defined by a genetic diagnosis (Alzheimer's disease) and who also have cognitive impairment, including MCI or mild, moderate, or severe dementia.

[0135] Vector delivery A method for treating Alzheimer's disease in a subject is provided herein, comprising the steps of (a) generating an AAV vector as defined above, comprising a nucleic acid sequence encoding an APOE2 Christchurch mutant polypeptide or a fragment thereof or an APOE3 Christchurch mutant polypeptide or a fragment thereof; and (b) delivering the AAV vector to a subject in need, wherein APOE2 or APOE3 is expressed at a therapeutically effective level by transduced cells.

[0136] The dosage and regimen can be determined by the physician and depend on the patient's age, sex, weight, and stage of the disease.

[0137] In some cases, the AAV vector or pharmaceutical composition is administered to the subject orally, rectally, mucosally, by inhalation, percutaneously, parenterally, intravenously, subcutaneously, intradermally, intramuscularly, intrapleurally, intracerebrally, intrathecally, intracerebrally, intravenously, intranasally, intraaurally, intraocularly, intraocularly, intraocularly, intraocularly, intraocularly, intralymphally, intracisionally, intravitreally.

[0138] In some cases, the AAV vector or pharmaceutical composition is administered to the central nervous system (CNS) of the target. In some cases, the administration is C1-C2 administration. C1-C2 administration refers to the administration of the AAV vector between the first two cervical vertebrae. In some cases, C1-C2 administration is CT-guided. In some cases, the administration is intracisor major (ICM). In some cases, ICM administration is performed when C1-C2 administration is not feasible. In some cases, factors limiting C1-C2 administration include the vagus artery and / or limited dorsal subarachnoid space (i.e., <2 mm) along the needle trajectory. In some embodiments, the AAV vector is administered to the hippocampus of the target, for example, by intrahippocampal injection or intrahippocampal infusion.

[0139] In some cases, the administration of the AAV vector or pharmaceutical composition is performed over a period of at least approximately 1 minute, at least approximately 2 minutes, at least approximately 3 minutes, at least approximately 4 minutes, at least approximately 5 minutes, at least approximately 10 minutes, at least approximately 20 minutes, or at least approximately 30 minutes. In some cases, the IV infusion is performed over a period of 60 minutes. In some cases, the C1-C2 administration is performed at a rate of approximately 1 mL / min, approximately 2 mL / min, approximately 3 mL / min, approximately 4 mL / min, approximately 5 mL / min, approximately 6 mL / min, approximately 7 mL / min, approximately 8 mL / min, approximately 9 mL / min, or approximately 10 mL / min.

[0140] In some cases, the amount of AAV vector administered is approximately 5 mL, 10 mL, 15 mL, 20 mL, or 25 mL.

[0141] In some contexts, the AAV vector or pharmaceutical composition of this disclosure is administered to a subject in a therapeutically effective dose. In some contexts, the dose is approximately 5.0 × 10⁻⁶ 9 Genome copy (gc) / 1 ml (mL) of cerebrospinal fluid (CSF) (gc / mL) ~ approximately 5.0 × 10⁻¹⁴ 12 The CSF is gc / mL. In some situations, the dose is approximately 1.4 × 10⁻⁶. 10The CSF is gc / mL. In some situations, the dose is approximately 4.4 × 10⁻⁶. 10 The CSF is gc / mL. In some situations, the dose is approximately 1.4 × 10⁻⁶. 11 It is gc / mL CSF.

[0142] In some situations, the dosage is approximately 1.0 × 10⁻⁶. 9 gc / mL CSF, approximately 1.1×10 9 gc / mL CSF, approximately 1.2×10 9 gc / mL CSF, approximately 1.3×10 9 gc / mL CSF, approximately 1.4×10 9 gc / mL CSF, approximately 1.5×10 9 gc / mL CSF, approximately 1.6×10 9 gc / mL CSF, approximately 1.7×10 9 gc / mL CSF, approximately 1.8×10 9 gc / mL CSF, approximately 1.9×10 9 gc / mL CSF, approximately 2.0×10 9 gc / mL CSF, approximately 2.1×10 9 gc / mL CSF, approximately 2.2×10 9 gc / mL CSF, approximately 2.3×10 9 gc / mL CSF, approximately 2.4×10 9 gc / mL CSF, approximately 2.5×10 9 gc / mL CSF, approximately 2.6×10 9 gc / mL CSF, approximately 2.7×10 9 gc / mL CSF, approximately 2.8×10 9 gc / mL CSF, approximately 2.9×10 9 gc / mL CSF, approximately 3.0×10 9 gc / mL CSF, approximately 3.1×10 9 gc / mL CSF, approximately 3.2×10 9 gc / mL CSF, approximately 3.3×10 9 gc / mL CSF, approximately 3.4×10 9 gc / mL CSF, approximately 3.5×10 9 gc / mL CSF, approximately 3.6×10 9 gc / mL CSF, approximately 3.7×10 9gc / mL CSF, approximately 3.8 × 10 9 gc / mL CSF, approximately 3.9 × 10 9 gc / mL CSF, approximately 4.0 × 10 9 gc / mL CSF, approximately 4.1 × 10 9 gc / mL CSF, approximately 4.2 × 10 9 gc / mL CSF, approximately 4.3 × 10 9 gc / mL CSF, approximately 4.4 × 10 9 gc / mL CSF, approximately 4.5 × 10 9 gc / mL CSF, approximately 4.6 × 10 9 gc / mL CSF, approximately 4.7 × 10 9 gc / mL CSF, approximately 4.8 × 10 9 gc / mL CSF, approximately 4.9 × 10 9 gc / mL CSF, approximately 5.0 × 10 9 gc / mL CSF, approximately 5.1 × 10 9 gc / mL CSF, approximately 5.2 × 10 9 gc / mL CSF, approximately 5.3 × 10 9 gc / mL CSF, approximately 5.4 × 10 9 gc / mL CSF, approximately 5.5 × 10 9 gc / mL CSF, approximately 5.6 × 10 9 gc / mL CSF, approximately 5.7 × 10 9 gc / mL CSF, approximately 5.8 × 10 9 gc / mL CSF, approximately 5.9 × 10 9 gc / mL CSF, approximately 6.0 × 10 9 gc / mL CSF, approximately 6.1 × 10 9 gc / mL CSF, approximately 6.2 × 10 9 gc / mL CSF, approximately 6.3 × 10 9 gc / mL CSF, approximately 6.4 × 10 9 gc / mL CSF, approximately 6.5 × 10 9 gc / mL CSF, approximately 6.6 × 10 9 gc / mL CSF, approximately 6.7 × 10 9 gc / mL CSF, approximately 6.8 × 10 9 gc / mL CSF, approximately 6.9 × 10 9 gc / mL CSF, approximately 7.0 × 10 9gc / mL CSF, approximately 7.1×10 9 gc / mL CSF, approximately 7.2×10 9 gc / mL CSF, approximately 7.3×10 9 gc / mL CSF, approximately 7.4×10 9 gc / mL CSF, approximately 7.5×10 9 gc / mL CSF, approximately 7.6×10 9 gc / mL CSF, approximately 7.7×10 9 gc / mL CSF, approximately 7.8×10 9 gc / mL CSF, approximately 7.9×10 9 gc / mL CSF, approximately 8.0×10 9 gc / mL CSF, approximately 8.1×10 9 gc / mL CSF, approximately 8.2×10 9 gc / mL CSF, approximately 8.3×10 9 gc / mL CSF, approximately 8.4×10 9 gc / mL CSF, approximately 8.5×10 9 gc / mL CSF, approximately 8.6×10 9 gc / mL CSF, approximately 8.7×10 9 gc / mL CSF, approximately 8.8×10 9 gc / mL CSF, approximately 8.9×10 9 gc / mL CSF, approximately 9.0×10 9 gc / mL CSF, approximately 9.1×10 9 gc / mL CSF, approximately 9.2×10 9 gc / mL CSF, approximately 9.3×10 9 gc / mL CSF, approximately 9.4×10 9 gc / mL CSF, approximately 9.5×10 9 gc / mL CSF, approximately 9.6×10 9 gc / mL CSF, approximately 9.7×10 9 gc / mL CSF, approximately 9.8×10 9 gc / mL CSF, or approximately 9.9×10 9 gc / mL CSF, or any dosage in between.

[0143] In some situations, the dosage is approximately 1.0×10 10 gc / mL CSF, approximately 1.1×1010 gc / mL CSF, approximately 1.2 × 10 10 gc / mL CSF, approximately 1.3 × 10 10 gc / mL CSF, approximately 1.4 × 10 10 gc / mL CSF, approximately 1.5 × 10 10 gc / mL CSF, approximately 1.6 × 10 10 gc / mL CSF, approximately 1.7 × 10 10 gc / mL CSF, approximately 1.8 × 10 10 gc / mL CSF, approximately 1.9 × 10 10 gc / mL CSF, approximately 2.0 × 10 10 gc / mL CSF, approximately 2.1 × 10 10 gc / mL CSF, approximately 2.2 × 10 10 gc / mL CSF, approximately 2.3 × 10 10 gc / mL CSF, approximately 2.4 × 10 10 gc / mL CSF, approximately 2.5 × 10 10 gc / mL CSF, approximately 2.6 × 10 10 gc / mL CSF, approximately 2.7 × 10 10 gc / mL CSF, approximately 2.8 × 10 10 gc / mL CSF, approximately 2.9 × 10 10 gc / mL CSF, approximately 3.0 × 10 10 gc / mL CSF, approximately 3.1 × 10 10 gc / mL CSF, approximately 3.2 × 10 10 gc / mL CSF, approximately 3.3 × 10 10 gc / mL CSF, approximately 3.4 × 10 10 gc / mL CSF, approximately 3.5 × 10 10 gc / mL CSF, approximately 3.6 × 10 10 gc / mL CSF, approximately 3.7 × 10 10 gc / mL CSF, approximately 3.8 × 10 10 gc / mL CSF, approximately 3.9 × 10 10 gc / mL CSF, approximately 4.0 × 10 10 gc / mL CSF, approximately 4.1 × 10 10 gc / mL CSF, approximately 4.2 × 10 10 gc / mL CSF, approximately 4.3 × 10 10 gc / mL CSF, approximately 4.4 × 10 10gc / mL CSF, approximately 4.5 × 10 10 gc / mL CSF, approximately 4.6 × 10 10 gc / mL CSF, approximately 4.7 × 10 10 gc / mL CSF, approximately 4.8 × 10 10 gc / mL CSF, approximately 4.9 × 10 10 gc / mL CSF, approximately 5.0 × 10 10 gc / mL CSF, approximately 5.1 × 10 10 gc / mL CSF, approximately 5.2 × 10 10 gc / mL CSF, approximately 5.3 × 10 10 gc / mL CSF, approximately 5.4 × 10 10 gc / mL CSF, approximately 5.5 × 10 10 gc / mL CSF, approximately 5.6 × 10 10 gc / mL CSF, approximately 5.7 × 10 10 gc / mL CSF, approximately 5.8 × 10 10 gc / mL CSF, approximately 5.9 × 10 10 gc / mL CSF, approximately 6.0 × 10 10 gc / mL CSF, approximately 6.1 × 10 10 gc / mL CSF, approximately 6.2 × 10 10 gc / mL CSF, approximately 6.3 × 10 10 gc / mL CSF, approximately 6.4 × 10 10 gc / mL CSF, approximately 6.5 × 10 10 gc / mL CSF, approximately 6.6 × 10 10 gc / mL CSF, approximately 6.7 × 10 10 gc / mL CSF, approximately 6.8 × 10 10 gc / mL CSF, approximately 6.9 × 10 10 gc / mL CSF, approximately 7.0 × 10 10 gc / mL CSF, approximately 7.1 × 10 10 gc / mL CSF, approximately 7.2 × 10 10 gc / mL CSF, approximately 7.3 × 10 10 gc / mL CSF, approximately 7.4 × 10 10 gc / mL CSF, approximately 7.5 × 10 10 gc / mL CSF, approximately 7.6 × 10 10 gc / mL CSF, approximately 7.7 × 10 10gc / mL CSF, approximately 7.8×10 10 gc / mL CSF, approximately 7.9×10 10 gc / mL CSF, approximately 8.0×10 10 gc / mL CSF, approximately 8.1×10 10 gc / mL CSF, approximately 8.2×10 10 gc / mL CSF, approximately 8.3×10 10 gc / mL CSF, approximately 8.4×10 10 gc / mL CSF, approximately 8.5×10 10 gc / mL CSF, approximately 8.6×10 10 gc / mL CSF, approximately 8.7×10 10 gc / mL CSF, approximately 8.8×10 10 gc / mL CSF, approximately 8.9×10 10 gc / mL CSF, approximately 9.0×10 10 gc / mL CSF, approximately 9.1×10 10 gc / mL CSF, approximately 9.2×10 10 gc / mL CSF, approximately 9.3×10 10 gc / mL CSF, approximately 9.4×10 10 gc / mL CSF, approximately 9.5×10 10 gc / mL CSF, approximately 9.6×10 10 gc / mL CSF, approximately 9.7×10 10 gc / mL CSF, approximately 9.8×10 10 gc / mL CSF, or approximately 9.9 × 10 10 gc / mL CSF, or any dose in between.

[0144] In some situations, approximately 1.0 × 10 11 gc / mL CSF, approximately 1.1×10 11 gc / mL CSF, approximately 1.2×10 11 gc / mL CSF, approximately 1.3×10 11 gc / mL CSF, approximately 1.4×10 11 gc / mL CSF, approximately 1.5×10 11 gc / mL CSF, approximately 1.6×10 11 gc / mL CSF, approximately 1.7×10 11 gc / mL CSF, approximately 1.8×10 11gc / mL CSF, approximately 1.9 × 10 11 gc / mL CSF, approximately 2.0 × 10 11 gc / mL CSF, approximately 2.1 × 10 11 gc / mL CSF, approximately 2.2 × 10 11 gc / mL CSF, approximately 2.3 × 10 11 gc / mL CSF, approximately 2.4 × 10 11 gc / mL CSF, approximately 2.5 × 10 11 gc / mL CSF, approximately 2.6 × 10 11 gc / mL CSF, approximately 2.7 × 10 11 gc / mL CSF, approximately 2.8 × 10 11 gc / mL CSF, approximately 2.9 × 10 11 gc / mL CSF, approximately 3.0 × 10 11 gc / mL CSF, approximately 3.1 × 10 11 gc / mL CSF, approximately 3.2 × 10 11 gc / mL CSF, approximately 3.3 × 10 11 gc / mL CSF, approximately 3.4 × 10 11 gc / mL CSF, approximately 3.5 × 10 11 gc / mL CSF, approximately 3.6 × 10 11 gc / mL CSF, approximately 3.7 × 10 11 gc / mL CSF, approximately 3.8 × 10 11 gc / mL CSF, approximately 3.9 × 10 11 gc / mL CSF, approximately 4.0 × 10 11 gc / mL CSF, approximately 4.1 × 10 11 gc / mL CSF, approximately 4.2 × 10 11 gc / mL CSF, approximately 4.3 × 10 11 gc / mL CSF, approximately 4.4 × 10 11 gc / mL CSF, approximately 4.5 × 10 11 gc / mL CSF, approximately 4.6 × 10 11 gc / mL CSF, approximately 4.7 × 10 11 gc / mL CSF, approximately 4.8 × 10 11 gc / mL CSF, approximately 4.9 × 10 11 gc / mL CSF, approximately 5.0 × 10 11 gc / mL CSF, approximately 5.1 × 10 11gc / mL CSF, approximately 5.2 × 10 11 gc / mL CSF, approximately 5.3 × 10 11 gc / mL CSF, approximately 5.4 × 10 11 gc / mL CSF, approximately 5.5 × 10 11 gc / mL CSF, approximately 5.6 × 10 11 gc / mL CSF, approximately 5.7 × 10 11 gc / mL CSF, approximately 5.8 × 10 11 gc / mL CSF, approximately 5.9 × 10 11 gc / mL CSF, approximately 6.0 × 10 11 gc / mL CSF, approximately 6.1 × 10 11 gc / mL CSF, approximately 6.2 × 10 11 gc / mL CSF, approximately 6.3 × 10 11 gc / mL CSF, approximately 6.4 × 10 11 gc / mL CSF, approximately 6.5 × 10 11 gc / mL CSF, approximately 6.6 × 10 11 gc / mL CSF, approximately 6.7 × 10 11 gc / mL CSF, approximately 6.8 × 10 11 gc / mL CSF, approximately 6.9 × 10 11 gc / mL CSF, approximately 7.0 × 10 11 gc / mL CSF, approximately 7.1 × 10 11 gc / mL CSF, approximately 7.2 × 10 11 gc / mL CSF, approximately 7.3 × 10 11 gc / mL CSF, approximately 7.4 × 10 11 gc / mL CSF, approximately 7.5 × 10 11 gc / mL CSF, approximately 7.6 × 10 11 gc / mL CSF, approximately 7.7 × 10 11 gc / mL CSF, approximately 7.8 × 10 11 gc / mL CSF, approximately 7.9 × 10 11 gc / mL CSF, approximately 8.0 × 10 11 gc / mL CSF, approximately 8.1 × 10 11 gc / mL CSF, approximately 8.2 × 10 11 gc / mL CSF, approximately 8.3 × 10 11 gc / mL CSF, approximately 8.4 × 10 11gc / mL CSF, approximately 8.5×10 11 gc / mL CSF, approximately 8.6×10 11 gc / mL CSF, approximately 8.7×10 11 gc / mL CSF, approximately 8.8×10 11 gc / mL CSF, approximately 8.9×10 11 gc / mL CSF, approximately 9.0×10 11 gc / mL CSF, approximately 9.1×10 11 gc / mL CSF, approximately 9.2×10 11 gc / mL CSF, approximately 9.3×10 11 gc / mL CSF, approximately 9.4×10 11 gc / mL CSF, approximately 9.5×10 11 gc / mL CSF, approximately 9.6×10 11 gc / mL CSF, approximately 9.7×10 11 gc / mL CSF, approximately 9.8×10 11 gc / mL CSF, or approximately 9.9 × 10 11 gc / mL CSF, or any dose in between.

[0145] In some situations, the dosage is approximately 1.0 × 10⁻⁶. 12 gc / mL CSF, approximately 1.1×10 12 gc / mL CSF, approximately 1.2×10 12 gc / mL CSF, approximately 1.3×10 12 gc / mL CSF, approximately 1.4×10 12 gc / mL CSF, approximately 1.5×10 12 gc / mL CSF, approximately 1.6×10 12 gc / mL CSF, approximately 1.7×10 12 gc / mL CSF, approximately 1.8×10 12 gc / mL CSF, approximately 1.9×10 12 gc / mL CSF, approximately 2.0×10 12 gc / mL CSF, approximately 2.1×10 12 gc / mL CSF, approximately 2.2×10 12 gc / mL CSF, approximately 2.3×10 12 gc / mL CSF, approximately 2.4×10 12 gc / mL CSF, approximately 2.5×1012 gc / mL CSF, approximately 2.6 × 10 12 gc / mL CSF, approximately 2.7 × 10 12 gc / mL CSF, approximately 2.8 × 10 12 gc / mL CSF, approximately 2.9 × 10 12 gc / mL CSF, approximately 3.0 × 10 12 gc / mL CSF, approximately 3.1 × 10 12 gc / mL CSF, approximately 3.2 × 10 12 gc / mL CSF, approximately 3.3 × 10 12 gc / mL CSF, approximately 3.4 × 10 12 gc / mL CSF, approximately 3.5 × 10 12 gc / mL CSF, approximately 3.6 × 10 12 gc / mL CSF, approximately 3.7 × 10 12 gc / mL CSF, approximately 3.8 × 10 12 gc / mL CSF, approximately 3.9 × 10 12 gc / mL CSF, approximately 4.0 × 10 12 gc / mL CSF, approximately 4.1 × 10 12 gc / mL CSF, approximately 4.2 × 10 12 gc / mL CSF, approximately 4.3 × 10 12 gc / mL CSF, approximately 4.4 × 10 12 gc / mL CSF, approximately 4.5 × 10 12 gc / mL CSF, approximately 4.6 × 10 12 gc / mL CSF, approximately 4.7 × 10 12 gc / mL CSF, approximately 4.8 × 10 12 gc / mL CSF, approximately 4.9 × 10 12 gc / mL CSF, approximately 5.0 × 10 12 gc / mL CSF, approximately 5.1 × 10 12 gc / mL CSF, approximately 5.2 × 10 12 gc / mL CSF, approximately 5.3 × 10 12 gc / mL CSF, approximately 5.4 × 10 12 gc / mL CSF, approximately 5.5 × 10 12 gc / mL CSF, approximately 5.6 × 10 12 gc / mL CSF, approximately 5.7 × 10 12 gc / mL CSF, approximately 5.8 × 10 12gc / mL CSF, approximately 5.9 × 10 12 gc / mL CSF, approximately 6.0 × 10 12 gc / mL CSF, approximately 6.1 × 10 12 gc / mL CSF, approximately 6.2 × 10 12 gc / mL CSF, approximately 6.3 × 10 12 gc / mL CSF, approximately 6.4 × 10 12 gc / mL CSF, approximately 6.5 × 10 12 gc / mL CSF, approximately 6.6 × 10 12 gc / mL CSF, approximately 6.7 × 10 12 gc / mL CSF, approximately 6.8 × 10 12 gc / mL CSF, approximately 6.9 × 10 12 gc / mL CSF, approximately 7.0 × 10 12 gc / mL CSF, approximately 7.1 × 10 12 gc / mL CSF, approximately 7.2 × 10 12 gc / mL CSF, approximately 7.3 × 10 12 gc / mL CSF, approximately 7.4 × 10 12 gc / mL CSF, approximately 7.5 × 10 12 gc / mL CSF, approximately 7.6 × 10 12 gc / mL CSF, approximately 7.7 × 10 12 gc / mL CSF, approximately 7.8 × 10 12 gc / mL CSF, approximately 7.9 × 10 12 gc / mL CSF, approximately 8.0 × 10 12 gc / mL CSF, approximately 8.1 × 10 12 gc / mL CSF, approximately 8.2 × 10 12 gc / mL CSF, approximately 8.3 × 10 12 gc / mL CSF, approximately 8.4 × 10 12 gc / mL CSF, approximately 8.5 × 10 12 gc / mL CSF, approximately 8.6 × 10 12 gc / mL CSF, approximately 8.7 × 10 12 gc / mL CSF, approximately 8.8 × 10 12 gc / mL CSF, approximately 8.9 × 10 12 gc / mL CSF, approximately 9.0 × 10 12 gc / mL CSF, approximately 9.1 × 10 12gc / mL CSF, approximately 9.2×10 12 gc / mL CSF, approximately 9.3×10 12 gc / mL CSF, approximately 9.4×10 12 gc / mL CSF, approximately 9.5×10 12 gc / mL CSF, approximately 9.6×10 12 gc / mL CSF, approximately 9.7×10 12 gc / mL CSF, approximately 9.8×10 12 gc / mL CSF, or approximately 9.9 × 10 12 gc / mL CSF, or any dose in between.

[0146] In some situations, the dosage is approximately 1.0 × 10⁻⁶. 13 gc / mL CSF, approximately 1.1×10 13 gc / mL CSF, approximately 1.2×10 13 gc / mL CSF, approximately 1.3×10 13 gc / mL CSF, approximately 1.4×10 13 gc / mL CSF, approximately 1.5×10 13 gc / mL CSF, approximately 1.6×10 13 gc / mL CSF, approximately 1.7×10 13 gc / mL CSF, approximately 1.8×10 13 gc / mL CSF, approximately 1.9×10 13 gc / mL CSF, approximately 2.0×10 13 gc / mL CSF, approximately 2.1×10 13 gc / mL CSF, approximately 2.2×10 13 gc / mL CSF, approximately 2.3×10 13 gc / mL CSF, approximately 2.4×10 13 gc / mL CSF, approximately 2.5×10 13 gc / mL CSF, approximately 2.6×10 13 gc / mL CSF, approximately 2.7×10 13 gc / mL CSF, approximately 2.8×10 13 gc / mL CSF, approximately 2.9×10 13 gc / mL CSF, approximately 3.0×10 13 gc / mL CSF, approximately 3.1×10 13 gc / mL CSF, approximately 3.2×1013 gc / mL CSF, approximately 3.3 × 10 13 gc / mL CSF, approximately 3.4 × 10 13 gc / mL CSF, approximately 3.5 × 10 13 gc / mL CSF, approximately 3.6 × 10 13 gc / mL CSF, approximately 3.7 × 10 13 gc / mL CSF, approximately 3.8 × 10 13 gc / mL CSF, approximately 3.9 × 10 13 gc / mL CSF, approximately 4.0 × 10 13 gc / mL CSF, approximately 4.1 × 10 13 gc / mL CSF, approximately 4.2 × 10 13 gc / mL CSF, approximately 4.3 × 10 13 gc / mL CSF, approximately 4.4 × 10 13 gc / mL CSF, approximately 4.5 × 10 13 gc / mL CSF, approximately 4.6 × 10 13 gc / mL CSF, approximately 4.7 × 10 13 gc / mL CSF, approximately 4.8 × 10 13 gc / mL CSF, approximately 4.9 × 10 13 gc / mL CSF, approximately 5.0 × 10 13 gc / mL CSF, approximately 5.1 × 10 13 gc / mL CSF, approximately 5.2 × 10 13 gc / mL CSF, approximately 5.3 × 10 13 gc / mL CSF, approximately 5.4 × 10 13 gc / mL CSF, approximately 5.5 × 10 13 gc / mL CSF, approximately 5.6 × 10 13 gc / mL CSF, approximately 5.7 × 10 13 gc / mL CSF, approximately 5.8 × 10 13 gc / mL CSF, approximately 5.9 × 10 13 gc / mL CSF, approximately 6.0 × 10 13 gc / mL CSF, approximately 6.1 × 10 13 gc / mL CSF, approximately 6.2 × 10 13 gc / mL CSF, approximately 6.3 × 10 13 gc / mL CSF, approximately 6.4 × 10 13 gc / mL CSF, approximately 6.5 × 10 13gc / mL CSF, approximately 6.6 × 10 13 gc / mL CSF, approximately 6.7 × 10 13 gc / mL CSF, approximately 6.8 × 10 13 gc / mL CSF, approximately 6.9 × 10 13 gc / mL CSF, approximately 7.0 × 10 13 gc / mL CSF, approximately 7.1 × 10 13 gc / mL CSF, approximately 7.2 × 10 13 gc / mL CSF, approximately 7.3 × 10 13 gc / mL CSF, approximately 7.4 × 10 13 gc / mL CSF, approximately 7.5 × 10 13 gc / mL CSF, approximately 7.6 × 10 13 gc / mL CSF, approximately 7.7 × 10 13 gc / mL CSF, approximately 7.8 × 10 13 gc / mL CSF, approximately 7.9 × 10 13 gc / mL CSF, approximately 8.0 × 10 13 gc / mL CSF, approximately 8.1 × 10 13 gc / mL CSF, approximately 8.2 × 10 13 gc / mL CSF, approximately 8.3 × 10 13 gc / mL CSF, approximately 8.4 × 10 13 gc / mL CSF, approximately 8.5 × 10 13 gc / mL CSF, approximately 8.6 × 10 13 gc / mL CSF, approximately 8.7 × 10 13 gc / mL CSF, approximately 8.8 × 10 13 gc / mL CSF, approximately 8.9 × 10 13 gc / mL CSF, approximately 9.0 × 10 13 gc / mL CSF, approximately 9.1 × 10 13 gc / mL CSF, approximately 9.2 × 10 13 gc / mL CSF, approximately 9.3 × 10 13 gc / mL CSF, approximately 9.4 × 10 13 gc / mL CSF, approximately 9.5 × 10 13 gc / mL CSF, approximately 9.6 × 10 13 gc / mL CSF, approximately 9.7 × 10 13 gc / mL CSF, approximately 9.8 × 10 13gc / mL CSF, or approximately 9.9 × 10 13 gc / mL CSF, or any dose in between.

[0147] In some contexts, the therapeutically effective dose of the AAV vectors of this disclosure is administered as a fixed dose. In some contexts, the fixed dose is expressed as the total number of genomic copies (gc).

[0148] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 10 gc~approx. 1.0×10 16 It is administered at a fixed dose of gc. In some cases, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 13 gc~approx. 1.0×10 15 It is administered at a fixed dose of GC.

[0149] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 10 gc~approx. 9.0×10 10 It is administered at a fixed dose of gc. In some cases, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 10 gc, approx. 1.4×10 10 gc, approx. 1.5×10 10 gc, approx. 2.0×10 10 gc, approx. 2.1×10 10 gc, approx. 2.2×10 10 gc, approx. 2.3×10 10 gc, approx. 2.4×10 10 gc, approx. 2.5×10 10 gc, approx. 2.6×10 10 gc, approx. 2.7×10 10 gc, approx. 2.8×10 10 gc, approx. 2.9×10 10 gc, approx. 3.0×10 10 gc, approx. 4.0×10 10 gc, approx. 5.0×10 10 gc, approx. 6.0×10 10 gc, approx. 7.0×10 10 gc, approx. 8.0×10 10 gc, or approximately 9.0 × 10 10It is administered in a fixed dose of gc, or any dose in between. In some cases, the AAV vector of this disclosure is approximately 2.0 × 10⁻⁶. 10 It is administered at a fixed dose of GC.

[0150] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 11 gc~approx. 9.0×10 11 It is administered at a fixed dose of gc. In some cases, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 11 gc, approx. 1.4×10 11 gc, approx. 1.5×10 11 gc, approx. 2.0×10 11 gc, approx. 2.1×10 11 gc, approx. 2.2×10 11 gc, approx. 2.3×10 11 gc, approx. 2.4×10 11 gc, approx. 2.5×10 11 gc, approx. 2.6×10 11 gc, approx. 2.7×10 11 gc, approx. 2.8×10 11 gc, approx. 2.9×10 11 gc, approx. 3.0×10 11 gc, approx. 4.0×10 11 gc, approx. 5.0×10 11 gc, approx. 6.0×10 11 gc, approx. 7.0×10 11 gc, approx. 8.0×10 11 gc, or approximately 9.0 × 10 11 It is administered in a fixed dose of gc, or any dose in between.

[0151] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 12 gc~approx. 9.0×10 12 It is administered at a fixed dose of gc. In some cases, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 12 gc, approx. 1.4×10 12 gc, approx. 1.5×10 12 gc, approx. 2.0×10 12 gc, approx. 2.1×10 12 gc, approx. 2.2×10 12gc, approx. 2.3×10 12 gc, approx. 2.4×10 12 gc, approx. 2.5×10 12 gc, approx. 2.6×10 12 gc, approx. 2.7×10 12 gc, approx. 2.8×10 12 gc, approx. 2.9×10 12 gc, approx. 3.0×10 12 gc, approx. 4.0×10 12 gc, approx. 5.0×10 12 gc, approx. 6.0×10 12 gc, approx. 7.0×10 12 gc, approx. 8.0×10 12 gc, or approximately 9.0 × 10 12 It is administered in a fixed dose of gc, or any dose in between.

[0152] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 13 gc~approx. 9.0×10 13 It is administered at a fixed dose of gc. In some cases, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 13 gc, approx. 1.4×10 13 gc, approx. 1.5×10 13 gc, approx. 2.0×10 13 gc, approx. 2.1×10 13 gc, approx. 2.2×10 13 gc, approx. 2.3×10 13 gc, approx. 2.4×10 13 gc, approx. 2.5×10 13 gc, approx. 2.6×10 13 gc, approx. 2.7×10 13 gc, approx. 2.8×10 13 gc, approx. 2.9×10 13 gc, approx. 3.0×10 13 gc, approx. 4.0×10 13 gc, approx. 5.0×10 13 gc, approx. 6.0×10 13 gc, approx. 7.0×10 13 gc, approx. 8.0×10 13 gc, or approximately 9.0 × 10 13 It is administered in a fixed dose of gc, or any dose in between.

[0153] In some scenarios, the AAV vectors of the present disclosure are about 1.0×10 9 gc, about 1.1×10 9 gc, about 1.2×10 9 gc, about 1.3×10 9 gc, about 1.4×10 9 gc, about 1.5×10 9 gc, about 1.6×10 9 gc, about 1.7×10 9 gc, about 1.8×10 9 gc, about 1.9×10 9 gc, about 2.0×10 9 gc, about 2.1×10 9 gc, about 2.2×10 9 gc, about 2.3×10 9 gc, about 2.4×10 9 gc, about 2.5×10 9 gc, about 2.6×10 9 gc, about 2.7×10 9 gc, about 2.8×10 9 gc, about 2.9×10 9 gc, about 3.0×10 9 gc, about 3.1×10 9 gc, about 3.2×10 9 gc, about 3.3×10 9 gc, about 3.4×10 9 gc, about 3.5×10 9 gc, about 3.6×10 9 gc, about 3.7×10 9 gc, about 3.8×10 9 gc, about 3.9×10 9 gc, about 4.0×10 9 gc, about 4.1×10 9 gc, about 4.2×10 9 gc, about 4.3×10 9 gc, about 4.4×10 9 gc, about 4.5×10 9 gc, about 4.6×10 9 gc, about 4.7×10 9 gc, about 4.8×10 9 gc, about 4.9×10 9 gc, about 5.0×10 9gc, approximately 5.1 × 10 9 gc, approximately 5.2 × 10 9 gc, approximately 5.3 × 10 9 gc, approximately 5.4 × 10 9 gc, approximately 5.5 × 10 9 gc, approximately 5.6 × 10 9 gc, approximately 5.7 × 10 9 gc, approximately 5.8 × 10 9 gc, approximately 5.9 × 10 9 gc, approximately 6.0 × 10 9 gc, approximately 6.1 × 10 9 gc, approximately 6.2 × 10 9 gc, approximately 6.3 × 10 9 gc, approximately 6.4 × 10 9 gc, approximately 6.5 × 10 9 gc, approximately 6.6 × 10 9 gc, approximately 6.7 × 10 9 gc, approximately 6.8 × 10 9 gc, approximately 6.9 × 10 9 gc, approximately 7.0 × 10 9 gc, approximately 7.1 × 10 9 gc, approximately 7.2 × 10 9 gc0, approximately 7.3 × 10 9 gc, approximately 7.4 × 10 9 gc, approximately 7.5 × 10 9 gc, approximately 7.6 × 10 9 gc, approximately 7.7 × 10 9 gc, approximately 7.8 × 10 9 gc, approximately 7.9 × 10 9 gc, approximately 8.0 × 10 9 gc, approximately 8.1 × 10 9 gc, approximately 8.2 × 10 9 gc, approximately 8.3 × 10 9 gc, approximately 8.4 × 10 9 gc, approximately 8.5 × 10 9 gc, approximately 8.6 × 10 9 gc, approximately 8.7 × 10 9 gc, approximately 8.8 × 10 9 gc, approximately 8.9 × 10 9 gc, approximately 9.0 × 10 9 gc, approximately 9.1 × 10 9 gc, approximately 9.2 × 10 9 gc, approximately 9.3 × 10 9 gc, approximately 9.4 × 109 gc, approx. 9.5×10 9 gc, approx. 9.6×10 9 gc, approx. 9.7×10 9 gc, approx. 9.8×10 9 gc, or approximately 9.9 × 10 9 It is administered in the total dose of gc, or any dose in between.

[0154] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 10 gc, approx. 1.1×10 10 gc, approx. 1.2×10 10 gc, approx. 1.3×10 10 gc, approx. 1.4×10 10 gc, approx. 1.5×10 10 gc, approx. 1.6×10 10 gc, approx. 1.7×10 10 gc, approx. 1.8×10 10 gc, approx. 1.9×10 10 gc, approx. 2.0×10 10 gc, approx. 2.1×10 10 gc, approx. 2.2×10 10 gc, approx. 2.3×10 10 gc, approx. 2.4×10 10 gc, approx. 2.5×10 10 gc, approx. 2.6×10 10 gc, approx. 2.7×10 10 gc, approx. 2.8×10 10 gc, approx. 2.9×10 10 gc, approx. 3.0×10 10 gc, approx. 3.1×10 10 gc, approx. 3.2×10 10 gc, approx. 3.3×10 10 gc, approx. 3.4×10 10 gc, approx. 3.5×10 10 gc, approx. 3.6×10 10 gc, approx. 3.7×10 10 gc, approx. 3.8×10 10 gc, approx. 3.9×10 10 gc, approx. 4.0×10 10 gc, approx. 4.1×10 10 gc, approx. 4.2×10 10 gc, approx. 4.3×10 10gc, approximately 4.4 × 10 10 gc, approximately 4.5 × 10 10 gc, approximately 4.6 × 10 10 gc, approximately 4.7 × 10 10 gc, approximately 4.8 × 10 10 gc, approximately 4.9 × 10 10 gc, approximately 5.0 × 10 10 gc, approximately 5.1 × 10 10 gc, approximately 5.2 × 10 10 gc, approximately 5.3 × 10 10 gc, approximately 5.4 × 10 10 gc, approximately 5.5 × 10 10 gc, approximately 5.6 × 10 10 gc, approximately 5.7 × 10 10 gc, approximately 5.8 × 10 10 gc, approximately 5.9 × 10 10 gc, approximately 6.0 × 10 10 gc, approximately 6.1 × 10 10 gc, approximately 6.2 × 10 10 gc, approximately 6.3 × 10 10 gc, approximately 6.4 × 10 10 gc, approximately 6.5 × 10 10 gc, approximately 6.6 × 10 10 gc, approximately 6.7 × 10 10 gc, approximately 6.8 × 10 10 gc, approximately 6.9 × 10 10 gc, approximately 7.0 × 10 10 gc, approximately 7.1 × 10 10 gc, approximately 7.2 × 10 10 gc, approximately 7.3 × 10 10 gc, approximately 7.4 × 10 10 gc, approximately 7.5 × 10 10 gc, approximately 7.6 × 10 10 gc, approximately 7.7 × 10 10 gc, approximately 7.8 × 10 10 gc, approximately 7.9 × 10 10 gc, approximately 8.0 × 10 10 gc, approximately 8.1 × 10 10 gc, approximately 8.2 × 10 10 gc, approximately 8.3 × 10 10 gc, approximately 8.4 × 10 10 gc, approximately 8.5 × 10 10 gc, approximately 8.6 × 10 10 gc, approximately 8.7 × 1010 gc, approx. 8.8×10 10 gc, approx. 8.9×10 10 gc, approx. 9.0×10 10 gc, approx. 9.1×10 10 gc, approx. 9.2×10 10 gc, approx. 9.3×10 10 gc, approx. 9.4×10 10 gc, approx. 9.5×10 10 gc, approx. 9.6×10 10 gc, approx. 9.7×10 10 gc, approx. 9.8×10 10 gc, or approximately 9.9 × 10 10 It is administered in the total dose of gc, or any dose in between.

[0155] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 11 gc, approx. 1.1×10 11 gc, approx. 1.2×10 11 gc, approx. 1.3×10 11 gc, approx. 1.4×10 11 gc, approx. 1.5×10 11 gc, approx. 1.6×10 11 gc, approx. 1.7×10 11 gc, approx. 1.8×10 11 gc, approx. 1.9×10 11 gc, approx. 2.0×10 11 gc, approx. 2.1×10 11 gc, approx. 2.2×10 11 gc, approx. 2.3×10 11 gc, approx. 2.4×10 11 gc, approx. 2.5×10 11 gc, approx. 2.6×10 11 gc, approx. 2.7×10 11 gc, approx. 2.8×10 11 gc, approx. 2.9×10 11 gc, approx. 3.0×10 11 gc, approx. 3.1×10 11 gc, approx. 3.2×10 11 gc, approx. 3.3×10 11 gc, approx. 3.4×10 11 gc, approx. 3.5×10 11 gc, approx. 3.6×10 11gc, approximately 3.7 × 10 11 gc, approximately 3.8 × 10 11 gc, approximately 3.9 × 10 11 gc, approximately 4.0 × 10 11 gc, approximately 4.1 × 10 11 gc, approximately 4.2 × 10 11 gc, approximately 4.3 × 10 11 gc, approximately 4.4 × 10 11 gc, approximately 4.5 × 10 11 gc, approximately 4.6 × 10 11 gc, approximately 4.7 × 10 11 gc, approximately 4.8 × 10 11 gc, approximately 4.9 × 10 11 gc, approximately 5.0 × 10 11 gc, approximately 5.1 × 10 11 gc, approximately 5.2 × 10 11 gc, approximately 5.3 × 10 11 gc, approximately 5.4 × 10 11 gc, approximately 5.5 × 10 11 gc, approximately 5.6 × 10 11 gc, approximately 5.7 × 10 11 gc, approximately 5.8 × 10 11 gc, approximately 5.9 × 10 11 gc, approximately 6.0 × 10 11 gc, approximately 6.1 × 10 11 gc, approximately 6.2 × 10 11 gc, approximately 6.3 × 10 11 gc, approximately 6.4 × 10 11 gc, approximately 6.5 × 10 11 gc, approximately 6.6 × 10 11 gc, approximately 6.7 × 10 11 gc, approximately 6.8 × 10 11 gc, approximately 6.9 × 10 11 gc, approximately 7.0 × 10 11 gc, approximately 7.1 × 10 11 gc, approximately 7.2 × 10 11 gc, approximately 7.3 × 10 11 gc, approximately 7.4 × 10 11 gc, approximately 7.5 × 10 11 gc, approximately 7.6 × 10 11 gc, approximately 7.7 × 10 11 gc, approximately 7.8 × 10 11 gc, approximately 7.9 × 10 11 gc, approximately 8.0 × 1011 gc, approx. 8.1×10 11 gc, approx. 8.2×10 11 gc, approx. 8.3×10 11 gc, approx. 8.4×10 11 gc, approx. 8.5×10 11 gc, approx. 8.6×10 11 gc, approx. 8.7×10 11 gc, approx. 8.8×10 11 gc, approx. 8.9×10 11 gc, approx. 9.0×10 11 gc, approx. 9.1×10 11 gc, approx. 9.2×10 11 gc, approx. 9.3×10 11 gc, approx. 9.4×10 11 gc, approx. 9.5×10 11 gc, approx. 9.6×10 11 gc, approx. 9.7×10 11 gc, approx. 9.8×10 11 gc, or approximately 9.9 × 10 11 It is administered in the total dose of gc, or any dose in between.

[0156] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 12 gc, approx. 1.1×10 12 gc, approx. 1.2×10 12 gc, approx. 1.3×10 12 gc, approx. 1.4×10 12 gc, approx. 1.5×10 12 gc, approx. 1.6×10 12 gc, approx. 1.7×10 12 gc, approx. 1.8×10 12 gc, approx. 1.9×10 12 gc, approx. 2.0×10 12 gc, approx. 2.1×10 12 gc, approx. 2.2×10 12 gc, approx. 2.3×10 12 gc, approx. 2.4×10 12 gc, approx. 2.5×10 12 gc, approx. 2.6×10 12 gc, approx. 2.7×10 12 gc, approx. 2.8×10 12 gc, approx. 2.9×10 12gc, approximately 3.0 × 10 12 gc, approximately 3.1 × 10 12 gc, approximately 3.2 × 10 12 gc, approximately 3.3 × 10 12 gc, approximately 3.4 × 10 12 gc, approximately 3.5 × 10 12 gc, approximately 3.6 × 10 12 gc, approximately 3.7 × 10 12 gc, approximately 3.8 × 10 12 gc, approximately 3.9 × 10 12 gc, approximately 4.0 × 10 12 gc, approximately 4.1 × 10 12 gc, approximately 4.2 × 10 12 gc, approximately 4.3 × 10 12 gc, approximately 4.4 × 10 12 gc, approximately 4.5 × 10 12 gc, approximately 4.6 × 10 12 gc, approximately 4.7 × 10 12 gc, approximately 4.8 × 10 12 gc, approximately 4.9 × 10 12 gc, approximately 5.0 × 10 12 gc, approximately 5.1 × 10 12 gc, approximately 5.2 × 10 12 gc, approximately 5.3 × 10 12 gc, approximately 5.4 × 10 12 gc, approximately 5.5 × 10 12 gc, approximately 5.6 × 10 12 gc, approximately 5.7 × 10 12 gc, approximately 5.8 × 10 12 gc, approximately 5.9 × 10 12 gc, approximately 6.0 × 10 12 gc, approximately 6.1 × 10 12 gc, approximately 6.2 × 10 12 gc, approximately 6.3 × 10 12 gc, approximately 6.4 × 10 12 gc, approximately 6.5 × 10 12 gc, approximately 6.6 × 10 12 gc, approximately 6.7 × 10 12 gc, approximately 6.8 × 10 12 gc, approximately 6.9 × 10 12 gc, approximately 7.0 × 10 12 gc, approximately 7.1 × 10 12 gc, approximately 7.2 × 10 12 gc, approximately 7.3 × 1012 gc, approx. 7.4×10 12 gc, approx. 7.5×10 12 gc, approx. 7.6×10 12 gc, approx. 7.7×10 12 gc, approx. 7.8×10 12 gc, approx. 7.9×10 12 gc, approx. 8.0×10 12 gc, approx. 8.1×10 12 gc, approx. 8.2×10 12 gc, approx. 8.3×10 12 gc, approx. 8.4×10 12 gc, approx. 8.5×10 12 gc, approx. 8.6×10 12 gc, approx. 8.7×10 12 gc, approx. 8.8×10 12 gc, approx. 8.9×10 12 gc, approx. 9.0×10 12 gc, approx. 9.1×10 12 gc, approx. 9.2×10 12 gc, approx. 9.3×10 12 gc, approx. 9.4×10 12 gc, approx. 9.5×10 12 gc, approx. 9.6×10 12 gc, approx. 9.7×10 12 gc, approx. 9.8×10 12 gc, or approximately 9.9 × 10 12 It is administered in the total dose of gc, or any dose in between.

[0157] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 13 gc, approx. 1.1×10 13 gc, approx. 1.2×10 13 gc, approx. 1.3×10 13 gc, approx. 1.4×10 13 gc, approx. 1.5×10 13 gc, approx. 1.6×10 13 gc, approx. 1.7×10 13 gc, approx. 1.8×10 13 gc, approx. 1.9×10 13 gc, approx. 2.0×10 13 gc, approx. 2.1×10 13 gc, approx. 2.2×10 13gc, approximately 2.3 × 10 13 gc, approximately 2.4 × 10 13 gc, approximately 2.5 × 10 13 gc, approximately 2.6 × 10 13 gc, approximately 2.7 × 10 13 gc, approximately 2.8 × 10 13 gc, approximately 2.9 × 10 13 gc, approximately 3.0 × 10 13 gc, approximately 3.1 × 10 13 gc, approximately 3.2 × 10 13 gc, approximately 3.3 × 10 13 gc, approximately 3.4 × 10 13 gc, approximately 3.5 × 10 13 gc, approximately 3.6 × 10 13 gc, approximately 3.7 × 10 13 gc, approximately 3.8 × 10 13 gc, approximately 3.9 × 10 13 gc, approximately 4.0 × 10 13 gc, approximately 4.1 × 10 13 gc, approximately 4.2 × 10 13 gc, approximately 4.3 × 10 13 gc, approximately 4.4 × 10 13 gc, approximately 4.5 × 10 13 gc, approximately 4.6 × 10 13 gc, approximately 4.7 × 10 13 gc, approximately 4.8 × 10 13 gc, approximately 4.9 × 10 13 gc, approximately 5.0 × 10 13 gc, approximately 5.1 × 10 13 gc, approximately 5.2 × 10 13 gc, approximately 5.3 × 10 13 gc, approximately 5.4 × 10 13 gc, approximately 5.5 × 10 13 gc, approximately 5.6 × 10 13 gc, approximately 5.7 × 10 13 gc, approximately 5.8 × 10 13 gc, approximately 5.9 × 10 13 gc, approximately 6.0 × 10 13 gc, approximately 6.1 × 10 13 gc, approximately 6.2 × 10 13 gc, approximately 6.3 × 10 13 gc, approximately 6.4 × 10 13 gc, approximately 6.5 × 10 13 gc, approximately 6.6 × 1013 gc, approx. 6.7×10 13 gc, approx. 6.8×10 13 gc, approx. 6.9×10 13 gc, approx. 7.0×10 13 gc, approx. 7.1×10 13 gc, approx. 7.2×10 13 gc, approx. 7.3×10 13 gc, approx. 7.4×10 13 gc, approx. 7.5×10 13 gc, approx. 7.6×10 13 gc, approx. 7.7×10 13 gc, approx. 7.8×10 13 gc, approx. 7.9×10 13 gc, approx. 8.0×10 13 gc, approx. 8.1×10 13 gc, approx. 8.2×10 13 gc, approx. 8.3×10 13 gc, approx. 8.4×10 13 gc, approx. 8.5×10 13 gc, approx. 8.6×10 13 gc, approx. 8.7×10 13 gc, approx. 8.8×10 13 gc, approx. 8.9×10 13 gc, approx. 9.0×10 13 gc, approx. 9.1×10 13 gc, approx. 9.2×10 13 gc, approx. 9.3×10 13 gc, approx. 9.4×10 13 gc, approx. 9.5×10 13 gc, approx. 9.6×10 13 gc, approx. 9.7×10 13 gc, approx. 9.8×10 13 gc, or approximately 9.9 × 10 13 It is administered in the total dose of gc, or any dose in between.

[0158] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 14 gc, approx. 1.1×10 14 gc, approx. 1.2×10 14 gc, approx. 1.3×10 14 gc, approx. 1.4×10 14 gc, approx. 1.5×10 14gc, approximately 1.6 × 10 14 gc, approximately 1.7 × 10 14 gc, approximately 1.8 × 10 14 gc, approximately 1.9 × 10 14 gc, approximately 2.0 × 10 14 gc, approximately 2.1 × 10 14 gc, approximately 2.2 × 10 14 gc, approximately 2.3 × 10 14 gc, approximately 2.4 × 10 14 gc, approximately 2.5 × 10 14 gc, approximately 2.6 × 10 14 gc, approximately 2.7 × 10 14 gc, approximately 2.8 × 10 14 gc, approximately 2.9 × 10 14 gc, approximately 3.0 × 10 14 gc, approximately 3.1 × 10 14 gc, approximately 3.2 × 10 14 gc, approximately 3.3 × 10 14 gc, approximately 3.4 × 10 14 gc, approximately 3.5 × 10 14 gc, approximately 3.6 × 10 14 gc, approximately 3.7 × 10 14 gc, approximately 3.8 × 10 14 gc, approximately 3.9 × 10 14 gc, approximately 4.0 × 10 14 gc, approximately 4.1 × 10 14 gc, approximately 4.2 × 10 14 gc, approximately 4.3 × 10 14 gc, approximately 4.4 × 10 14 gc, approximately 4.5 × 10 14 gc, approximately 4.6 × 10 14 gc, approximately 4.7 × 10 14 gc, approximately 4.8 × 10 14 gc, approximately 4.9 × 10 14 gc, approximately 5.0 × 10 14 gc, approximately 5.1 × 10 14 gc, approximately 5.2 × 10 14 gc, approximately 5.3 × 10 14 gc, approximately 5.4 × 10 14 gc, approximately 5.5 × 10 14 gc, approximately 5.6 × 10 14 gc, approximately 5.7 × 10 14 gc, approximately 5.8 × 10 14 gc, approximately 5.9 × 1014 gc, approx. 6.0×10 14 gc, approx. 6.1×10 14 gc, approx. 6.2×10 14 gc, approx. 6.3×10 14 gc, approx. 6.4×10 14 gc, approx. 6.5×10 14 gc, approx. 6.6×10 14 gc, approx. 6.7×10 14 gc, approx. 6.8×10 14 gc, approx. 6.9×10 14 gc, approx. 7.0×10 14 gc, approx. 7.1×10 14 gc, approx. 7.2×10 14 gc, approx. 7.3×10 14 gc, approx. 7.4×10 14 gc, approx. 7.5×10 14 gc, approx. 7.6×10 14 gc, approx. 7.7×10 14 gc, approx. 7.8×10 14 gc, approx. 7.9×10 14 gc, approx. 8.0×10 14 gc, approx. 8.1×10 14 gc, approx. 8.2×10 14 gc, approx. 8.3×10 14 gc, approx. 8.4×10 14 gc, approx. 8.5×10 14 gc, approx. 8.6×10 14 gc, approx. 8.7×10 14 gc, approx. 8.8×10 14 gc, approx. 8.9×10 14 gc, approx. 9.0×10 14 gc, approx. 9.1×10 14 gc, approx. 9.2×10 14 gc, approx. 9.3×10 14 gc, approx. 9.4×10 14 gc, approx. 9.5×10 14 gc, approx. 9.6×10 14 gc, approx. 9.7×10 14 gc, approx. 9.8×10 14 gc, or approximately 9.9 × 10 14 It is administered in the total dose of gc, or any dose in between.

[0159] In some scenarios, the AAV vectors of the present disclosure are about 1.0×10 15 gc, about 1.1×10 15 gc, about 1.2×10 15 gc, about 1.3×10 15 gc, about 1.4×10 15 gc, about 1.5×10 15 gc, about 1.6×10 15 gc, about 1.7×10 15 gc, about 1.8×10 15 gc, about 1.9×10 15 gc, about 2.0×10 15 gc, about 2.1×10 15 gc, about 2.2×10 15 gc, about 2.3×10 15 gc, about 2.4×10 15 gc, about 2.5×10 15 gc, about 2.6×10 15 gc, about 2.7×10 15 gc, about 2.8×10 15 gc, about 2.9×10 15 gc, about 3.0×10 15 gc, about 3.1×10 15 gc, about 3.2×10 15 gc, about 3.3×10 15 gc, about 3.4×10 15 gc, about 3.5×10 15 gc, about 3.6×10 15 gc, about 3.7×10 15 gc, about 3.8×10 15 gc, about 3.9×10 15 gc, about 4.0×10 15 gc, about 4.1×10 15 gc, about 4.2×10 15 gc, about 4.3×10 15 gc, about 4.4×10 15 gc, about 4.5×10 15 gc, about 4.6×10 15 gc, about 4.7×10 15 gc, about 4.8×10 15 gc, about 4.9×10 15 gc, about 5.0×10 15 gc, about 5.1×10 15gc, approximately 5.2 × 10 15 gc, approximately 5.3 × 10 15 gc, approximately 5.4 × 10 15 gc, approximately 5.5 × 10 15 gc, approximately 5.6 × 10 15 gc, approximately 5.7 × 10 15 gc, approximately 5.8 × 10 15 gc, approximately 5.9 × 10 15 gc, approximately 6.0 × 10 15 gc, approximately 6.1 × 10 15 gc, approximately 6.2 × 10 15 gc, approximately 6.3 × 10 15 gc, approximately 6.4 × 10 15 gc, approximately 6.5 × 10 15 gc, approximately 6.6 × 10 15 gc, approximately 6.7 × 10 15 gc, approximately 6.8 × 10 15 gc, approximately 6.9 × 10 15 gc, approximately 7.0 × 10 15 gc, approximately 7.1 × 10 15 gc, approximately 7.2 × 10 15 gc, approximately 7.3 × 10 15 gc, approximately 7.4 × 10 15 gc, approximately 7.5 × 10 15 gc, approximately 7.6 × 10 15 gc, approximately 7.7 × 10 15 gc, approximately 7.8 × 10 15 gc, approximately 7.9 × 10 15 gc, approximately 8.0 × 10 15 gc, approximately 8.1 × 10 15 gc, approximately 8.2 × 10 15 gc, approximately 8.3 × 10 15 gc, approximately 8.4 × 10 15 gc, approximately 8.5 × 10 15 gc, approximately 8.6 × 10 15 gc, approximately 8.7 × 10 15 gc, approximately 8.8 × 10 15 gc, approximately 8.9 × 10 15 gc, approximately 9.0 × 10 15 gc, approximately 9.1 × 10 15 gc, approximately 9.2 × 10 15 gc, approximately 9.3 × 10 15 gc, approximately 9.4 × 10 15 gc, approximately 9.5 × 1015 gc, approx. 9.6×10 15 gc, approx. 9.7×10 15 gc, approx. 9.8×10 15 gc, or approximately 9.9 × 10⁻⁶ 15 It is administered in the total dose of gc, or any dose in between.

[0160] In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 14 gc~approx. 9.0×10 14 It is administered at a fixed dose of gc. In some aspects, the AAV vector of this disclosure is approximately 1.0 × 10⁻⁶. 14 gc, approx. 1.4×10 14 gc, approx. 1.5×10 14 gc, approx. 2.0×10 14 gc, approx. 2.1×10 14 gc, approx. 2.2×10 14 gc, approx. 2.3×10 14 gc, approx. 2.4×10 14 gc, approx. 2.5×10 14 gc, approx. 2.6×10 14 gc, approx. 2.7×10 14 gc, approx. 2.8×10 14 gc, approx. 2.9×10 14 gc, approx. 3.0×10 14 gc, approx. 4.0×10 14 gc, approx. 5.0×10 14 gc, approx. 6.0×10 14 gc, approx. 7.0×10 14 gc, approx. 8.0×10 14 gc, or approximately 9.0 × 10 14 It is administered in a fixed dose of gc, or any dose in between. In some cases, the AAV vector of this disclosure is approximately 1.4 × 10⁻⁶. 14 It is administered at a fixed dose of GC.

[0161] Any dosage form of the AAV vectors of this disclosure or a method for calculating such dosage form is contemplated herein. In some cases, the dosage is based on brain mass and / or volume. In some cases, the dosage is based on the body weight of the subject. In some cases, the dosage is calculated using the qPCR titer method. In some cases, the dosage is calculated using the ddPCR titer method.

[0162] In some cases, the therapeutically effective dose can be tailored to each AAV capsid serotype. In other cases, the therapeutically effective dose can be tailored to account for differences in cardiac tropism for different AAV capsid serotypes.

[0163] In some scenarios, a single dose of the AAV vector is administered to the subject. In some scenarios, a second, third, fourth, or fifth dose of the AAV vector is administered to the subject. In some scenarios, the second and subsequent doses of the AAV vector may differ from the first dose.

[0164] In some cases, the dose is measured by a quantitative polymerase chain reaction (qPCR) titer. In other cases, the dose is measured by a droplet digital polymerase chain reaction (ddPCR) titer.

[0165] In some cases, immunosuppressants are further administered to the subject along with the administration of the AAV vector. The immunosuppressant may be any immunosuppressant and / or corticosteroid known in the art. The immunosuppressant may be administered at any schedule or interval and in any amount. The immunosuppressant may be administered to improve patient safety, minimize the recipient's immune response to AAV-based therapy, and / or enhance the therapeutic efficacy of AAV-based therapy. In some cases, prednisone is further administered to the subject along with the administration of the AAV vector.

[0166] In some situations, prednisone is administered at the following dosages: 40 mg once daily, one week prior to administration of the AAV virus vector; 40 mg once daily for the first two weeks after administration of the AAV virus vector; 30 mg once daily for three weeks after administration of the AAV virus vector; 20 mg once daily for four weeks after administration of the AAV virus vector; 10 mg once daily for 5 weeks after administration of the AAV virus vector; 5 mg once daily for 6 weeks after administration of the AAV virus vector; For the first seven weeks after administration of the AAV virus vector, 2.5 mg once daily; and 2.5 mg every other day for 8 weeks after administration of the AAV virus vector.

[0167] In some cases, if evidence of an immune response in the recipient is observed after administration of the AAV vector, the immunosuppressant dose can be kept constant (i.e., not tapered as described above) or even increased. In some cases, the maximum dose of prednisone is approximately 60 mg. Once evidence of the recipient's immune response has subsided or decreased, tapering of the immunosuppressant can be initiated.

[0168] In some cases, proton pump inhibitors are administered to the patient in addition to prednisone.

[0169] In some aspects, this disclosure relates to a vector comprising a nucleic acid encoding APOE2 for use in the treatment or prevention of Alzheimer's disease in a subject, wherein the AAV vector is delivered to the subject in need, and APOE2 is expressed at a therapeutically effective level by the transduced cells.

[0170] In certain embodiments, the disclosure relates to a vector comprising a nucleic acid encoding APOE2 for reversing the symptoms of Alzheimer's disease in a subject in need, wherein the AAV vector is delivered to the subject in need, and APOE2 is expressed at a therapeutically effective level by transduced cells.

[0171] Nonviral vectors In certain embodiments, the use of vectors according to this disclosure is a nonviral vector. Typically, a nonviral vector may be a plasmid containing a nucleic acid sequence encoding the APOE2 gene or a variant thereof.

[0172] Pharmaceutical composition In some aspects, this disclosure relates to a pharmaceutical composition for preventing or treating Alzheimer's disease in a subject where such treatment is needed, comprising a therapeutically effective amount of an AAV vector containing a nucleic acid encoding the APOE2 Christchurch mutation.

[0173] This disclosure provides a pharmaceutical composition comprising APOE2 Christchurch mutant rAAV virus particles; the rAAV virus particles comprise an AAVrh10 capsid protein and an APOE2 Christchurch mutant rAAV vector.

[0174] This disclosure provides a pharmaceutical composition comprising APOE3 Christchurch mutant rAAV virus particles; the rAAV virus particles comprise the AAVrh10 capsid protein and the APOE3 Christchurch mutant rAAV vector.

[0175] In some aspects, the pharmaceutical composition is at least about 1.5 × 10 9 gc / mL ~ approx. 1.5×10 11 It contains gc / mL. In some cases, the pharmaceutical composition contains at least about 1.5 × 10⁻⁶ gc / mL. 10 gc / mL, 1.5 × 10 11 gc / mL ~ approx. 1.5×10 15 It contains gc / mL. In some cases, the pharmaceutical composition contains at least about 1.5 × 10⁻⁶ gc / mL. 13 Contains gc / mL

[0176] In some cases, the pharmaceutical composition contains less than approximately 30% empty rAAV capsid.

[0177] In some cases, the pharmaceutical compositions disclosed herein contain less than about 30% empty rAAV capsids. In some cases, the pharmaceutical compositions disclosed herein contain less than about 25% empty rAAV capsids, less than about 20% empty rAAV capsids, or less than about 15% empty rAAV capsids. In some cases, the pharmaceutical compositions contain less than about 10%, less than about 8% empty rAAV capsids, less than 7%, less than about 5%, less than about 3%, or less than 1% empty rAAV capsids. In some cases, the pharmaceutical compositions contain about 1% to about 10% empty rAAV capsids. In some cases, the pharmaceutical composition contains approximately 2% to 30% empty rAAV capsids, approximately 2% to 25% empty rAAV capsids, approximately 2% to 20% empty rAAV capsids, approximately 2% to 15% empty rAAV capsids, and approximately 2% to 10% empty rAAV capsids. In some cases, the pharmaceutical composition contains approximately 2% to 8% empty rAAV capsids. In some cases, the pharmaceutical composition contains approximately 6% or less empty rAAV capsids, approximately 5% empty rAAV capsids, approximately 4% empty rAAV capsids, approximately 3% empty rAAV capsids, approximately 2% empty rAAV capsids, or approximately 1% empty rAAV capsids. In some cases, the number of empty rAAV capsids is below the detection limit.

[0178] In some cases, the percentage of empty rAAV capsids is determined as a percentage of total rAAV capsids, for example, using analytical ultracentrifugation (AUC). In some cases, AUC is sedimentation velocity AUV (SV-AUC). In some cases, these low percentages of empty rAAV particles improve the efficacy of the treatment and / or reduce adverse events (e.g., inflammatory response, liver injury) after administration to a subject, compared to administration of a composition having a high percentage of empty rAAV particles. In some cases, the methods for preparing the rAAV compositions disclosed herein provide these low percentages of empty rAAV particles compared to the level of empty rAAV particles produced by other methods, e.g., by methods that do not use the generation and / or purification methods described herein. In some cases, the pharmaceutical compositions disclosed herein contain at least 80% complete rAAV particles. In some aspects, the pharmaceutical composition contains at least 85% complete rAAV particles, at least 90% complete rAAV particles, or at least 95% complete rAAV particles.

[0179] “Therapeutic effective dose” means the amount of the AAV vector of this disclosure sufficient to treat Alzheimer’s disease with a reasonable benefit / risk ratio applicable to any medical treatment.

[0180] It will be understood that the single dose or total daily dose of the compounds and compositions disclosed herein is to be determined by the attending physician within the bounds of appropriate medical judgment. A specific therapeutically effective dose level for any particular patient is likely to depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the patient's age, weight, overall health, sex, and diet; the timing, route of administration, and elimination rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific polypeptide used; and similar factors well known in the medical field. For example, starting with a dose of the compound at a level lower than necessary to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved is well within the scope of the art. However, the daily dose of the product can vary widely per adult per day. The therapeutically effective dose of the vectors to be administered according to this disclosure, as well as the number of viral or nonviral particles and / or the dose for treating pathological conditions using the pharmaceutical compositions according to this disclosure, are thought to depend on a number of factors, including the patient's age and condition, the severity of the disturbance or disorder, the method and frequency of administration, and the specific peptides used.

[0181] The pharmaceutical compositions comprising AAV vectors provided herein may be in any form suitable for a selected mode of administration, such as C1-C2 administration or ICM administration.

[0182] The pharmaceutical compositions of this disclosure for administration to the CNS can be administered to animals and humans in unit dose forms, either alone or in combination with other active ingredients, as mixtures with conventional pharmaceutical supports.

[0183] In one aspect, a pharmaceutical composition comprises a pharmaceutically acceptable vehicle for an injectable formulation. These may be dry, particularly lyophilized, compositions that enable the formation of an injectable solution, in particular with the addition of isotonic sterile saline (such as monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, or mixtures of such salts), or optionally, sterilized water or physiological saline.

[0184] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid. It must be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi.

[0185] Solutions containing the compounds of this disclosure as free bases or pharmacokinetically acceptable salts can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0186] The AAV vectors according to this disclosure can be formulated into neutral or salt forms. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0187] The carrier may also be a solvent or dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it may be preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectable composition can be provided by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0188] Sterile injectable solutions are prepared by incorporating active polypeptides, optionally together with some of the other components listed above, in a suitable solvent in appropriate amounts, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a base dispersion medium and other components from those listed above. For sterile powders for preparing sterile injectable solutions, exemplary preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredients + any further desired ingredients from those solutions that have been previously sterilized by filtration.

[0189] Once formulated, the solution is expected to be administered in a manner suitable for the formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as the injectable solution type described above, but drug-releasing capsules can also be used.

[0190] Multiple doses can also be administered.

[0191] In some aspects, the pharmaceutical compositions comprising the rAAV viral vector of this disclosure are formulated with more excipients suitable for administration to the target subject in need by any appropriate method of administration. In some aspects, one or more excipients include phosphate buffer and salts. In some aspects, one or more excipients include Tris buffer and salts.

[0192] In some situations, Tris buffer is used at concentrations ranging from approximately 0.01 mM to approximately 100 mM. In other situations, Tris buffer is used at concentrations of approximately 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM, or any concentration in between.

[0193] In some situations, phosphate buffer contains monobasic potassium phosphate and dibasic sodium phosphate. In some situations, monobasic potassium phosphate is used at concentrations ranging from approximately 0.01 mM to approximately 100 mM. In some situations, monobasic potassium phosphate is used at concentrations of approximately 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.25 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, or 30 mM, or any concentration in between. In some situations, monobasic potassium phosphate is used at a concentration of approximately 1 mM. In some situations, dibasic sodium phosphate is used at concentrations ranging from approximately 0.01 mM to approximately 100 mM. In some situations, dibasic sodium phosphate is used at concentrations of approximately 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, or 30 mM, or any concentration in between. In some situations, dibasic sodium phosphate is used at a concentration of approximately 3 mM.

[0194] In some cases, salt is sodium chloride. In some cases, sodium chloride is concentrated at a concentration of approximately 0.01 mM to 1 M. In some cases, sodium chloride is concentrated at a concentration of approximately 25 mM to 300 mM. In some cases, sodium chloride is concentrated at a concentration of approximately 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 155 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some cases, sodium chloride is concentrated at a concentration of approximately 200 mM.

[0195] In some situations, pharmaceutical compositions are formulated at a pH suitable for administration to the target. In some situations, the pH of the pharmaceutical composition is approximately 6.0 to approximately 9.0. In some situations, the pH of the pharmaceutical composition is approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, or approximately 8.0. In some situations, the pH of the pharmaceutical composition is approximately 7.4. In some situations, the pH of the pharmaceutical composition is approximately 7.6.

[0196] In some aspects, the formulation further contains sucrose. In some aspects, the formulation contains sucrose in concentrations of about 0.1% to 5% or any concentration in between. In some aspects, the formulation contains sucrose in concentrations of about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or about 2.0%. In some aspects, the formulation contains about 1% sucrose.

[0197] In some cases, the formulation further contains magnesium chloride (MgCl2). In some cases, the magnesium chloride is present in concentrations of approximately 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, or 30 mM, or any concentration in between. In some cases, the magnesium chloride is present in a concentration of approximately 1 mM.

[0198] In some cases, the formulation further contains poloxamer. In some cases, poloxamer is poloxamer 188. In some cases, poloxamer 188 is present in concentrations of approximately 0.001% to approximately 1%, or any concentration in between. In some cases, poloxamer 188 is present in concentrations of approximately 0.001%, approximately 0.002%, approximately 0.003%, approximately 0.004%, approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, or approximately 0.1%.

[0199] In some cases, the formulation contains approximately 20 mM Tris, approximately 0.01% Poloxamer 188, approximately 1% Sucrose, approximately 200 mM NaCl, and approximately 1 mM MgCl2 at a pH of approximately 7.6.

[0200] The pharmaceutical compositions of this disclosure can be administered to a subject in any amount deemed appropriate by the attending physician, and may vary depending on the specific requirements of each individual subject. In some cases, the pharmaceutical compositions of this disclosure, including pre-packaged compositions, can be diluted in an appropriate amount of fluid prior to administration. In some cases, when the pharmaceutical compositions of this disclosure are diluted, the amount of diluted pharmaceutical composition administered to a subject is equivalent to the dose of rAAV virus particles provided herein. In other words, if the amount of pharmaceutical composition is increased and thereby the concentration of rAAV virus particles is diluted, the patient is still considered to receive the same total number of either genomic copies of the rAAV vector or rAAV virus capsids as specified by the dose disclosed herein.

[0201] In some cases, the volume of the CSF in question determines the final amount of the pharmaceutical composition. In some cases, the volume of the CSF is measured by magnetic resonance imaging (MRI).

[0202] In some situations, the pharmaceutical composition is administered in a total volume of approximately 0.5 mL, 1 mL, 2 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL, or any amount in between.

[0203] APOE2 AAV virus vector manufacturing This disclosure provides rAAV vectors encoding APOE2 containing the polypeptide Christchurch mutation and methods for generating rAAV viral vectors. In some aspects, the rAAV viral vectors are derived from cell lysates produced by the cell culture and purification methods described herein.

[0204] This disclosure provides rAAV vectors encoding APOE3 containing the polypeptide Christchurch mutation and methods for generating rAAV viral vectors. In some aspects, the rAAV viral vectors are derived from cell lysates produced by the cell culture and purification methods described herein.

[0205] This disclosure provides a method for producing a cell lysate containing an rAAV viral vector, comprising the steps of: (i) obtaining a culture vessel containing HEK293T cells in culture medium; (ii) transfecting HEK293T cells in transfection medium with a first plasmid encoding an APOE2 Christchurch mutant AAV vector or an APOE3 Christchurch mutant AAV vector and a second plasmid encoding AAV Rep protein and AAV Cap protein, wherein the ratio of the second plasmid to the first plasmid is 2:1; (iii) culturing the transfected HEK293T cells in culture medium under conditions that the transfected HEK293T cells produce a recombinant adeno-associated virus (rAAV) viral vector encoding an APOE2 polypeptide; (iv) recovering the transfected HEK293T cells; and (v) lysing the transfected HEK293T cells to produce a cell lysate containing an rAAV viral vector.

[0206] The culture media described herein may be any culture medium capable of culturing mammalian cells, including HEK293T cells, but are not limited to this. In some aspects, the culture medium comprises Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells can be expanded for any length of time required to produce the desired cells. In some aspects, HEK293T cells are obtained after expansion culture for approximately 2 to 5 days.

[0207] Transfection can be carried out in any suitable culture medium. In some cases, the transfection medium contains serum-free DMEM and polyethyleneimine (PEI).

[0208] The transfection of the two plasmids can be carried out in any order. In some cases, the transfection of the first plasmid and the second plasmid can be performed simultaneously.

[0209] In some aspects, prior to transfection, HEK293T cells were approximately 2.0 × 10⁶ 4 ~Approx. 2.0×10 6 cells / cm 2 They are present in the culture vessel at a density of approximately 2.0 × 10⁶. In some aspects, HEK293T cells are present at a density of approximately 2.0 × 10⁶. 5 cells / cm 2 It exists at that density.

[0210] After transfection, cells can be cultured for any appropriate length of time to produce the desired quantity of APOE2 rAAV viral vector or APOE3 rAAV viral vector. In some cases, transfected cells are cultured for approximately 1 to 7 days. In other cases, transfected cells are cultured for approximately 3 days.

[0211] After harvesting the transfected cells, the cells containing the APOE2 Christchurch mutant rAAV virus vector or the APOE3 Christchurch mutant rAAV virus vector are lysed to produce cell lysates containing the APOE2 Christchurch mutant rAAV virus vector or the APOE3 Christchurch mutant rAAV virus vector. Cell lysis can be carried out by any suitable method, including freeze-thaw, mechanical lysis, and lysis with chemical agents such as surfactants. In some cases, HEK293T cells are lysed via at least about four consecutive freeze-thaw cycles to produce cell lysates.

[0212] The cell lysates of this disclosure may be processed to remove any residual uncapsidized virus or cellular DNA. In some aspects, the cell lysates are further processed with recombinant nucleases to digest any viral DNA or cellular DNA. In some aspects, the recombinant nuclease is a benzonase. In some aspects, DNA digestion is carried out in the presence of magnesium chloride.

[0213] After DNA digestion, the lysate containing the APOE2 Christchurch mutant rAAV virus vector or the APOE3 Christchurch mutant rAAV virus vector is clarified. Clarification can be carried out according to any method known in the art. In some cases, clarification is carried out by centrifugation or ultracentrifugation. Clarification of the cell lysate removes cell debris and undestroyed cells.

[0214] After clarification, the number of rAAV vectors or rAAV viral vectors in the cell lysate can be quantified. In some cases, the cell lysate contains approximately 1.0 × 10⁶ per milliliter. 9 ~Approx. 5.0×10 14 Includes genome copy (GC).

[0215] This disclosure further provides a method for producing an APOE2 Christchurch mutant rAAV pharmaceutical composition or an APOE3 Christchurch mutant rAAV pharmaceutical composition, comprising the steps of: (i) obtaining a cell lysate containing an rAAV viral vector encoding an APOE2 Christchurch mutant polypeptide or an APOE3 Christchurch mutant polypeptide; (ii) contacting the cell lysate containing the APOE2 Christchurch mutant polypeptide or an rAAV viral vector encoding an APOE2 Christchurch mutant polypeptide with a density gradient and subjecting the density gradient to centrifugation; (iii) contacting the cell lysate containing the rAAV viral vector encoding an APOE2 Christchurch mutant polypeptide or an APOE3 Christchurch mutant polypeptide with a chromatography column; (iv) eluting rAAV viral particles from the column; and (v) concentrating the eluted rAAV viral particles in a formulation buffer via ultrafiltration, thereby producing an APOE2 Christchurch mutant rAAV pharmaceutical composition or an APOE2 Christchurch mutant rAAV pharmaceutical composition.

[0216] The density gradient of this disclosure may be any suitable density gradient. The density gradient provides a means for separating components in a mixture by their size and / or molecular weight. Using density gradient purification, empty AAV capsids can be separated from AAV capsids containing APOE2 Christchurch mutant rAAV vectors or APOE3 Christchurch mutant rAAV vectors. In some aspects, the density gradient is an iodixanol density gradient.

[0217] In some cases, the iodixanol gradient includes a stepped density gradient containing (i) an iodixanol solution of approximately 10% to 20%; (ii) an iodixanol solution of approximately 20% to 30%; (iii) an iodixanol solution of approximately 40% to 50%; and (iv) an iodixanol solution of approximately 50% to 60%. In some cases, the iodixanol gradient includes a stepped density gradient containing (i) an iodixanol solution of approximately 15%; (ii) an iodixanol solution of approximately 25%; (iii) an iodixanol solution of approximately 40%; and (iv) an iodixanol solution of approximately 54%.

[0218] In some situations, alternative purification methods can be used instead of density gradient purification. In some situations, chromatography can be used. In some situations, ion exchange chromatography can be used.

[0219] Chromatography can be used to purify the rAAV viral vector after density gradient purification. In some cases, the chromatography is anion exchange chromatography. In some cases, the anion exchange column is a Q Sepharose high performance strong quaternary ammonium anion exchange resin column.

[0220] After chromatographic purification, the APOE2 Christchurch mutant rAAV virus vector or the APOE3 Christchurch mutant rAAV virus vector is exchanged for a formulation buffer via ultrafiltration, thereby producing the pharmaceutical composition of this disclosure. In some aspects, the formulation buffer contains phosphate-buffered saline (PBS).

[0221] In some cases, the pharmaceutical composition, after ultracentrifugation, is approximately 1.0 × 10⁶ per milliliter. 10 ~Approx. 5.0×10 13Contains genome copies (viral genome). In some cases, the pharmaceutical composition, after ultracentrifugation, is approximately 1.5 × 10⁶ per milliliter. 13 Includes genome copies (vial genomes).

[0222] This disclosure provides methods for generating rAAV vectors and rAAV viral vectors encoding APOE2 or APOE3 polypeptides containing Christchurch mutations. In some aspects, rAAV vectors and rAAV viral vectors encoding APOE2 or APOE3 polypeptides containing Christchurch mutations can be generated in a baculovirus system using a viral expression construct and a payload construct vector. In certain embodiments, the baculovirus system comprises a baculovirus expression vector (BEV) and / or baculovirus-infected insect cells (BIIC). In certain embodiments, the baculovirus system comprises a baculovirus expression vector (BEV) and / or baculovirus-infected Sf9 insect cells. In certain embodiments, the viral expression construct or payload construct of this disclosure may be a bacmid, which is also known as a baculovirus plasmid or recombinant baculovirus genome. In certain embodiments, the viral expression construct or payload construct of this disclosure may be a polynucleotide incorporated into a bacmid by homologous recombination (transposon donor / acceptor system) by standard molecular biological techniques known to those skilled in the art and performed by those skilled in the art. Transfection of separate viral replication cell populations can produce two or more groups of baculoviruses (BEVs) (e.g., two, three), one or more groups that may contain a viral expression construct (expressed BEV), and one or more groups that may contain a payload construct (payloaded BEV). The baculoviruses can be used to infect virus-producing cells for the purpose of producing AAV particles or viral vectors.

[0223] Baculoviruses are composed of several essential proteins, such as replication proteins, coat proteins, and capsid proteins, which are essential for baculovirus function and replication. Therefore, the baculovirus genome contains several essential gene nucleotide sequences that encode these essential proteins. As an unrestricted example, the genome may include essential gene regions containing essential gene nucleotide sequences that encode essential proteins for the baculovirus construct. Examples of essential proteins include the GP64 baculovirus coat protein, the VP39 baculovirus capsid protein, or other similar essential proteins for the baculovirus construct.

[0224] Baculovirus expression vectors (BEVs) for generating AAV particles in insect cells, including, but not limited to, armyworm (Spodoptera frugiperda) (Sf9) cells, provide high-titer viral vector products. Recombinant baculoviruses encoding the viral expression construct and payload construct initiate generative infection of viral vector-replicating cells. Infectious baculovirus particles released from primary infection secondarily infect further cells in culture, exponentially infecting the entire cell culture population with numerous infection cycles, a function of the initial infection multiplicity. For details regarding the generation and use of BEVs and viral particles, see Urabe, M. et al. J Virol. 2006 Feb;80(4): 1874-85, the contents of which are incorporated entirely herein by reference. The generation of AAV particles using baculoviruses in insect cell systems can address the known genetic and physical instabilities of baculoviruses.

[0225] Listed aspects Enumerated aspect 1. In the direction from 5' to 3', A first AAV ITR sequence containing SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:23; Enhancer sequence including SEQ ID NO:3; Promoter sequence containing SEQ ID NO:4; Chimera Intron containing SEQ ID NO:5; Nucleic acid sequences encoding apolipoprotein 2 (APOE2) polypeptide or apolipoprotein 3 (APOE3) polypeptide containing the Christchurch mutation, including SEQ ID NO:6 or SEQ ID NO:20; Poly-A sequence including SEQ ID NO:7; and A second ITR sequence containing SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:23 A recombinant adeno-associated virus (rAAV) vector containing this virus.

[0226] Enumerated aspect 2. The Christchurch mutation is R154S mutation related to unprocessed APOE polypeptide; or R136S mutation in mature APOE polypeptide lacking signal peptide An rAAV vector according to embodiment 1, including the above.

[0227] Embodiment 3. An rAAV vector comprising any one of the embodiments described above, comprising the nucleic acid sequences described in SEQ ID NO:8, SEQ ID NO:21, and SEQ ID NO:22.

[0228] Embodiment 4 listed above. An rAAV vector of any one of the embodiments, packaged as an rAAV viral vector containing an AAV capsid protein.

[0229] Embodiment 5 listed above. An rAAV vector of any one of the embodiments, wherein the AAV capsid protein is AAV1 capsid protein, AAV2 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAV13 capsid protein, AAVPHP.B capsid protein, AAVrh74 capsid protein, or AAVrh10 capsid protein.

[0230] Embodiment 6 listed above. An rAAV vector according to any one of the embodiments, wherein the AAV capsid protein is the AAVrh10 capsid protein.

[0231] Enumerated aspect 7. A pharmaceutical composition comprising the rAAV viral vector of aspect 4.

[0232] Embodiment 8, enumerated. A method for treating Alzheimer's disease in a human subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to Embodiment 7.

[0233] Enumerated aspect 9. The therapeutically effective amount of the vector is about 1 × 10⁻⁶ 10 ~Approx. 1×10 16 The method of embodiment 8, which involves genome copying.

[0234] Enumerated aspect 10. The method of aspect 8, wherein the subject is an APOE2 / APOE4 heterozygote, an APOE4 / APOE4 homozygote, or an APOE3 / APOE4 heterozygote.

[0235] Enumerated aspect 11. The method of aspect 8, wherein the composition is administered systemically, intracervically, via the large cisterna, or via CI-C2 administration.

[0236] Enumerated aspect 12. The pharmaceutical composition comprises about 5.0 × 10 9 gc / mL CSF ~ approx. 5.0×1012 The method according to embodiment 8, administered in a dose of gc / mL CSF.

[0237] Enumerated aspect 13. The pharmaceutical composition is i) Approximately 1.4 × 10 10 gc / mL CSF, ii) Approximately 4.4×10 10 gc / mL CSF, or iii) Approximately 1.4×10 11 gc / mL CSF The method according to embodiment 8, administered in the specified dose.

[0238] Enumerated aspect 14. The pharmaceutical composition is approximately 1.4 × 10 14 The method according to embodiment 8, administered in a dose of gc.

[0239] Embodiment 15 listed. The method of Embodiment 8, wherein the pharmaceutical composition is administered in a total volume of about 5 mL, about 10 mL, about 15 mL, or about 20 mL.

[0240] Embodiment 16. The method of Embodiment 8, wherein the subject experiences an increase of at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100% in the expression of APOE2 Christchurch.

[0241] Enumerated aspect 17. The method of aspect 8, wherein the manifestation of APOE2 Christchurch or APOE3 Christchurch occurs in the central nervous system.

[0242] Embodiment 18. The method of Embodiment 8, wherein the expression of APOE2 Christchurch or APOE3 Christchurch is measured in cerebrospinal fluid (CSF).

[0243] Embodiment 19. The method of Embodiment 8, wherein, after administration of the pharmaceutical composition, the expression level of at least one of T-tau and P-tau is reduced in the subject compared to baseline before administration.

[0244] Embodiment 20 listed. The method of Embodiment 19, wherein the expression levels of T-tau and / or P-tau are reduced by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100%.

[0245] Enumerated aspect 21. After administration of the pharmaceutical composition, amyloid beta 42 / amyloid beta 40 (Aβ 42 / 40 The method of embodiment 8, wherein the ratio of ) increases.

[0246] Enumerated aspect 22. The Aβ 42 / 40 The method of embodiment 21, wherein the ratio increases by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100%.

[0247] Enumerated aspect 23. The method of aspect 8, wherein the subject is administered an immunosuppressant prior to treatment with the pharmaceutical composition.

[0248] Enumerated aspect 24. The method of aspect 23, wherein the immunosuppressant is prednisone.

[0249] Enumerated aspect 25. The prednisone is 40 mg once daily, one week prior to administration of the AAV virus vector; 40 mg once daily for the first two weeks after administration of the AAV virus vector; 30 mg once daily for three weeks after administration of the AAV virus vector; 20 mg once daily for four weeks after administration of the AAV virus vector; 10 mg once daily for 5 weeks after administration of the AAV virus vector; 5 mg once daily for 6 weeks after administration of the AAV virus vector; For the first seven weeks after administration of the AAV virus vector, 2.5 mg once daily; and For 8 weeks after administration of the AAV virus vector, take 2.5 mg every other day. The method according to embodiment 24, administered in the specified dosage. [Examples]

[0250] The present invention is further illustrated by the following figures and examples. However, these examples and figures should not be construed as limiting the scope of this disclosure.

[0251] Figure 1C shows the experimental design of the studies described in Examples 1 and 2.

[0252] Example 1: Heparin binding of APOE variant Heparan sulfate proteoglycans (HSPGs) have been linked to tau pathogenesis; it has been suggested that HSPGs enable tau fibrils to attach to neurons, thereby promoting the uptake and propagation of toxic forms of tau. APOE variants bind differentially to heparin, with APOE4 binding most strongly to heparin, followed by APOE3, and finally APOE2. Previous studies have shown that APOE3 contains Christchurch mutations that bind to heparin even more weakly than APOE2. Therefore, protection of the APOE3 Christchurch variant may be associated with the loss of binding to heparan sulfate proteoglycans (HSPGs), which are thought to propagate tau tangles.

[0253] method The heparin binding of APOE isoforms was evaluated as described by Futamura et al. (J Biol Chem 2005;280(7):5414-22). Plasmid transfection with a plasmid / PEI (Polyplus, France) complex in serum-free DMEM generated hemagglutinin (HA) 3'-tagged human (h)APOE variants, including hAPOE2, hAPOE2Ch, hAPOE3, hAPOE3Ch, and hAPOE4, in HEK293T cells. After 72 hours, cell medium was collected every 24 hours and supplied with fresh serum-free DMEM three times. Pooled transfected cell medium was centrifuged at 1500 × g to remove cell debris. APOE levels in each sample were quantified by hAPOE-specific ELISA (ab108813, Abcam, UK). To evaluate heparin binding of each isoform, APOE-containing medium was diluted 5-fold in binding buffer (20 mM Tris-HCl, pH 7.5), loaded onto a heparin-binding column (7040703, Cytiva, Marlborough, MA), and maintained at 23°C for 1 hour before use. The column was washed with 5 ml of 20 mM Tris-HCl, pH 7.5 buffer. Each APOE variant (1 ml, 50 μg / ml) in 20 mM Tris-HCl, pH 7.5 was passed through the column five times for recycling. The column was then washed five times with the same buffer. A NaCl gradient of 20 mM Tris-HCl (0.025–1 M, 1 ml per gradient step) was passed through the column, and 1 ml fractions were collected and analyzed for APOE using human APOE-specific ELISA. Three independent experiments were performed for each hAPOE isoform. Based on untransfected cells under the same transfection conditions, the background APOE level for each fraction was subtracted. The quantified binding characteristics of each APOE isoform from the three experiments were averaged and plotted as a function of the NaCl concentration eluted from the column.

[0254] AAV Vector All vectors were based on clade E non-human primate AAVrh.10 serotypes, including AAVrh.10hAPOE2 (encoding a human APOE2 variant), AAVrh.10hAPOE2Ch (encoding human APOE2 Christchurch), and AAVrh.10 Null (as a control; identical but lacking a translatable sequence). Expression cassettes for all vectors in this study used a cytomegalovirus enhancer fused to a chicken beta-actin promoter (CAG) promoter. For some experiments, a hemagglutinin (HA) tag was added to the 3' end of the coding sequence. AAVrh.10 vectors were generated, purified, and characterized as previously described in Rosenberg et al., (Hum Gene Ther Clin Dev 2018;29(1):24-47). HEK293T cells were transfected with expression cassette plasmids (pAAV-hAPOE2, pAAV-hAPOE2Ch, pAAV-null) and adenovirus / AAVrh.10 helper plasmid (pPAK-MArh.10) expressing the AAVrh.10 cap gene and AAV2 rep gene required for viral replication and capsid formation. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 5% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin, and maintained at 37°C with 5% CO2. Cells were seeded in CellSTACKS (Corning, Tewksbury, MA) at 70-80% confluence for 24 hours and transfected with PEIpro lipid reagent (Polyplus, France). After incubation at 37°C for 72 hours, cells were harvested and lysed by five freeze / thaw cycles. Cell lysates were treated with 50 U / mL benzonase (E1014, Milpore, Billerica, MA) at 37°C for 30 minutes to remove any contaminating genomic DNA. Crude virus lysates were centrifuged at 3300 × g and then purified by discontinuous iodixanol density gradient followed by QHP ion exchange chromatography.The vector was concentrated in phosphate-buffered saline, pH 7.4 (PBS), using a Millipore spin column at 13,000 × g for 15 minutes. The purified vector was filtered and sterilized, tested for bacterial and fungal contamination, and tested for endotoxins. The vector genome titer was determined by TaqMan qPCR (Applied Biosystems, Foster City, CA) using a CAG promoter-specific primer-probe set. The purified AAVrh.10 vector was digested with proteinase K at 70°C for 1 hour in the presence of 0.5% sodium dodecyl sulfate and 25 mM ethylenediamine tetraacetate, followed by protease inactivation at 95°C for 15 minutes. Calibration curves were then constructed using the vector as a template for TaqMan analysis with known copy number AAV plasmid DNA standards. Prior to use, all vectors were tested for expression in an in vivo efficacy assay, as previously described in De et al. (Hum Gene Ther Methods 2018;29(3):146-155).

[0255] result APOE primarily binds to and interacts with heparin via residues 136-150 (Figure 1A). Among common APOE variants, APOE4 has the highest affinity for heparin, APOE3 has an intermediate affinity, and APOE2 has the lowest affinity (see Arboleda-Velasquez et al., Med 2019;25(11):1680-1683; Futamura et al. J Biol Chem 2005;280(7):5414-22). Arboleda-Velasquez et al. showed that the Christchurch variant (APOE3Ch), which possesses the APOE3 variant, has reduced heparin binding compared to APOE2 due to a change in the amino acid at position 136 from arginine to serine. Here, the heparin binding data for APOE variants (APOE4>E3>E2>E3Ch) was replicated compared to the therapeutic E2Ch variant. Interestingly, the E2Ch variant was observed to have lower binding affinity than all other isoforms, including E3Ch (Figure 1B).

[0256] The APOE2 Christchurch variant was found to bind to heparin even more weakly than the APOE3 Christchurch variant. This suggests that the APOE2 Christchurch variant may have a greater protective effect against tau formation and Alzheimer's disease.

[0257] Example 2: In vivo study of an AAV vector encoding the APOE2 Christchurch polypeptide We evaluated the hypothesis that AAVrh.10-mediated expression of the human APOE2 allele (AAVrh.10hAPOE2Ch) combined with the Christchurch mutation would effectively protect against the onset of Alzheimer's disease.

[0258] method Mouse model APOE4 target replacement mice (TRE4; mouse ApoE4 knockout, human APOE4 knock-in) were obtained from Duke University Medical Center. APP.PSEN1 (amyloid, B6C3-Tg / APPswe, PSEN1dE9 / 85#004462 / 034829 MMRRC) and P301S (tau, B6;C3-Tg / Prnp-MAPT*P301S / PS19Vle / J #008169) were both obtained from Jackson Laboratories (Bar Harbor, ME). Both strains were crossed with TRE4 mice to produce APP.PSEN1 / TRE4 (humanized APOE4, amyloid mouse) and P301S / TRE4 (humanized APOE4, tau mouse) strains. Colonies of each mouse were maintained and expanded at the Belfer Gene Therapy Core Facility. Genotype was confirmed by analysis of tail snips or ear punches (Transnetyx, Cordova, TN). APP.PSEN1 / TRE4 mice were treated at 2.5 months and evaluated at 5.5–6 months. P301 / PSEN1 / TRE4 mice were treated at 5.5 months and evaluated at 8.5–9 months.

[0259] Vector administration and experimental group Intrahippocampal injection of AAV vector was performed by stereotactic fixation surgery as previously described (see Zhao et al., Neurobiol Aging 2016;44(159-172)). The animals were anesthetized with isoflurane and positioned on a stereotactic fixation frame (Harvard Apparatus, Holliston, MA). After skin incision, a needle-sized burr hole was made using a high-speed drill. Vector (2 × 10⁶ per 2 μl dose) 10Gc) was administered bilaterally to the hippocampus, with 2 μl administered over 10 minutes at a rate of 0.2 μl / min using a 33-gauge needle (Hamilton, Reno NV) and syringe pump (KD Scientific, Holliston, MA) (stereotactic coordinates: 1.7 mm anterior-posterior from the bregma; 1.2 mm medial-lateral from the bregma; and 1.7 mm subdural-dorsal-ventral). The injection needle was left in place for 4 minutes to minimize reflux and then slowly withdrawn. Postoperatively, mice were housed in separate cages for 3 months, and their overall health and body weight were monitored daily throughout the experiment.

[0260] Amyloid mice were treated at 2.5 months of age, and tau mice were treated at 5.5 months of age. Three months after treatment, all mice were evaluated for behavior over two weeks (see below for details), then sacrificed by CO2 gas asphyxiation and perfused transcardially with 0.3% heparinized saline (2500 IU / mL). Brains were rapidly collected and each was divided along the sagittal plane, and one hemisphere was processed for histological and immunohistochemical analysis. The isolated hemispheres were microscopically dissected under a stereomicroscope. The meninges were removed, and midbrain tissue was extracted to visualize the hippocampus. Based on macroscopic morphology, the hippocampus was carefully removed without stripping any cortex, then rapidly frozen on dry ice and stored at -80°C for biochemical analysis.

[0261] Amyloid mice were administered AAV vectors expressing E2-HA (n=13, 8M / 5F), E2CH-HA (n=13, 7M / 6F), E2 (n=22, 12M / 10F), E2Ch (n=24, 12M / 12F), null (n=27, 17M / 10F), or PBS (n=34, 14M / 20F). Tau mice were administered AAV vectors expressing E2-HA (n=11, 6M / 5F), E2CH-HA (n=9, 5M / 4F), E2 (n=19, 10M / 9F), E2CH (n=23, 14M / 9F), null (n=20, 10M / 10F), or PBS (n=20, 10M / 10F). To avoid injection-side bias, different hemispheres were randomly selected for histological and other analyses.

[0262] Quantification of vector genome copy, transgene expression, and APOE protein levels. To quantify vector-related parameters, dissected brains were thawed and homogenized in ice-cold sterile Tris-buffered saline, pH 7.4 (TBS). Three equal volumes of homogenates were used for the isolation of DNA, RNA, and protein. DNA isolation was performed using the DNeasy tissue / blood DNA isolation kit according to the manufacturer's instructions (Qiagen, Valencia, CA). RNA isolation was performed using Qiazol (Qiagen, Valencia, CA), and the final RNA was enriched on an RNeasy mini-elution column (Qiagen, Valencia, CA). DNA and RNA concentrations were determined using Nanodrop (ThermoFisher Scientific, Waltham, MA). All RNA samples were converted to cDNA using a high-capacity reverse transcription kit (ThermoFisher Scientific). hAPOE mRNA levels were measured using primer / probe:forward. TIFF2026516699000002.tif3128 Reverse Quantification was performed by RT-PCR using TIFF2026516699000003.tif3128. Total protein content was determined by the Bradford assay (see Kielkopf et al. Cold Spring Harb Protoc 2020;2020(4):102269). APOE levels were measured by human APOE-specific ELISA (ab108813, Abcam, UK) and normalized to total protein.

[0263] Quantification of insoluble and soluble Aβ42 and Aβ40 levels A continuous extraction method was used to quantify insoluble and soluble Aβ42 and Aβ40 peptides. Homogenized hippocampal extract in TBS was centrifuged at 100,000 × g at 4°C for 1 hour. The TBS soluble supernatant was divided equally into separate 0.6 ml tubes, frozen in liquid nitrogen, and stored at -80°C. The pellet was washed with 200 μl of TBS buffer and centrifuged at 14,000 × g at 4°C for 5 minutes, and the washings were discarded. The pellet was resuspended in 15 times (weight / tissue volume) of TBS buffer containing 1% Triton X-100 (TBSTX), gently mixed by rotation at 4°C for 30 minutes, and then subjected to a second centrifugation at 100,000 × g at 4°C for 1 hour. The TBSTX soluble supernatant was divided equally into separate 0.6 ml tubes, frozen in liquid nitrogen, and stored at -80°C. The pellet was washed with TBSTX buffer, and the TBSTX buffer was discarded. The TBSTX-insoluble pellet was resuspended in 400 μl of 5M guanidine HCl, mixed by rotation at 23°C for 6 hours, and centrifuged at 16,000 × g for 30 minutes. The peptides resuspended in guanidine HCl (insoluble fraction) were divided equally and frozen. The total protein content in the TBS fraction was quantified using the Bradford assay (see Kielkopf et al. Cold Spring Harb Protoc 2020;2020(4):102269). Due to interference between Triton X-100 and the final buffer of the insoluble fraction in 1% sodium dodecyl sulfate (SDS), total protein was quantified using a transition metal-based protein assay evaluated at 660 nm (polyhydroxybenzenesulfonphthalein type dye, 22660, ThermoFisher Scientific) (see Antharavally et al., Anal Biochem 2009;385(2):342-5). The levels of Aβ42 (KHB3441, ThermoFisher Scientific) and Aβ40 (KHB3481, ThermoFisher Scientific) in the soluble and insoluble fractions were quantified using a commercially available ELISA kit according to the manufacturer's instructions. The total amounts of Aβ42 and Aβ40 were normalized to the total protein content.The TBSTX fraction and the guanidine HCl fraction were considered together as an insoluble fraction, while the TBS fraction was considered a soluble fraction.

[0264] Quantification of insoluble and soluble total tau and tau phosphate levels. Increased levels of tau phosphate phosphorylated at residues such as Thr181, Thr231, and Ser396 are associated with neurofibrillary tangle formation and neuronal dysfunction (see Shi et al., Nature 2017;549(7673):523-527). Total tau levels reflect the overall tau load and, like tau solubility, serve as markers of neurodegeneration (Limorenko et al., Chem Soc Rev 2022;51(2):513-565). A sequential extraction method was used to quantify insoluble and soluble tau. Hippocampal extracts homogenized with TBS were centrifuged at 100,000 × g at 4°C for 1 hour. The TBS soluble supernatant was divided equally into separate 0.6 ml tubes, frozen in liquid nitrogen, and stored at -80°C. The pellet was washed with 200 μl of TBS buffer and centrifuged at 14,000 × g at 4°C for 5 minutes. The pellet was resuspended in 15 times (weight / tissue volume) of TBS buffer containing 1% Triton X-100 (TBSTX), gently mixed at 4°C for 30 minutes, and then subjected to a second centrifugation at 100,000 × g at 4°C for 1 hour. The TBSTX soluble supernatant was divided equally into separate 0.6 ml tubes, frozen in liquid nitrogen, and stored at -80°C. The pellet was washed with TBSTX buffer. The pellet was resuspended in 400 μl of 5 M guanidine HCl, gently mixed at 23°C for 6 hours, and centrifuged at 16,000 × g for 30 minutes. The fraction resuspended in guanidine HCl (insoluble fraction) was divided equally and frozen. Proteins in the insoluble fraction were precipitated using ethanol-chloroform precipitation (Wessel et al. Anal Biochem 1984;138(1):141-3). The total protein content in the TBS fraction was quantified using the Bradford assay (Kielkopf et al. Cold Spring Harb Protoc 2020;2020(4):102269).Due to interference between Triton X-100 and the final buffer of the insoluble fraction in 1% SDS, total protein was quantified using a transition metal-based protein assay evaluated at 660 nm (polyhydroxybenzenesulfonphthalein type dye, 22660, ThermoFisher Scientific) (Antharavally et al., Anal Biochem 2009;385(2):342-5). Total tau and phosphate tau levels in the soluble and insoluble fractions were assayed using ELISA kits (KHB0041 and KHB7031, respectively, ThermoFisher Scientific) according to the manufacturer's instructions. The total amounts of total tau and p-tau were normalized to the total protein content. The TBSTX fraction and guanidine HCl fraction were considered together as the insoluble fraction, and the TBS fraction was considered as the soluble fraction.

[0265] Histological evaluation After necropsy, one hemisphere of the mouse brain was immediately fixed with 4% paraformaldehyde in PBS, embedded in paraffin, and serially sectioned at 5 μm in the sagittal plane (Histoserv, Germantown, MD). Sections identified as hippocampal by macroscopic morphology were deparaffinized using xylene and a series of stepwise ethanol washes, followed by treatment with a low pH antigen retrieval solution (00-4955-58, ThermoFisher Scientific) at 88°C for 20 minutes. The sections were then blocked overnight in a humid chamber at 4°C using SuperBlock Buffer (37515, ThermoFisher Scientific). The sections were then incubated with primary and secondary antibodies at room temperature for 2 hours, as described below.

[0266] MOAB-2. To evaluate beta-amyloid loading in amyloid mice, a MOAB-2 antibody that specifically binds to the N-terminus of beta-amyloid peptides was used (Youmans et al. Mol Neurodegener 2012;7(8)). Staining was performed using MOAB-2 antibody (NBP2-13075, MOAB-2, Novus Biologicals). After incubation with the antibody, sections were washed four times in Tris-buffered saline / 0.01% tween 20 (TBS-T), and for counterstaining, 4',6-diamidino-2-phenylindole (DAPI) solution was used. The sections were incubated for 30 minutes with goat anti-mouse IgG Alexa Flour 488 conjugate antibody (A28175, ThermoFisher Scientific) diluted 1:1,000 in 20% SuperBlock in TBS. The slides were covered with coverslips using EMS-Mount mounting medium (Electron Microscopy Sciences), and then imaged using an EVOS fluorescence microscope (ThermoFisher Scientific).

[0267] Glial fiber acidic proteins (GFAP). GFAP is an intermediate filament protein highly expressed in astrocytes that provides structural support and stability to astrocyte protrusions (Parhizkar et al. Semin Immunol 2022;59:101594). Because GFAP staining specifically targets astrocytes, it was used for visualization and quantification of astrogliosis in amyloid mice and tau mice. After incubation with primary chicken anti-GFAP antibody (ab4674, Abcam), sections were washed four times in TBS-T and incubated for 30 minutes with goat anti-chicken IgY H&L (Alexa Fluor 488, ab150169, Abcam, Waltham MA) diluted 1:1,000 in 20% SuperBlock in TBS, using DAPI for counterstaining. Together with Dabco (Electron Microscopy Sciences), we used EMS Shield Mount mounting medium to place coverslips on the slides, and then imaged them using an EVOS fluorescence microscope (ThermoFisher Scientific).

[0268] Iba-1. Iba-1 is a calcium-binding protein highly expressed by microglia and localized in the cytoplasm and protrusions (Moser et al. iScience 2021;24(11):103238). Iba-1 staining was used to visualize and quantify microglial activation in amyloid and tau mice. After incubation with primary rabbit anti-Iba-1 antibody (019-19741, Fujifilm, Santa Clara, CA), sections were washed four times in TBS-T and incubated for 30 minutes with goat anti-rabbit IgG (A11035, ThermoFisher Scientific) conjugated in Alexa Flour 564 diluted 1:1,000 in 20% SuperBlock in TBS, using DAPI for counterstaining. Slides were covered with coverslips using EMS Shield Mount mounting medium (Electron Microscopy Science) and imaged using an EVOS fluorescence microscope (ThermoFisher Scientific).

[0269] AT8. Total tau loading and the presence of neurofibrillary tangles (intracellular aggregates of abnormal tau protein) were assessed in tau mice using AT8 antibody (Koutsodendris et al., Nat Aging 2023;3(3):275-296). The AT8 antibody specifically targets epitopes phosphorylated at the amino acid residues Ser202 and Thr205 of the tau protein (MN1020, AT8, ThermoFisher Scientific). After incubation with the primary antibody, sections were washed four times in TBS-T and incubated for 30 minutes with goat anti-mouse IgG (A28175, ThermoFisher Scientific) conjugated in Alexa Flour 488 diluted 1:1,000 in 20% SuperBlock in TBS using DAPI for counterstaining. Slides were covered with coverslips using EMS-Mount mounting medium and imaged using EVOS fluorescence microscopy.

[0270] X-34. X-34 (SML1954, Sigma Aldrich) is a small molecule dye, a fluorescent derivative of Congo red, that has affinity for Aβ aggregates and produces a clear fluorescent signal (Ulrich et al., J Exp Med 2018;215(4):1047-1058). X-34 staining was used to assess total amyloid loading to quantify amyloid aggregates in amyloid mice (Styren et al. J Histochem Cytochem 2000;48(9):1223-32). After continuous ethanol washing, slides were incubated in PBS for 5 minutes. Sections were immersed for 5 minutes in a 40% ethanol / 60% distilled H2O solution of 1 mM X-34 (adjusted to pH 10 by adding 1N NaOH). Sections were then washed in PBS, briefly immersed five times in tap water, and transferred to 80% 0.2 g% NaOH for 2 minutes. Cover slips were placed on the sections using DPX mounting medium (Electron Microscopy Science).

[0271] Quantification of histological parameters To quantify histological parameters, sections for histological examination were selected from tissue blocks including the hippocampus. All sections were used, but randomly assigned to tissue staining to avoid bias. Those performing staining were blinded to the experimental conditions. After staining, immunopositivity assessment was fully automated using Qupath software with default settings and parameters to identify and quantify immunopositive cells. The staining intensity of X34 was measured using Haralick's texture feature to evaluate the co-occurrence matrix on a black background (Lofstedt et al., PLoS One 2019;14(2):e0212110). Grid-based sampling was used to randomly select starting points within the hippocampus and systematically sample image regions at regular intervals. This selection approach prevented bias and provided representative sampling across the entire region of interest (Zhao et al., Nat Commun 2020;11(1):4275).

[0272] Behavioral evaluation Behavior was evaluated over a two-week period in amyloid mice aged 5.5–6 months and 3–3.5 months post-therapy, and in tau mice aged 8.5–9 months and 3–3.5 months post-therapy. Neurological screening and behavioral defects were assessed using four behavioral tests during the experimental period, including nesting, Y-maze, novel object recognition, and Burns maze. In all behavioral tests except nesting, all behavioral equipment was disinfected with 70% ethanol between animals to avoid any olfactory cues. Behavioral assays were performed on animals using blinded identification and data analysis with fully automated software (AnyMaze, v7.0, 2022, Stoelting, San Diego, CA) to ensure unbiased assessment without subjective input (Bailoo et al., J Neurosci Methods 2010;188(1):45-52). The nesting assay was an exception to the automated method, with nesting behavior assessments scored by three or four blinded observers.

[0273] Nesting: Nesting is an innate behavior in mice and is considered an early indicator of behavioral deficit (Dorninger et al., Bio Protoc 2020;10(24)). Two weeks prior to necropsy, the animals' bedding was changed to square cotton sheets, and three or four blinded observers evaluated them after 24 hours (Samaey et al., Front Aging Neurosci 2019;11:335). The nesting behavior of animals was quantified (using a 5-point scale) based on how nesting materials were used, and scored as follows: no nesting behavior (score of 1), minimal involvement and organization in manipulating nesting materials and constructing nest structures (score of 2), partial involvement and organization in manipulating nesting materials and constructing nest structures (score of 3), nearly complete nest structure (score of 4), and excellent performance (score of 5) characterized by meticulous collection and placement resulting in a well-organized and stable nest structure (Samaey et al., Front Aging Neurosci 2019;11:335).

[0274] Y maze: The Y-maze measures spatial working memory and tests animals' innate curiosity in exploring new environments and their ability to distinguish between familiar and novel spatial cues (Kraeuter et al., Pre-Clinical Models: Techniques and Protocols. (Guest PC. ed.) Springer New York: New York, NY; 2019; pp. 105-111). Animals were placed on a Y-maze platform (Stoelting, IL) and recorded for 5 minutes. Consecutive novel arm entries were considered alternations. The alternation ratio, a measure of novel exploration behavior, was calculated using the total number of arm entries and alternations, assessed by AnyMaze software. In addition, the total number of entries and alternations were also assessed to avoid the potential influence of significant immobility time on alternation behavior.

[0275] Novel object recognition: The novel object recognition task assesses working memory and is based on the mice's spontaneous tendency to explore novel objects rather than familiar ones (Lueptow et al., J Vis Exp 2017;126). The novel object recognition task was conducted on the Novel Object Recognition Test Platform (Stoelting, IL) over three days, each belonging to a distinct phase. On day 1, the habituation phase, each animal was allowed to freely explore an open arena (a white plastic box measuring 40cm x 40cm x 30cm high) for two 5-minute trials with a 5-minute interval between trials. On day 2, the proficiency phase, each animal was returned to the same arena with two identical objects in opposite corners for two 5-minute sessions. After a 24-hour retention period (the period during which the animals forget or remember the novelty of the objects), the test phase began on day 3, when the animals were returned to the arena with two objects in the same positions as the previous day, but one replaced with a novel object. To prevent object preference, mice were consistently placed in an arena facing the back wall and allowed to explore for 5 minutes. The discrimination index was calculated by dividing the time spent exploring a new object by the time spent exploring both objects. The AnyMaze software defined exploration as the mouse facing a new object within 3 cm, sniffing, climbing, or interacting with it.

[0276] Barnes' Maze: The Barnes maze tests hippocampus-dependent spatial memory by leveraging rodents' innate aversion to open space and their instinctive tendency to seek hiding places (Pitts et al., Bio Protoc 2018;8(5)). The apparatus includes 20 circular holes (50 mm in diameter) evenly spaced along the perimeter of the maze (Stoelting, IL). Only one hole provides access to a closed escape chamber (108 mm × 55 mm × 55 mm) beneath the maze floor. The test procedure and distal visual cues were modified to enhance visuospatial learning and memory. Bright ceiling lighting (10.8 × 10² lux) and white noise were used to stimulate the desire to escape. Animals were trained for 7 days to locate the escape holes using distal visual cues. The test was terminated when a mouse escaped through a hole or after 180 seconds. Spatial learning was assessed by calculating the latency to escape the maze by entering the appropriate hole. Escape descent time was evaluated during training days and probe testing. Data was recorded in a final test conducted 24 hours after the last training day by sealing the escape hole and recording the descent time using ANY-maze software with a diameter setting of 10-15 mm.

[0277] statistical analysis All experimental data were collected and analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). Linear quantitative data are presented as mean ± standard error of the mean. Logarithmic scale data are presented as geometric mean ± geometric standard deviation. Data normality was assessed by the Shapiro-Wilk test. One-way ANOVA and Kruskal-Wallis tests were used based on the data distribution. Multiple comparisons were performed using Dunn and Bonferroni post-hoc analyses. Some experiments were performed with HA-tagged vectors, and some without HA tagging. Statistical differences between HA / non-HA results and male / female results were analyzed by independent t-tests and Mann-Whitney U tests. Since there was no difference between the tagged and untagged evaluations, the data were combined. Since there was no difference in studies performed between males and females, data from both sexes were combined. For all evaluations, a p-value < 0.05 was considered statistical significance.

[0278] result AAV expression in vitro To ensure that the detection of vector-mediated APOE isoforms accurately detects each with equal sensitivity, the manufacturer provided data demonstrating that this ELISA kit detects APOE2 and APOE4 with equal sensitivity. The kit was also confirmed to detect APOE2Ch equally with APOE4 and APOE2 by assaying APOE in the medium of 293 cells transfected with equivalent amounts of plasmids otherwise (Figure 14).

[0279] In vivo AAV expression This study was designed to treat amyloid and tau mouse models that had early evidence of disease as indicated by histological and biochemical evaluations (Figures 15A, 15B, 15C, and 15D). Amyloid mice (APP.PSEN1 / TRE4) were treated at 2.5 months; at this point, these mice had evidence of amyloid deposition, and p-tau mice (P301S / TRE4) were treated at 5.5 months; at this point, these mice had evidence of p-tau aggregates. In relation to this, early disease was modeled.

[0280] To ensure that all experimental groups had similar AAV transduction, vector genomic copies, APOE mRNA, and APOE protein were assayed in the hippocampi of all study cohort controls in both amyloid and tau mice (Figures 2A–2F). No vector genomic copies were detected in the PBS cohort in either amyloid or tau mice (Figures 2A and 2B), and there were no significant differences in vector copies between amyloid mice treated with null, E2, and E2Ch (p>0.1, Figure 2A) and tau mice (p>0.2, Figure 2B). Human APOE transgene mRNA expression was quantified using specific primers / probes for hAPOE, measuring background levels in control mice and above-background levels in treated mice (Figures 2C and 2D). In amyloid mice (p>0.5, Figure 2C) or tau mice (p>0.9, Figure 2D), no significant difference in APOE mRNA levels was observed between the PBS group and the null group; i.e., both groups had similar background levels. Treatment with E2 and E2CH resulted in significantly higher mRNA levels in both amyloid and tau mice compared to the PBS (p<0.01, Figure 2C) and null (p<0.01, Figure 2D) cohorts, respectively. The PBS and null groups had similar background levels of hAPOE protein in amyloid and tau mice (p>0.9, Figures 2E and 2F). Mice treated with E2 and E2CH had similar (p>0.9) 3-5 times higher hAPOE protein levels compared to the background in both amyloid and tau mice (p<0.01, Figures 2E and 2F).

[0281] The effect of treatment on amyloid loading in amyloid mice The effects of treatment on amyloid loading were investigated by quantifying Aβ42 and Aβ40 levels in the hippocampus in the insoluble and soluble fractions (Figures 3A and 3B). In amyloid mice, no significant differences were observed between the PBS group and the null group in the insoluble or soluble fractions of Aβ42 (p>0.3 insoluble, p>0.9 soluble, Figure 3A) or Aβ40 (p>0.3 insoluble, p>0.9 soluble, Figure 3B). E2 treatment significantly suppressed insoluble and soluble Aβ42 levels by 79.7% (p<0.01) and 80.0% (p<0.01), respectively, and Aβ40 levels by 68.0% (p<0.01) and 68.1% (p<0.01), respectively, compared to the PBS group and the null group (Figures 3A and 3B). Furthermore, E2Ch significantly reduced insoluble and soluble Aβ42 levels by 80.5% (p<0.01) and 81.6% (p<0.01), respectively, and Aβ40 levels by 66.6% (p<0.01) and 71.0% (p<0.01), respectively, compared to the PBS and null groups. No significant differences were observed between E2 treatment and E2CH treatment in both Aβ42 and Aβ40 levels in the insoluble and soluble fractions (all comparisons p>0.8 (Figures 3A and 3B)).

[0282] The effect of treatment for tau loading in tau mouths Total tau and p-tau were quantified in each cohort using sequential extracts from hippocampal tissue (Figures 3C and 3D). Levels of total tau (p>0.3 insoluble, p>0.5 soluble, Figure 3C) and p-tau (p>0.9 insoluble, p>0.5 soluble, Figure 3D) were similar in the PBS and null groups. In contrast, total tau in the insoluble fraction was significantly lower in the E2 group (P<0.01 compared to PBS, but not compared to null, p>0.1, Figure 3C), but not in soluble total tau (p>0.9 compared to PBS, p>0.1 compared to null, Figure 3C) or in the insoluble and soluble fractions of p-tau (p>0.9 compared to PBS, p>0.4 and p>0.3 compared to null in both fractions; Figure 3D). In contrast, E2Ch administration reduced total tau levels by 40.5% in the insoluble fraction and 68.6% in the soluble fraction compared to PBS (p<0.01 insoluble, p<0.05 soluble; Figure 3C) and the null group (p<0.01 insoluble, p<0.05 soluble; Figure 3C). Furthermore, E2Ch significantly reduced p-tau levels by 36.2% in the insoluble fraction and 44.5% in the soluble fraction compared to the PBS group (p<0.01 insoluble, p<0.05 soluble; Figure 3D) and the null group (p<0.01 insoluble, p<0.01 soluble; Figure 3D) cohort. Importantly, significant differences were observed between E2CH treatment and E2 treatment for the evaluated amounts of soluble total tau (p<0.01, Figure 3C), soluble p-tau (p<0.05, Figure 3D), and insoluble p-tau (p<0.05, Figure 3D), demonstrating a clear superior effect of E2CH over E2 in reducing tau pathology.

[0283] The effect of treatment on lipid profile APOE2 overexpression may lead to serum hyperlipidemia; therefore, serum cholesterol and triglyceride levels were assessed as a function of treatment cohort in both tau and amyloid mice. There was no treatment-dependent deviation from baseline mild hyperlipidemia in the TRE4 mouse strain (Figures 13A and 13B).

[0284] To measure lipid efflux, which may be a mechanism of ApoE2 protection, we used spatial transcriptomics to examine lipid metabolism-related transcripts in hippocampal cells. For example, SGMS2 (sphingomyelin synthase 2), a key regulator of sphingomyelin and diacylglycerol metabolism, is typically decreased in AD, but SGMS2 is increased in both E2 treatment and E2Ch treatment. Furthermore, a reduction in LRP2 (LDL receptor-associated protein 2) is thought to be a causative factor in Alzheimer's disease (AD). The E2Ch cohort showed increased LRP2 expression (Figures 16A, 16B, 16C, and 16D).

[0285] Effects on amyloid aggregation, neuronal degeneration, and microglia / astroglial activation in amyloid mice Amyloid-beta plaque accumulation was examined using amyloid-beta staining detectable by MOAB-2 (see Youmans et al. Mol Neurodegener 2012;7(8)). The PBS and null groups showed similar levels of amyloid-beta staining (p>0.07, Figures 4A, 4B, and 4E). Both E2 (Figure 4C) and E2CH (Figure 4D) treatments led to a significant reduction in MOAB-2 detectable amyloid-beta (p<0.01 for E2 and p<0.01 for E2Ch compared to PBS and null (p<0.01 for both E2 and E2Ch compared to null), Figure 4E). No significant difference was observed between E2 treatment and E2Ch treatment (p>0.9, Figure 4E).

[0286] The presence of amyloid aggregates was assessed using X34 staining (see Styren et al. J Histochem Cytochem 2000;48(9):1223-32). Similar to MOAB-2 staining, the PBS (Figure 4F) and null (Figure 4G) groups showed similar X34 staining profiles (p>0.05, Figure 4J). Treatment with both E2 (Figure 4H) and E2Ch (Figure 4I) reduced X34 staining (p<0.01 for both E2 and E2Ch compared to PBS), indicating that both E2 and E2Ch mediated the suppression of amyloid aggregate accumulation in amyloid mice (p>0.4, Figure 4J).

[0287] Iba-1 immunostaining provides a measure of microglial activation (see Moser et al. iScience 2021;24(11):103238). Both the PBS (Figure 4K) and null (Figure 4L) groups showed strong staining profiles for Iba-1, indicating similar levels of microglial activation. In contrast, both E2 (Figure 4M) and E2CH (Figure 4N) treatments significantly inhibited Iba-1 immunopositivity compared to the PBS and null groups (p<0.01 for E2 and E2CH, Figure 4O). There was no difference between the E2 and E2Ch groups (p>0.9, Figure 4O). These results suggest that both E2 and E2Ch have similar abilities to suppress microglia-associated neuroinflammation associated with amyloid pathology.

[0288] GFAP immunostaining provides a further measure of neuroinflammation mediated by astroglial activation (see Parhizkar et al. Semin Immunol 2022;59). Both the PBS (4P) and null (Figure 4Q) groups showed strong staining with GFAP, indicating similar levels of astroglial activation. However, both E2 (Figure 4R) and E2CH (Figure 4S) treatments significantly inhibited GFAP immunopositivity compared to the PBS and null groups (p<0.01 for E2 and E2CH, Figure 4T). There was no significant difference between the E2 and E2CH groups (p>0.7, Figure 4T), suggesting that both E2 and E2CH treatments have the ability to suppress amyloid-related astroglial activation.

[0289] Effects of phosphorylated tau on the activation of microglia and astrocylia in tau mice AT8 staining detects phosphorylated tau (see Koutsodendris et al., Nat Aging 2023;3(3):275-296). Similar levels of staining were observed in both the PBS (Figure 5A) and null (Figure 5B) groups (p>0.9, Figure 5E). E2 did not induce any significant changes in AT8 staining compared to the control group (Figure 5C) (p>0.9, Figure 5E). However, E2CH (Figure 5D) significantly suppressed AT8 staining compared to the PBS, null, and E2 groups (p<0.01, p<0.01, and p<0.05, respectively, Figure 5E).

[0290] Both the PBS (Figure 5F) and null (Figure 5G) groups exhibited strong Iba-1 staining profiles in tau mice, indicating similar levels of microglial activation. However, treatment with E2CH (Figure 5I) significantly inhibited Iba-1 immunopositivity compared to the E2 group (p<0.01, Figures 5H and 5O), the PBS group, and the null group (both p<0.01, Figure 5J), supporting the conclusion that E2Ch treatment is effective in alleviating tau pathogenesis-associated neuronal inflammation, while E2 is not.

[0291] Both the PBS (Figure 5K) and the null (Figure 5L) groups showed strong staining with GFAP, indicating similar levels of astroglial activation. However, treatment with E2CH (Figure 5N) significantly inhibited GFAP immunopositivity compared to the E2 group (p<0.01, Figures 5M and 5O), the PBS group, and the null group (both p<0.01, Figure 5O). This indicates that E2CH treatment is effective in mitigating tau pathogenesis-related astroglial activation, while E2 is not.

[0292] Effects on behavior in amyloid mice and tau mice Nesting tests reflect overall health status, motor coordination, and cognitive function, and dysfunction in nesting behavior has been observed in animal models of Alzheimer's disease (see Torres-Lista et al., Behav Brain Res 2013;247(153-7)). Significant differences were observed between E2 treatment and PBS treatment in amyloid mice (p<0.03), but not between E2 and null, E2Ch and control, or E2 and E2Ch (all comparisons p>0.9, Figure 6A). In nesting tests in tau mice, E2Ch treatment, rather than E2 treatment, improved nesting scores compared to PBS and null controls (p<0.01), and the E2Ch group performed better than the E2 group (p<0.04, Figure 6B).

[0293] The Y-maze test assesses spatial working memory and spontaneous alternation behavior, measuring animals' ability to remember and navigate a series of arm choices within a maze (see Kraeuter et al., Pre-Clinical Models: Techniques and Protocols. (Guest PC. ed.) Springer New York: New York, NY; 2019; pp. 105-111). Impairment of spontaneous alternation has been associated with memory deficits and cognitive decline (see Lalonde et al., Neurosci Biobehav Rev 2002;26(1):91-104). To ensure the effectiveness of Y-maze performance, all groups were tested for the total number of entries; no significant differences were observed (all comparisons between amyloid mouse groups p>0.9, Figure 7A; all comparisons between tau mouse groups p>0.06, Figure 7C). In the Y-maze test for amyloid mice, both E2 and E2Ch treatment improved the alternation ratio compared to PBS (p<0.01 for E2 and E2Ch, Figure 6C) and the null group (p<0.01 for E2 and E2Ch), but no significant difference was observed between E2 and E2Ch treatment in amyloid mice (p>0.9, Figure 6C). In contrast, in the Y-maze test for tau mice, E2Ch showed a significantly higher alternation ratio compared to all other groups (P<0.01 compared to all groups, Figure 6D). Similarly, the assay was effective for tau mice as the number of entry was indistinguishable in all groups (Figure 7C), but tau mice were significantly different from E2 (p<0.04), PBS (p<0.02), and null (p<0.03), while there was no difference between E2 and control (PBS p>0.2, null p>0.1, Figure 7D).

[0294] The novel object recognition test is used to assess memory and the ability to distinguish between familiar and novel objects (Antunes et al., Cogn Process 2012;13(2):93-110). This test helps assess an animal's innate preference for novels and evaluate memory deficits and impairment recognition. In the novel object recognition test in amyloid mice, both E2 treatment and E2Ch treatment showed significantly higher discrimination indices compared to PBS (p<0.01 and p<0.04, respectively), and there was no significant difference between E2 and E2Ch (p>0.8, Figure 6E). However, in tau mice, E2CH treatment had a significantly higher discrimination index compared to PBS and null (both p<0.05), and was significantly more effective than treatment with E2 (p<0.01, Figure 6F).

[0295] For the Barnes maze, animals were trained for 7 days to identify visual cues for locating the escape hole. After 7 days of training, data from the last 24 hours were assayed as a measure of performance (Figures 8A and 8B). In amyloid mice, the Barnes maze test showed that both E2 and E2CH significantly reduced escape latency compared to PBS and null (p<0.01 for both E2 and E2CH, Figure 6G), demonstrating enhanced spatial learning and memory retention (see Gawel et al., Naunyn Schmiedebergs Arch Pharmacol 2019;392(1):1-18). There was no significant difference between E2 treatment and E2CH treatment in amyloid mice (p>0.9, Figure 6G). In tau mice, no significant effect was observed in the E2 group compared to the PBS and null groups (p>0.9). In contrast, in tau mice, E2Ch treatment significantly reduced escape latency compared to PBS, null, and E2 (all p<0.01, Figure 6H), and E2Ch was significantly superior to E2 (p<0.01). This is consistent with the concept that, in relation to tau pathology, E2Ch treatment is effective in enhancing spatial learning and memory retention, while E2 is not.

[0296] The effect of treatment on neuronal loss in amyloid mice and tau mice. In addition to the accumulation of amyloid plaques and tau tangles, mouse models of Alzheimer's disease are also characterized by neuronal loss, which must be protected for effective therapy. Quantitative imaging of hippocampal sections stained with NeuN (neurons) and Olig2 (myelin) was used to evaluate whether E2 therapy leads to neuronal rescue, and both stains showed a reduction in neurodegeneration (Figure 9). In amyloid mice, both E2 and E2Ch cohorts showed significantly higher NeuN staining compared to PBS and null controls (p<0.01), but no significant difference from each other (p>0.3) (Figure 9A). In contrast, only the E2Ch cohort showed significantly higher NeuN staining in tau mice (P<0.01 compared to all other cohorts). Similar results were observed for Olig2, confirming that these therapies protect against neuronal loss (Figure 9B).

[0297] The effect of treatment on neuronal health in amyloid mice and tau mice. To quantitatively measure gene expression at the cellular level, a pilot study was conducted using spatial transcriptomics on coronal sections from each cohort, with a total of 800 million reads from each sample. Brain regions and cell types were determined by standard methods. The data provided an indicator of the effect of treatment on neuronal health. Neurons were identified by standard marker genes, and the expression ratio for each gene in each cohort was determined compared to the corresponding PBS control (Figures 10A and 10B). This effect of gene transfer was greater in the tau mouse model than in the amyloid mouse model.

[0298] The effectiveness of treatment for microglial inflammation As an independent assessment of the benefits of APOE gene therapy using E2 and E2Ch vectors in both amyloid and tau models, microglial inflammation, as indicated by the level of inflammation expression, was evaluated. Anti-inflammatory markers, including Arg-1, CD163, CD68, CLEC7A, TREM2, and CD206 by RTqPCR, were selected based on data from Lu et al. Cell Rep 42, 112785 (2023). Data generated using these novel inflammatory and anti-inflammatory markers revealed that glial cells were less activated in the amyloid E2 cohort and E2Ch cohort, as well as the tau E2Ch cohort (Supplementary Figure 8). In addition to evaluation of mRNA levels of further inflammatory and anti-inflammatory markers, spatial transcriptomics using further markers of microglial activation was also used to compare genome-wide gene expression in E2 and E2Ch-treated animals with controls (Figures 11A and 11B). The data reveal a consistent pattern of reduction in injury-associated microglia (Figures 12A, 12B, 12C, and 12D). As an example of the results, which include a list of 14 genes reflecting injury-associated microglia (DAMs) and homeostatic microglia, E2 and E2Ch gene transfer was found to suppress the DAM phenotype and promote the homeostatic phenotype. This effect of gene transfer was greater in the tau mouse model than in the amyloid mouse model.

[0299] (Table 2) TIFF2026516699000004.tif51145TIFF2026516699000005.tif223145TIFF2026516699000006.tif223145TIFF2026516699000007.tif224145 TIFF2026516699000008.tif225145TIFF2026516699000009.tif225145TIFF2026516699000010.tif226145TIFF2026516699000011.tif188145

[0300] All publications, patents, and patent applications are incorporated herein by reference. While the foregoing specification has described the present invention in relation to certain preferred embodiments and has provided many details for illustrative purposes, it will be apparent to those skilled in the art that further embodiments are possible and that certain details herein can be modified substantially without departing from the fundamental principles of the invention.

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide containing the Christchurch mutation or an apolipoprotein 3 (APOE3) polypeptide containing the Christchurch mutation, wherein the rAAV vector comprises SEQ ID NO:8, SEQ ID NO:21, or SEQ ID NO:

22.

2. The aforementioned Christchurch mutation, R154S mutation related to unprocessed APOE polypeptide; or R136S mutation in mature APOE polypeptide lacking signal peptide The rAAV vector according to claim 1, comprising:

3. The rAAV vector according to any one of the claims, which is packaged as an rAAV viral vector containing an AAV capsid protein.

4. The rAAV vector according to any one of the claims, wherein the AAV capsid protein is AAV1 capsid protein, AAV2 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAV13 capsid protein, AAVPHP.B capsid protein, AAVrh74 capsid protein, or AAVrh10 capsid protein.

5. The rAAV vector according to any one of the claims, wherein the AAV capsid protein is the AAVrh10 capsid protein.

6. A pharmaceutical composition comprising the AAV virus vector according to claim 3.

7. A method for treating Alzheimer's disease in a human subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to claim 6.

8. The therapeutically effective dose of the aforementioned vector is approximately 1 × 10⁻⁶ 10 ~Approx. 1×10 16 The method according to claim 7, wherein the genome copy is obtained.

9. The method according to claim 7, wherein the subject is an APOE2 / APOE4 heterozygote, an APOE4 / APOE4 homozygote, or an APOE3 / APOE4 heterozygote.

10. The method according to claim 7, wherein the composition is administered systemically, intracervically, via the large cisterna, or via CI-C2 administration.

11. The aforementioned pharmaceutical composition contains approximately 5.0 × 10 9 gc / mL CSF ~ approx. 5.0×10 12 The method according to claim 7, administered in a dose of gc / mL CSF.

12. The aforementioned pharmaceutical composition i) Approximately 1.4×10 10 gc / mL CSF, ii) Approximately 4.4×10 10 gc / mL CSF, or iii) Approximately 1.4 × 10 11 gc / mL CSF The method according to claim 7, administered in the dose of [specify dose].

13. The aforementioned pharmaceutical composition contains approximately 1.4 × 10 14 The method according to claim 7, administered in a fixed dose of gc.

14. The method according to claim 7, wherein the pharmaceutical composition is administered in a total volume of about 5 mL, about 10 mL, about 15 mL, or about 20 mL.

15. The method according to claim 7, wherein the subject experiences an increase of at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100% in the expression of APOE2 Christchurch or APOE3 Christchurch.

16. The method according to claim 7, wherein the expression of APOE2 Christchurch or APOE3 Christchurch occurs in the central nervous system.

17. The method according to claim 7, wherein the expression of APOE2 Christchurch or APOE3 Christchurch is measured in cerebrospinal fluid (CSF).

18. The method according to claim 7, wherein, after administration of the pharmaceutical composition, the expression level of at least one of T-tau and P-tau is reduced in the subject compared to baseline before administration.

19. The method according to claim 18, wherein the expression levels of T-tau and / or P-tau are reduced by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100%.

20. The method according to claim 7, wherein the ratio of amyloid beta 42 / amyloid beta 40 (Aβ 42 / 40 ) increases after administration of the pharmaceutical composition.

21. The Aβ 42 / 40 The method according to claim 20, wherein the ratio increases by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, 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 100%.

22. The method according to claim 7, wherein the subject is administered an immunosuppressant prior to treatment with the pharmaceutical composition.

23. The method according to claim 22, wherein the immunosuppressant is prednisone.

24. The aforementioned prednisone, 40 mg once daily, one week prior to administration of the AAV virus vector; 40 mg once daily for the first two weeks after administration of the AAV virus vector; 30 mg once daily for three weeks after administration of the AAV virus vector; 20 mg once daily for four weeks after administration of the AAV virus vector; 10 mg once daily for 5 weeks after administration of the AAV virus vector; 5 mg once daily for 6 weeks after administration of the AAV virus vector; 2.5 mg once daily for 7 weeks after administration of the AAV virus vector; and For 8 weeks after administration of the AAV virus vector, take 2.5 mg every other day. The method according to claim 23, administered in the specified dose.

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