Methods and pharmaceutical compositions for treating and preventing alzheimer's disease

Abstract

The present disclosure provides methods and compositions for the treatment of Alzheimer's disease. The methods and compositions of the present disclosure comprise AAV vectors and AAV viral vectors comprising transgenic nucleic acid molecules comprising a nucleic acid sequence encoding an APOE2 polypeptide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 367,995, filed on July 8, 2022, U.S. Provisional Application No. 63 / 385,889, filed on December 2, 2022, and U.S. Provisional Application No. 63 / 496,792, filed on April 18, 2023. The contents of each of these U.S. provisional patent applications are hereby incorporated by reference in their entirety.

[0003] Submit sequence listing in XML format

[0004] The contents of the electronic sequence listing (LEXE_010_001WO_SeqList_ST26.xml; size: 28,886 bytes; and creation date: July 7, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to a method for preventing or treating Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an AAV vector comprising a nucleic acid encoding apolipoprotein 2 (APOE2). A method for producing an AAV viral vector encoding APOE2 is also provided. Background Art

[0006] The need for therapeutic agents that effectively treat or prevent Alzheimer's disease (AD) is long-standing, but remains unmet. An AAV vector encoding a human APOE2 gene is disclosed. The AAV vector is formulated for administration to an individual suffering from Alzheimer's disease or at risk of developing Alzheimer's disease. A method for producing an APOE2 AAV vector is also provided. To date, common APOE alleles (APOE4, APOE3, APOE2) are the main genetic risk modifiers for AD, wherein the APOE4 allele increases the risk and reduces the age of onset, while the APOE2 allele reduces the risk and significantly delays the age of onset. Extensive human genetic data indicate that APOE4 and APOE2 are codominant. APOE2 / APOE4 heterozygotes have the normal risk of APOE3 / APOE3 homozygotes, rather than the 4-fold higher risk of AD with APOE3 / APOE4 heterozygotes. APOE4 homozygotes have a significantly increased risk of developing AD (14.5-fold compared to APOE3 homozygotes), as well as an earlier age of onset of the disease (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 age-matched healthy controls. In contrast, APOE2 is a protective allele, reducing the risk of AD by approximately 50% (1.8-fold risk reduction) and significantly delaying the age of onset even in the presence of the APOE4 allele. Thus, roughly equal expression of APOE2 eliminates the deleterious effects of the E4 allele in humans.

[0007] Amyloid burden is associated with the neuropathology of AD. Both experimental animal and clinical studies have shown that APOE genotype also predicts the timing and amount of brain Aβ peptide deposition and amyloid burden (APOE4>APOE3>APOE2). In a model of AD-associated amyloidosis, intrahippocampal administration of APOE4 and APOE2 using gene transfer vectors increased and decreased brain Aβ / amyloid burden, respectively. Amyloid burden is one mechanism by which APOE isoforms exert their effects. Other mechanisms include the tau pathway.

[0008] Therefore, patients who are homozygous for APOE4 (APOE4 homozygotes) have the highest risk of developing AD, and APOE3 / APOE4 heterozygotes also have an increased risk of developing AD. The methods and compositions disclosed herein can be used to increase APOE2 expression in the CNS of APOE4 homozygous or APOE3 / APOE4 heterozygous subjects to convert their brains to brains that resemble APOE2 / APOE4 brains (i.e., low risk of developing AD), thereby reducing the risk of developing AD, treating symptoms of AD, and / or reversing symptoms of AD. Summary of the invention

[0009] The present disclosure provides a pharmaceutical composition comprising an APOE2 rAAV viral vector; wherein the rAAV viral vector comprises an AAVrh10 capsid protein and an APOE2 rAAV vector; wherein the pharmaceutical composition comprises at least about 1.0×10 11 genome copies (gc) / mL to about 1.0×10 14 gc / mL, and wherein the pharmaceutical composition comprises less than about 40% empty rAAV capsids.

[0010] In some aspects, the pharmaceutical composition comprises less than about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% empty rAAV capsids.

[0011] In some aspects, the pharmaceutical composition comprises at least about 1.5×10 13 gc / mL.

[0012] In some aspects, the APOE2 rAAV viral vector is formulated in about 1 mM potassium phosphate monobasic, 3 mM sodium phosphate dibasic, and about 155 mM sodium chloride (NaCl) at a pH of about 7.4.

[0013] In some aspects, the rAAV vector comprises, in the 5' to 3' direction: a first AAV ITR sequence; an enhancer sequence; a promoter sequence; a chimeric intron; a nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide; a polyA sequence; and a second ITR sequence.

[0014] In some aspects, the nucleic acid sequence encoding the APOE2 polypeptide comprises SEQ ID NO:5.

[0015] In some aspects, the first ITR sequence comprises the nucleic acid sequence shown in SEQ ID NO:1.

[0016] In some aspects, the second ITR sequence comprises the nucleic acid sequence shown in SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12.

[0017] In some aspects, the enhancer sequence comprises the nucleic acid sequence shown in SEQ ID NO:2.

[0018] In some aspects, the promoter sequence comprises the nucleic acid sequence shown in SEQ ID NO:3.

[0019] In some aspects, the polyA sequence comprises the nucleic acid sequence shown in SEQ ID NO:6.

[0020] In some aspects, the rAAV vector comprises the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:11.

[0021] In some aspects, the rAAV vector is packaged as a rAAV viral vector comprising an AAV capsid protein.

[0022] In some aspects, the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein, or an AAVrh10 capsid protein. In some aspects, the AAV capsid protein is an AAVrh10 capsid protein.

[0023] The present disclosure provides a pharmaceutical composition comprising an APOE2 rAAV viral vector; wherein the rAAV viral vector comprises an AAVrh10 capsid protein and an APOE2 rAAV vector; wherein the APOE2 rAAV vector comprises the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:11.

[0024] The present disclosure provides a method of treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition according to an embodiment of the present disclosure.

[0025] In some aspects, following administration, the subject experiences at least about a 5% increase in APOE2 expression relative to a pre-administration baseline.

[0026] In some aspects, the patient is homozygous for APOE4.

[0027] In some aspects, the pharmaceutical composition is administered by C1-C2 administration or intracisternal (ICM) administration.

[0028] In some aspects, the pharmaceutical composition is present in an amount of about 5.0×10 9 gc / mL CSF to approximately 5.0×10 12 gc / mL CSF. In some aspects, the pharmaceutical composition is administered at a dose of about: i) 1.4×10 10 gc / mL CSF, ii) 4.4×10 10 gc / mL CSF, iii) 5.0×10 10 gc / mL CSF, iv) 1.4×10 11 gc / mL CSF, v)1.6×1011 gc / mL CSF, or vi) 5.0×10 11 gc / mL CSF.

[0029] In some aspects, the pharmaceutical composition is administered in a total volume of about 5 mL, about 10 mL, about 15 mL, or about 20 mL.

[0030] In some aspects, the subject experiences 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% increase in APOE2 expression.

[0031] In some aspects, the APOE2 expression occurs in the central nervous system. In some aspects, the APOE2 expression is measured in cerebrospinal fluid (CSF).

[0032] In some aspects, after applying the pharmaceutical composition, the expression level of at least one of T-tau and P-tau is reduced in the subject relative to the baseline before application. In some aspects, the expression level of T-tau and / or P-tau is 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%.

[0033] In some aspects, after administration of the pharmaceutical composition, amyloid β42 / amyloid β40 (Aβ 42 / 40 ) ratio is increased. In some aspects, Aβ 42 / 40 The ratio is increased 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%.

[0034] In some aspects, before treatment with the pharmaceutical composition, an immunosuppressant is administered to the subject. In some aspects, the immunosuppressant is prednisone. In some aspects, prednisone is administered at the following doses: 40 mg once a day for 1 week before AAV viral vector administration; 40 mg once a day for the 1st to 2nd week after AAV viral vector administration; 30 mg once a day for the 3rd week after AAV viral vector administration; 20 mg once a day for the 4th week after AAV viral vector administration; 10 mg once a day for the 5th week after AAV viral vector administration; 5 mg once a day for the 6th week after AAV viral vector administration; 2.5 mg once a day for the 7th week after AAV viral vector administration; and 2.5 mg every other day for the 8th week after AAV viral vector administration.

[0035] The present disclosure provides a method for producing a cell lysate containing a rAAV viral vector, the method comprising: (i) transfecting a cell culture containing HEK293T cells with a first plasmid encoding an APOE2 AAV vector and a second plasmid encoding an AAV Rep protein and an AAV Cap protein in a transfection medium, wherein the ratio of the second plasmid to the first plasmid is 2:1; (ii) culturing the transfected HEK293T cells in a culture medium under conditions in which the transfected HEK293T cells produce a recombinant adeno-associated virus (rAAV) viral vector encoding APOE2; (iii) harvesting the transfected HEK293T cells; and (iv) lysing the transfected HEK293T cells to produce a cell lysate containing a rAAV viral vector.

[0036] In some aspects, the culture medium comprises Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). In some aspects, the transfection medium comprises serum-free DMEM and polyethyleneimine (PEI).

[0037] In some aspects, the HEK293T cells are obtained after about two days to about five days of expansion culture.

[0038] In some aspects, transfection of the first plasmid and the second plasmid occurs simultaneously.

[0039] In some aspects, the HEK293T cells are expressed at about 2.0×10 4 About 2.0×10 6 cells / cm 2 In some aspects, the transfected cells are cultured for about 3 days.

[0040] In some aspects, the HEK293T cells are lysed by at least about 4 consecutive freeze-thaw cycles to produce a cell lysate.

[0041] In some aspects, the cell lysate is further treated with a recombinant nuclease to digest any non-encapsulated DNA.

[0042] In some aspects, following DNA digestion, the cell lysate is clarified by ultracentrifugation.

[0043] In some aspects, the cell lysate comprises about 1.0×10 9 to about 5.0×10 14 genome copies (gc) / ml.

[0044] The present disclosure provides a method for producing an APOE2 rAAV pharmaceutical composition, the method comprising: (i) obtaining a cell lysate containing an rAAV viral vector encoding APOE2; (ii) contacting a density gradient with the cell lysate containing an rAAV viral vector encoding APOE2, and subjecting the density gradient to centrifugation; (iii) contacting an anion exchange column with the cell lysate containing an rAAV viral vector encoding APOE2; (iv) eluting the rAAV viral vector from the column; and (v) concentrating the eluted rAAV viral vector into a formulation buffer by ultrafiltration, thereby producing an APOE2 rAAV pharmaceutical composition.

[0045] In some aspects, the density gradient is an iodixanol density gradient.

[0046] In some aspects, the iodixanol gradient comprises a stepwise density gradient comprising: (i) about 10% to about 20% iodixanol solution; (ii) about 20% to about 30% iodixanol solution; (iii) about 40% to about 50% iodixanol solution; and (iv) about 50% to about 60% iodixanol solution.

[0047] In some aspects, the anion exchange column is a Q Sepharose high efficiency strong quaternary ammonium anion exchange resin column.

[0048] In some aspects, the formulation buffer comprises phosphate buffered saline (PBS).

[0049] In some aspects, after ultracentrifugation, the pharmaceutical composition comprises about 1.0×10 10 to about 5.0×10 13 viral genomes / ml.

[0050] The present disclosure provides an APOE2 rAAV pharmaceutical composition produced by the method of any embodiment of the present disclosure.

[0051] The present disclosure provides a cell lysate comprising a rAAV viral vector produced by the method of any embodiment of the present disclosure.

[0052] The present disclosure provides an rAAV vector comprising the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:11.

[0053] The present disclosure provides an rAAV vector, which comprises in the 5' to 3' direction: a first AAV ITR sequence; an enhancer sequence; a promoter sequence; a chimeric intron; a nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide; a polyA sequence; and a second ITR sequence.

[0054] Any of the above aspects or any other aspect described herein may be combined with any other aspect.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In the specification, the singular also includes the plural, unless the context clearly indicates otherwise; as an example, the terms "a / an" and "the" should be understood as singular or plural, and the term "or" should be understood as inclusive. For example, "element" means one or more elements.

[0056] Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All disclosures, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. It is not recognized that the references cited herein are prior art of the claimed invention. In the event of a conflict, this specification (including definitions) shall prevail. In addition, these materials, methods and examples are only illustrative and are not intended to be restrictive. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is a graph depicting APOE2 levels relative to APOE4 levels in patients treated with APOE2 rAAV vectors at the indicated time points.

[0058] Figure 2 is a graph depicting APOE2 levels in patients treated with APOE2 rAAV vectors at the indicated time points.

[0059] FIG3 is a graph depicting Aβ42 (Aβ42) at 12 months relative to pre-treatment baseline in patients treated with APOE2 rAAV vectors. Figure 3A )、T-tau( Figure 3B ) and P-tau( Figure 3C ) expression changes.

[0060] Figure 4A Graph depicting the amount of viral genome copies detected in the hippocampus of APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2+hemagglutinin (HA) tag (AAV-Rh10-E2HA). The y-axis depicts the number of viral genome copies per microgram of DNA. Data are expressed as geometric mean ± SD. Circles represent non-HA-tagged (AAV-Rh10-E2); triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0061] Figure 4B Graph depicting APOE mRNA levels detected in the hippocampus of APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). The y-axis represents transgene mRNA copies per microgram of RNA. Data are expressed as geometric mean ± SD. Circles represent non-HA-tagged (AAV-Rh10-E2); triangles represent HA (AAV-Rh10-E2HA)-tagged treatments.

[0062] Figure 4C is a graph depicting APOE protein levels detected in the hippocampus of APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or an APOE2 HA tag (AAV-Rh10-E2HA). The y-axis depicts APOE protein levels expressed as ng of APOE per microgram of total protein. Data are expressed as geometric mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2) and triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0063] Figure 5A Figure 3 Soluble and insoluble Aβ in the hippocampus of APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL) or APOE2 (AAV-Rh10-E2). 40 A series of graphs of protein levels. The y-axis depicts APOE levels expressed as Aβ per mg of total protein. 40 Data are expressed as geometric mean ± SEM.

[0064] Figure 5BFigure 3 Soluble and insoluble Aβ in the hippocampus of APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL) or APOE2 (AAV-Rh10-E2). 42 A series of graphs of protein levels. The y-axis depicts APOE levels expressed as Aβ per mg of total protein. 42 Data are expressed as geometric mean ± SEM.

[0065] Figure 6 Graph depicting the number of positive staining for amyloid beta in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). Data are presented as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2); triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0066] Figure 7 is a graph depicting fluoro jade positivity in the hippocampus of APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). Data are presented as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2), and triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0067] Figure 8 is a graph depicting GFAP positivity in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). Data are presented as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2), and triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0068] Fig. 9is a graph depicting Iba1 positivity in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). Data are presented as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2), and triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0069] Fig.10 Graph depicting X-34 staining intensity in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). Data are presented as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2), and triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0070] Fig.11 Graph depicting nested behavioral scores in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). Data are presented as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2), and triangles represent HA-tagged (AAV-Rh10-E2HA) treatments.

[0071] Fig.12 Graph depicting Y-maze performance (a measure of spatial working memory) in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL) or APOE2 (AAV-Rh10-E2). The Y axis depicts the number of entries. Data are expressed as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2) treatments.

[0072] Fig.13 Graph depicting Y-maze performance (a measure of spatial working memory) in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL) or APOE2 (AAV-Rh10-E2). The Y axis depicts the number of alternations. Data are expressed as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2) treatments.

[0073] Fig.14 Graph depicting Y-maze performance (a measure of spatial working memory) in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL) or APOE2 (AAV-Rh10-E2). The Y axis depicts the percentage of alternations. Data are expressed as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2) treatments.

[0074] Fig.15 is a graph depicting novel object recognition (a measure of working memory) in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL), APOE2 (AAV-Rh10-E2), or APOE2 HA tag (AAV-Rh10-E2HA). The Y axis depicts the discrimination index. Data are expressed as mean ± SEM. Circles represent non-HA labeled (AAV-Rh10-E2), and triangles represent HA labeled (AAV-Rh10-E2HA) treatments.

[0075] Fig.16 is a graph depicting Barn maze test performance (a measure of hippocampal-dependent spatial memory) in APP.PSEN1 / TRE4 mice after administration of AAVrh10 viral capsids encoding null (AAV-Rh10-NULL) or APOE2 (AAV-Rh10-E2). The Y axis depicts escape latency in seconds. Data are expressed as mean ± SEM. Circles represent non-HA-tagged (AAV-Rh10-E2) treatments. DETAILED DESCRIPTION

[0076] The present disclosure provides a method for treating or preventing Alzheimer's disease in a subject in need thereof. In some aspects, the subject with Alzheimer's disease is homozygous for APOE4 expression. In some aspects, the method comprises administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide or a fragment thereof. In some aspects, the vector is administered in a therapeutically effective amount in a range of 5.0×10 9 genome copies (gc) / mL CSF to about 5.0×10 12 In some aspects, the vector is administered by C1-C2 administration or intracisternal (ICM) administration. In some aspects, the rAAV vector comprises the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:11.

[0077] AAV vectors

[0078] In some aspects, the isolated nucleic acid sequence comprising a nucleic acid sequence encoding apolipoprotein 2 (APOE2) can be a recombinant AAV vector (rAAV vector).

[0079] As used herein, "rAAV vector" refers to a vector comprising, consisting essentially of, or consisting of one or more transgene sequences and one or more AAV inverted terminal repeats (ITRs). In some aspects, the rAAV vector contains one or more of the following: an enhancer, a promoter, at least one nucleic acid encoding at least one protein, an intron sequence, and a polyA sequence.

[0080] In some aspects, the present invention relates to a rAAV vector comprising a nucleic acid encoding apolipoprotein 2 (APOE2) for use in treating or preventing Alzheimer's disease in a subject in need thereof.

[0081] In some aspects, the present invention relates to a rAAV vector comprising a nucleic acid encoding apolipoprotein 2 (APOE2) for use in treating or preventing cardiomyopathy associated with Alzheimer's disease in a subject in need thereof.

[0082] In some aspects, the present invention relates to a rAAV vector comprising a nucleic acid encoding apolipoprotein 2 (APOE2) for use in reversing or stabilizing symptoms of Alzheimer's disease in a subject in need thereof.

[0083] In some aspects, the present invention relates to a rAAV vector comprising a nucleic acid encoding apolipoprotein 2 (APOE2) for use in improving symptoms of Alzheimer's disease in a subject in need thereof.

[0084] 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 APOE2; a polyA sequence; and a second ITR sequence.

[0085] In some aspects, the AAV vector encoding APOE2 of the present disclosure may comprise, consist essentially of, 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 therebetween) identical to SEQ ID NO: 8. In some aspects, the AAV vector encoding APOE2 of the present disclosure may comprise, consist essentially of, 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 therebetween) identical to SEQ ID NO: 11.

[0086] In some aspects, the enhancer sequence may comprise, consist essentially of, or consist of a human cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 (or any percentage therebetween).

[0087] In some aspects, the promoter sequence may comprise, consist essentially of, or consist of a chicken β-actin promoter sequence. The chicken β-actin promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 (or any percentage therebetween).

[0088] In some aspects, the chimeric intron sequence can comprise, consist essentially of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 (or any percentage therebetween).

[0089] In some aspects, the polyA sequence can comprise, consist essentially of, or consist of a β-globin polyA sequence. The β-globin polyA sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6 (or any percentage therebetween).

[0090] Nucleic acid sequence encoding APOE2

[0091] Apolipoprotein (APOE) is a protein involved in fat metabolism in the body. It is a family of proteins that bind fat and interact with low-density lipoprotein receptors (LDLR), which are important for the normal processing of lipoproteins rich in triglycerides. In peripheral tissues, APOE is produced by liver and macrophages, and mediates cholesterol metabolism. In the central nervous system, APOE is produced by astrocytes and cholesterol is transported to neurons by APO receptors, which are members of the LDLR family.

[0092] APOE is synthesized as a 317 amino acid protein, which is processed intracellularly to cleave the N-terminal 18 amino acid signal peptide (SEQ ID NO: 13), producing a mature protein of 299 amino acids in length. There are three major variants of APOE: APOE2, APOE3, and APOE4. The three variants differ from each other at two positions: residue 112 (including residue 130 of the signal peptide) and residue 158 (including residue 176 of 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 global population.

[0093] APOE2 is characterized by having a cysteine ​​at position 112 (including residue 130 of the signal peptide) and a cysteine ​​at position 158 (including residue 176 of the signal peptide). APOE3 is characterized by having a cysteine ​​at position 112 (including residue 130 of the signal peptide) and an arginine at position 158 (including residue 176 of the signal peptide). APOE4 is characterized by having an arginine at position 112 (including residue 130 of the signal peptide) and an arginine at position 158 (including residue 176 of the signal peptide).

[0094] Table 1: Summary of APOE variants: APOE2, APOE3, and APOE4.

[0095]

[0096] In some aspects, the nucleic acid sequence encoding the apolipoprotein 2 (APOE2) polypeptide may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5 (or any percentage therebetween).

[0097] In some aspects, the APOE2 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9 (or any percentage therebetween).

[0098] In some aspects, the mature APOE2 polypeptide lacking the signal peptide may comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:14 (or any percentage therebetween).

[0099] In some aspects, the APOE3 polypeptide comprising a signal peptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:17.

[0100] In some aspects, the mature APOE3 polypeptide lacking the signal peptide may comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15 (or any percentage therebetween).

[0101] In some aspects, the APOE4 polypeptide comprising a signal peptide may comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:18 (or any percentage therebetween).

[0102] In some aspects, the mature APOE4 polypeptide lacking the signal peptide may comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:16.

[0103] In some aspects, the present invention provides a nucleic acid sequence comprising SEQ ID NO: 8 or SEQ ID NO: 11 or a variant thereof for use in treating Alzheimer's disease.

[0104] Inverted terminal repeats

[0105] In some aspects, the first ITR may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 1, or its complement. In some aspects, the first ITR may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 7, or its complement. In some aspects, the first ITR may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 10, or its complement. In some aspects, the first ITR may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 12, or its complement.

[0106] In some aspects, the second ITR may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, or its complement (or any percentage therebetween). In some aspects, the second ITR may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, or its complement (or any percentage therebetween). In some aspects, the second ITR may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 10, or its complement. In some aspects, the second ITR may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 12, or its complement.

[0107] AAV vectors

[0108] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of a class of viruses associated with the name and belonging to the genus Dependo parvovirus of the family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses grown in cells, in which certain functions are provided by co-infected helper viruses. Basic information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication date of the reviews, because it is well known that the various serotypes are closely related in structure and function, even at the genetic level. (See, e.g., Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165-174, ed. JR Pattison; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further indicated by heteroduplex analysis, which shows extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing fragments at the ends, which correspond to "inverted terminal repeats" (ITRs). Similar infectivity patterns also indicate that the replication function of each serotype is under similar regulatory control. Multiple serotypes of the virus are known to be suitable for gene delivery; all known serotypes can infect cells from a variety of tissue types. At least 11 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes that can be used in the methods disclosed herein include any of the 11 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. AAV particles comprise, consist essentially of, or consist of the following three major viral proteins: VP1, VP2, and VP3. In some aspects, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, or AAVrh.10.

[0109] Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, and AAVrh.10). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8) containing a genome of one serotype and a capsid of another serotype. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (described in detail in Kerun et al. JCI Insight, 2019; 4(22): e131610), and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303).

[0110] AAV is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, including two 145-nucleotide inverted terminal repeats (ITRs). There are multiple AAV serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; AAV- The complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928. U.S. Patent No. 9,434,928 also provides the sequences of the capsid protein and the self-complementary genome. In one aspect, the AAV genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), combined with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep40) from the rep gene. Rep proteins have a variety of enzymatic properties that are ultimately responsible for replicating the viral genome.

[0111] The cap gene is expressed by the p40 promoter and encodes three capsid proteins, VP1, VP2 and VP3. Variable splicing and non-shared translation start sites are responsible for producing three related capsid proteins. More specifically, after the single mRNA of each of the VP1, VP2 and VP3 proteins is transcribed, it can be spliced ​​in two different ways: longer or shorter introns can be excised, resulting in the formation of two mRNA sets: 2.3kb and 2.6kb long mRNA sets. Longer introns are usually preferred, so the 2.3kb long mRNA can be called the main splice variant. This form lacks the first AUG codon, and the synthesis of VP1 protein starts from this codon, thereby causing the overall level of VP1 protein synthesis to decrease. The first AUG codon retained in the main splice variant is the start codon of VP3 protein. However, in the same open reading frame, the upstream of this codon is an ACG sequence (encoding threonine), which is surrounded by the optimal Kozak (translation start) environment. As described by Becerra SP et al. (December 1985), this results in lower levels of synthesis of the VP2 protein, which is actually the VP3 protein with an additional N-terminal residue, as is the case with VP1. "Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon". Proceedings of the National Academy of Sciences of the United States of America. 82(23):7919–23, Cassinotti P et al., (November 1988). "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced ​​mRNA coding for virus capsid protein 1". Virology. 167(1):176–84, Muralidhar S et al., (January 1994)."Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity". Journal of Virology. 68(1): 170–6 and Trempe JP, Carter BJ (September 1988). "Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein". Journal of Virology. 62(9): 3356–63, each of which is incorporated herein by reference. A single consensus polyA site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Microbiology and Immunology, 158: 97-129 (1992).

[0112] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein, which is unique to the VP1 protein. The VP2 portion is an amino acid sequence present in the VP1 protein, which is also present in the N-terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein, which is common to both the VP1 and VP2 proteins.

[0113] The VP3 protein can be further divided into discrete variable surface regions I-IX (VR-I-IX). Each of the variable surface regions (VRs) can comprise or contain specific amino acid sequences that, alone or in combination with specific amino acid sequences in each of the other VRs, can confer a unique infection phenotype to a particular serotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific tropism relative to other AAV serotypes), as described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9", J. Virol., Vol. 86(12): 6947-6958, June 2012, the contents of which are incorporated herein by reference.

[0114] AAV has unique characteristics, which makes it attractive as a vector for delivering exogenous DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells, allowing the possibility of targeting many different tissues in vivo. In addition, AAV transduces slowly dividing and non-dividing cells, and can basically last the life of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA in a plasmid, which makes the construction of recombinant genomes possible. In addition, since the signals for directing AAV replication and genome encapsidation are contained in the ITR of the AAV genome, some or all of the internal approximately 4.3kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with exogenous DNA to generate AAV vectors. Rep protein and cap protein can be provided in trans. Another notable feature of AAV is that it is an extremely stable and robust virus. It is easy to withstand the conditions for inactivating adenovirus (56°C to 65°C for several hours), making the refrigeration of AAV less important. AAV can even be lyophilized. Finally, AAV-infected cells do not tolerate repeated infection.

[0115] AAV viral vector

[0116] The AAV vector of the present disclosure can be packaged as an AAV viral vector.

[0117] "rAAV viral vector" refers to a viral particle composed of at least one rAAV capsid protein and an encapsidated polynucleotide AAV vector. Therefore, the production of rAAV viral vectors necessarily includes the production of rAAV vectors. The term "viral capsid" or "capsid" refers to the protein shell or coating of a viral particle. The capsid plays a role in encapsidating, protecting, transporting and releasing the viral genome into the host cell. The capsid is generally composed of oligomeric structural subunits of a protein ("capsid protein"). As used herein, the term "encapsidation" means encapsidated in a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2 and VP3.

[0118] rAAV viral vectors useful in the practice of the present invention can be constructed using methods well known in the field of molecular biology. Generally, AAV viral vectors carrying transgenes are assembled from polynucleotides encoding transgenes, appropriate regulatory elements, and elements necessary to produce viral proteins that mediate cell transduction.

[0119] The term "gene transfer" or "gene delivery" refers to a method or system for reliably inserting foreign DNA into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of the host cell.

[0120] Examples of viral vectors include, but are not limited to, adenoviral, retroviral, lentiviral, herpes viral, and adeno-associated viral (AAV) vectors.

[0121] Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found in, for example, WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056, and WO94 / 19478.

[0122] In one embodiment, an adeno-associated virus (AAV) vector is employed.

[0123] In other embodiments, the rAAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74 and AAVrh10 or any other serotype of AAV that can infect humans, monkeys or other species.

[0124] In an exemplary embodiment, the rAAV vector is AAVrh10.

[0125] "rAAV vector" means a vector derived from an adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74 and AAVrh10. The AAV vector may have one or more AAV wild-type genes that are completely or partially deleted, preferably rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are essential for the rescue, replication and packaging of AAV virions. Therefore, the AAV vector is defined herein as comprising at least those sequences required for viral replication and packaging (e.g., functional ITR) in cis. ITR is not necessarily a wild-type nucleotide sequence, and can be, for example, changed by insertion, deletion or replacement of nucleotides, as long as the sequence provides functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to provide at least control elements as components operably linked in the direction of transcription, including a transcription initiation region, a DNA of interest (ie, the APOE2 gene), and a transcription termination region.

[0126] Select control elements that are functional in mammalian cells. The resulting construct containing operably linked components is combined with a functional AAV ITR sequence (5' and 3'). "Adeno-associated virus inverted terminal repeats" or "AAV ITRs" means regions recognized in the art found at each end of the AAV genome, which together act as the origin of DNA replication and as a viral packaging signal in cis. The AAV ITR, together with the AAV rep coding region, provides effective excision and rescue from the nucleotide sequence inserted between the two flanking ITRs, and the integration of the nucleotide sequence into the genome of mammalian cells. In some aspects, the ITR sequence of the present disclosure may include the deletion of one or more nucleotides at one or more positions of the ITR sequence. In some aspects, the nucleotides deleted in the ITR sequence can be repaired in vivo or during vector replication. The nucleotide sequence of the AAV ITR region is known. For AAV-2 sequences, see, for example, Kotin, 1994; Berns, KI "Parvoviridae and their Replication", Fundamental Virology, 2nd edition, (edited by BNFields and DMKnipe). As used herein, "AAV ITR" does not necessarily comprise a wild-type nucleotide sequence, but may be altered, for example, by insertion, deletion or substitution of nucleotides. In addition, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, and the like. In addition, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector are not necessarily identical or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing the excision and rescue of the sequence of interest from the host cell genome or vector, and allowing heterologous sequences to be integrated into the recipient cell genome when the AAV Rep gene product is present in the cell. In addition, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, and the like. Furthermore, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV expression vector do not have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing excision and rescue of the sequence of interest from the host cell genome or vector, and allowing integration of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0127] Particularly preferred are vectors derived from AAV serotypes that have tropism and high transduction efficiency for cells of the mammalian myocardium, particularly cardiomyocytes and cardiomyocyte progenitor cells. A review and comparison of the transduction efficiency of different serotypes is provided in Cearley CN et al., 2008. In other non-limiting examples, preferred vectors include vectors derived from any serotype such as AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9 or AAVrh10, which have also been shown to transduce cardiomyocytes.

[0128] The selected nucleotide sequence is operably linked to control elements that direct its transcription or expression in the subject.Such control elements may comprise control sequences that are normally associated with the selected gene.

[0129] Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from coding mammalian or viral genes. Examples include, but are not limited to, phosphoglycerate kinase (PKG) promoter, CAG, MCK (muscle creatine kinase), SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (AdMLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoter, hybrid promoter, etc. The promoter can be human or from other species, including from mice. In addition, sequences derived from non-viral genes (such as mouse metallothionein genes) can also be used in the present invention. Such promoter sequences can be commercially available from, for example, Stratagene (San Diego, CA).

[0130] Examples of heterologous promoters include the CMV promoter.

[0131] Examples of inducible promoters include the DNA response elements for ecdysone, tetracycline, hypoxia, and aufin.

[0132] AAV expression vectors carrying a DNA molecule of interest bound by AAV ITRs can be constructed by inserting the selected sequence directly into the AAV genome from which the major AAV open reading frame ("ORF") has been excised. Other portions of the AAV genome may also be deleted as long as sufficient ITR portions are retained to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published on January 23, 1992) and WO 93 / 03769 (published on 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, the AAV ITR can be excised from the viral genome or from an AAV vector containing the viral genome 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 have been described in, for example, U.S. Patent No. 5,139,941. In particular, several AAV vectors with accession numbers 53222, 53223, 53224, 53225 and 53226 available from the American Type Culture Collection ("ATCC") are described. In addition, chimeric genes can be produced synthetically to include AAV ITR sequences arranged at 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expressing chimeric gene sequences in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, for example, Edge, 1981; Nambair et al., 1984; Jay et al., 1984. To produce AAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. Many transfection techniques are generally known in the art. See, for example, Graham et al., 1973; Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Habor 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-speed microprojectile bombardment (Klein et al., 1987).

[0133] The AAV viral vector of the present disclosure comprises: i) the AAV vector described herein; and ii) an AAV capsid protein.

[0134] In some aspects, the AAV capsid protein can be any AAV capsid protein. In some aspects, the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein, or an AAVrh.10 capsid protein. In some aspects, the AAV capsid protein is an AAVrh.10 capsid protein.

[0135] Inverted terminal repeats

[0136] "Inverted terminal repeats" or "ITRs" refer to regions recognized in the art found at each end of the AAV genome that together act as an origin of DNA replication and as a packaging signal for the virus in cis. The AAV ITRs, together with the AAVrep coding region, provide efficient excision and rescue from nucleotide sequences inserted between the two flanking ITRs, as well as integration of the nucleotide sequences into the genome of mammalian cells. The nucleotide sequences of the AAV ITR regions are known. For AAV-2 sequences, see, for example, Kotin, 1994; Berns, KI "Parvoviridae and their Replication", Fundamental Virology, 2nd edition, (edited by BNFields and DMKnipe). As used herein, "AAV ITRs" do not necessarily comprise wild-type nucleotide sequences, but may be altered, for example, by insertion, deletion or substitution of nucleotides. In addition, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like. In addition, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector do not have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing excision and rescue of the sequence of interest from the host cell genome or vector, and allowing the heterologous sequence to be integrated into the recipient cell genome when the AAV Rep gene product is present in the cell. In addition, the AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV1, AA2, AAV3, AAV4, AAV5, AAV6, etc. In addition, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV expression vector do not have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing excision and rescue of the sequence of interest from the host cell genome or vector, and allowing the DNA molecule to be integrated into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0137] In some aspects, the AAV ITR sequence can comprise any AAV ITR sequence known in the art. In some aspects, the AAV ITR sequence can be an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, an AAVrh74 ITR sequence, or an AAVrh10 ITR sequence.

[0138] Thus, in some aspects, the AAV ITR sequence may comprise, consist essentially of, or consist of an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, an AAVrh74 ITR sequence, or an AAVrh10 ITR sequence.

[0139] In some aspects, the rAAV vectors of the present disclosure may comprise, consist essentially of, or consist of an AAV2 ITR sequence. In some aspects, the rAAV vectors of the present disclosure may comprise, consist essentially of, or consist of an AAV2 ITR sequence or a modified AAV2 ITR sequence.

[0140] Promoter and enhancer sequences

[0141] As used herein, the terms "promoter" and "promoter sequence" mean a control sequence, which is a region of a polynucleotide sequence in which the initiation and rate of transcription of a coding sequence, such as a gene or transgene, is controlled. For example, a promoter can be constitutive, inducible, repressible, or tissue-specific. A promoter can contain genetic elements to which regulatory proteins and molecules, such as RNA polymerases and transcription factors, can bind.

[0142] The selected nucleotide sequence, such as a nucleotide sequence encoding Friedreich's ataxia protein, is operably linked to control elements that direct its transcription or expression in the subject. Such control elements may include control sequences that are normally associated with the selected gene.

[0143] Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from coding mammalian or viral genes. Examples include, but are not limited to, phosphoglycerate kinase (PKG) promoter, CAG, MCK (muscle creatine kinase), SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (AdMLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), chicken β-actin (CBA) promoter, Rous sarcoma virus (RSV) promoter, synthetic promoter, hybrid promoter, etc. The promoter can be human or from other species, including from mice. In addition, sequences derived from non-viral genes (such as mouse metallothionein genes) can also be used in the present invention. Such promoter sequences can be commercially available from, for example, Stratagene (SanDiego, CA).

[0144] Examples of heterologous promoters include the CMV promoter.

[0145] Examples of inducible promoters include the DNA response elements for ecdysone, tetracycline, hypoxia, and aufin.

[0146] Enhancer is a regulatory element that increases the expression of a target sequence." Promoter / enhancer " is a polynucleotide containing a sequence that can provide both promoter function and enhancer function. For example, the long terminal repeat sequence of a retrovirus contains both promoter function and enhancer function. Enhancer / promoter can be "endogenous" or "exogenous" or "heterologous". "Endogenous" enhancer / promoter is an enhancer / promoter naturally connected to a given gene in a genome. "Exogenous" or "heterologous" enhancer / promoter is placed in juxtaposition with a gene by gene manipulation (i.e., molecular biotechnology) or synthetic technology, so that the transcription of the gene is guided by the connected enhancer / promoter. Non-limiting examples of enhancers / promoters connected in the methods, compositions and constructs provided herein include the CMV enhancer connected to the CBA promoter. It is understood in the art that enhancers can be operated within a certain distance, regardless of their orientation relative to the endogenous or heterologous promoter position. Thus, it is further understood that an enhancer that operates at a distance from a promoter is therefore "operably linked" to the promoter, regardless of its position in the vector or its orientation relative to the promoter's position.

[0147] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene (i.e., a transgene) that is under the control of a promoter to which it is spatially linked. A promoter can be located 5' (upstream) or 3' (downstream) of a gene under its control. A promoter can be located 5' (upstream) of a gene under its control. The distance between a promoter and a gene can be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Variations in the distance between a promoter and a gene can be accommodated without loss of promoter function.

[0148] In some aspects, the enhancer sequence may comprise, consist essentially of, or consist of a human cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 (or any percentage therebetween).

[0149] In some aspects, the promoter sequence may comprise, consist essentially of, or consist of a chicken β-actin promoter sequence. The chicken β-actin promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 (or any percentage therebetween).

[0150] Treatment

[0151] The present disclosure provides a method for treating or preventing Alzheimer's disease (AD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a rAAV vector comprising a nucleic acid sequence encoding an APOE2 polypeptide or a fragment thereof.

[0152] The present disclosure provides a method for treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an APOE2 rAAV viral vector.

[0153] The present disclosure provides a method for treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an APOE2 rAAV viral vector, wherein following administration, the subject experiences an increase in APOE2 expression.

[0154] The subject of the present disclosure may express any combination of APOE variants, including APOE2 homozygotes, APOE3 homozygotes, APOE4 homozygotes, APOE2 / APOE4 heterozygotes, APOE2 / APOE3 heterozygotes, or APOE3 / APOE4 heterozygotes. In some aspects, the subject of the present disclosure is APOE4 homozygotes.

[0155] The subject of the present disclosure may have a certain range of cognitive impairment associated with AD, including no impairment, mild cognitive impairment (CI), or mild, moderate dementia, or severe dementia. In some aspects, the subject of the present disclosure is at least 50 years old. In some aspects, the subject of the present disclosure can be any age.

[0156] In some aspects, the subject of the present disclosure has CSF biomarkers consistent with Alzheimer's disease. In some aspects, the subject of the present disclosure is positive as determined by positron emission tomography (PET) targeting amyloid.

[0157] In some aspects, the subject experiences an increase in the expression of APOE2 relative to the baseline before application. The quantification of APOE2 expression can be performed according to any method known in the art. In some aspects, the subject experiences 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% increase relative to the APOE2 expression of the baseline before application.

[0158] APOE2 expression can occur anywhere in the central nervous system, including any area of ​​the brain and cerebrospinal fluid (CSF).

[0159] APOE2, APOE3 and APOE4 expression 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 blot, chromatography or chromatography-linked mass spectrometry (ie, LC-MS).

[0160] In some aspects, APOE2 expression is reflected as the percentage of APOE4 expression and / or total APOE expression. In some aspects, APOE2 expression is calculated as total APOE2 expression divided by APOE4 expression. This ratio can be multiplied by 100 to produce a percentage. In some aspects, APOE2 expression is calculated as total APOE2 expression divided by total APOE expression (total expression of APOE2\APOE 3 and / or APOE4). This ratio can be multiplied by 100 to produce a percentage. The evaluation of APOE expression can be evaluated according to any suitable protein quantification method.

[0161] In individuals with AD in the prodromal and later stages, hippocampal volume decreases over time. Hippocampal volume is often associated with cognitive and functional changes over time, making it a useful marker for disease progression. In some aspects, after treatment with the APOE2 pharmaceutical composition disclosed herein, the subject experiences an increase in hippocampal volume. In some aspects, after treatment with the APOE2 pharmaceutical composition disclosed herein, the hippocampal volume of the subject remains unchanged. In some aspects, after treatment with the APOE2 pharmaceutical composition disclosed herein, the subject experiences a decrease in the rate of decline in hippocampal volume. In some aspects, hippocampal volume decline is assessed by brain MRI.

[0162] Cognitive assessment

[0163] Cognitive improvement after administration of the APOE2 pharmaceutical composition can be assessed according to any method known in the art. Such assessments may include, but are not limited to, clinical dementia rating (CDR), Alzheimer's disease assessment scale-cognitive assessment (ADAS-Cog13), or a mini-mental state examination.

[0164] The Clinical Dementia Rating (CDR) scale is a dementia staging system rated by clinicians, which tracks the progress of cognitive impairment in 6 categories (memory, orientation, judgment and problem solving, community affairs, family and hobbies and personal care). Each category is scored on a 5-point scale, with none=0, doubtful=0.5, mild=1, moderate=2 and severe=3. Global CDR scoring is established by clinical scoring rules, and there is a value of 0 (no dementia), 0.5 (suspicious dementia), 1 (mild dementia), 2 (moderate dementia) and 3 (severe dementia). CDR-SB is obtained by adding the grades in each of the 6 categories, and the range is between 0 and 18, and wherein higher scoring represents larger damage.

[0165] CDR-SB will be administered at the screening / baseline visit prior to administration of the APOE2 pharmaceutical composition of the present disclosure. These assessments will be used as a measure of clinical efficacy.

[0166] 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 structural performance. Higher scores indicate greater impairment.

[0167] The MMSE is a brief 30-point questionnaire used to assess cognitive impairment, where lower scores indicate greater impairment. The MMSE assesses 11 categories of cognition, including orientation to time, memory, attention, concentration, naming, repetition, comprehension, and the ability to create sentences and copy two intersecting polygons.

[0168] Biomarker testing

[0169] After administering the APOE2 pharmaceutical composition of the present disclosure, APOE2 expression can be evaluated in any region of the CNS. In some aspects, APOE2 expression is evaluated in the brain. In some aspects, APOE2 expression is evaluated in CSF. Amyloid beta (Aβ) 42, Aβ42 / 40, T-tau and P-tau are considered to be core AD biomarkers. It is believed that T-tau and P-tau increase after amyloid brain accumulation (amyloid cascade hypothesis). The accumulation of amyloid and tau is associated with inflammation in the brain at different times during disease progression, amyloid drives inflammation during early accumulation (MCI cases), and tau after amyloid load in the brain is close to AD levels (prodromal AD). Aβ42 is reduced in the CSF of AD participants, and T-tau and P-tau increase. Although not a core CSF biomarker, amyloid beta 40 is also evaluated.

[0170] In some aspects, after administration of the pharmaceutical composition, amyloid β42 (Aβ 42) , amyloid β40 (Aβ 40) The expression level of at least one of t-tau and p-tau is reduced in the subject relative to the pre-administration baseline. In some aspects, after administration of the pharmaceutical composition, amyloid beta 42 (Aβ 42) , amyloid β40 (Aβ 40) The expression level of at least one of t-tau and p-tau is increased in the subject relative to the pre-administration baseline. In some aspects, after administration of the pharmaceutical composition, amyloid beta 42 (Aβ 42) , amyloid β40 (Aβ 40) The expression level of at least one of , T-tau and P-tau remains constant in the subject relative to a pre-administration baseline.

[0171] In some aspects, Aβ 42 , Aβ 40 In some embodiments, the expression level of T-tau and / or P-tau is 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%.

[0172] In some aspects, after administration of the pharmaceutical composition, Aβ 42 In some aspects, after administration of the pharmaceutical composition, Aβ 42 In some aspects, after administration of the pharmaceutical composition, Aβ42 In some aspects, after administration of the pharmaceutical composition, Aβ 42 The changes in expression levels are subject specific.

[0173] In some aspects, after administration of the pharmaceutical composition, amyloid β42 / amyloid β40 (Aβ 42 / 40 ) ratio is altered. In some aspects, after administration of the pharmaceutical composition, Aβ 42 / 40 In some aspects, after administration of the pharmaceutical composition, Aβ 42 / 40 In some aspects, after administration of the pharmaceutical composition, Aβ 42 / 40 Variations in the ratios are subject-specific.

[0174] In some aspects, Aβ 42 / 40 The ratio is increased 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%.

[0175] In a specific embodiment, the gene encoded by the nucleic acid sequence in the AAV vector is the APOE2 gene.

[0176] As used herein, the term "preventing" or "prevention" in its broadest sense refers to preventing a disease or disorder from occurring in a subject who has not yet been diagnosed with the disease or disorder or does not have any clinical symptoms.

[0177] As used herein, the terms "treating" or "treatment", as used herein, mean reversing, alleviating, or inhibiting the progression of the disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition. "Therapeutically effective amount" refers to the minimum amount of an active agent necessary to confer a therapeutic benefit on a subject. For example, a "therapeutically effective amount" for a patient is an amount that induces, ameliorates, stabilizes, slows the progression of, or otherwise causes an improvement in a pathological symptom, disease progression, or physiological condition associated with or resistant to death from a disorder.

[0178] As used herein, the term "subject" refers to a mammal, such as a rodent, a feline, a canine, and a primate. In some aspects, the subject according to the present invention is a human. In the context of the present invention, a "subject in need thereof" refers to a subject, preferably a human, and more particularly a subject suffering from Alzheimer's disease.

[0179] As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a specific polypeptide or protein after transcription and translation.

[0180] As used herein, the term "coding sequence", "sequence encoding a specific protein" or "coding nucleic acid" refers to a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0181] In a specific embodiment, the present invention relates to a method for preventing or treating Alzheimer's disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition comprising a nucleic acid encoding APOE2.

[0182] In a specific embodiment, the present invention is directed to a method of treating Alzheimer's disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition comprising a nucleic acid encoding APOE2.

[0183] In a specific embodiment, the present invention is directed to a method for reversing or stabilizing symptoms of Alzheimer's disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV vector, an AAV viral vector, or a pharmaceutical composition comprising a nucleic acid encoding APOE2.

[0184] As used herein, the term "asymptomatic" or "presymptomatic" refers to a subject who has the disease (Alzheimer's disease) as defined by the genetic diagnosis but has no detectable clinical symptoms.

[0185] As used herein, the term symptomatic refers to a subject who has the disease as defined by genetic diagnosis (Alzheimer's disease) and has cognitive impairment (including MCI or mild, moderate or severe dementia).

[0186] Vector delivery

[0187] Provided herein is a method for treating Alzheimer's disease in a subject in need thereof, the method comprising: (a) providing an AAV vector as defined above, which comprises a nucleic acid sequence encoding an APOE2 polypeptide or a fragment thereof; and (b) delivering the AAV vector to a subject in need thereof, whereby APOE2 is expressed by the transduced cells at a therapeutically effective level.

[0188] The preferred dosage and regimen can be determined by a physician and depends on the subject's age, sex, weight, and stage of disease.

[0189] In some aspects, the AAV vector is administered to the central nervous system (CNS) of the subject. In some aspects, administration is C1-C2 administration. C1-C2 administration refers to the administration of the AAV vector between the first two vertebrae of the cervical spine. In some aspects, C1-C2 administration is CT-guided. In some aspects, administration is intracisternal (ICM). In some aspects, ICM administration is performed when C1-C2 administration is not feasible. In some aspects, factors limiting C1-C2 administration include abnormal arteries along the needle trajectory and / or limited dorsal subarachnoid space (i.e. <2mm).

[0190] In some aspects, AAV vector administration occurs within at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, or at least about 30 minutes. In some aspects, IV infusion occurs within 60 minutes. In some aspects, C1-C2 administration occurs at a rate of about 1 mL / minute, about 2 mL / minute, about 3 mL / minute, about 4 mL / minute, about 5 mL / minute, about 6 mL / minute, about 7 mL / minute, about 8 mL / minute, about 9 mL / minute, or about 10 mL / minute.

[0191] In some aspects, the volume of the AAV vector administered is about 5 mL, about 10 mL, about 15 mL, about 20 mL, or about 25 mL.

[0192] In some aspects, the AAV vector of the present disclosure is administered to a subject at a therapeutically effective dose. In some aspects, the dose is about 5.0×10 9 genome copies (gc) / mL (mL) of cerebrospinal fluid (CSF) (gc / mL) to about 5.0×10 12 gc / mL CSF. In some aspects, the dose is about 1.4×10 10 gc / mL CSF. In some aspects, the dose is about 4.4×10 10 gc / mL CSF. In some aspects, the dose is about 5.0×10 10 gc / mL CSF. In some aspects, the dose is about 1.4×10 11gc / mL CSF. In some aspects, the dose is about 1.6×10 11 gc / mL CSF. In some aspects, the dose is about 5.0×10 11 gc / mL CSF.

[0193] In some aspects, the dose is about 1.0×10 10 gc / ml CSF, about 1.1×10 10 gc / ml CSF, about 1.2×10 10 gc / ml CSF, about 1.3×10 10 gc / ml CSF, about 1.4×10 10 gc / ml CSF, about 1.5×10 10 gc / ml CSF, about 1.6×10 10 gc / ml CSF, about 1.7×10 10 gc / ml CSF, about 1.8×10 10 gc / ml CSF, about 1.9×10 10 gc / mlCSF, about 2.0×10 10 gc / ml CSF, about 2.1×10 10 gc / ml CSF, about 2.2×10 10 gc / ml CSF, about 2.3×10 10 gc / ml CSF, about 2.4×10 10 gc / ml CSF, about 2.5×10 10 gc / ml CSF, about 2.6×10 10 gc / ml CSF, about 2.7×10 10 gc / ml CSF, about 2.8×10 10 gc / ml CSF, about 2.9×10 10 gc / ml CSF, about 3.0×10 10 gc / mlCSF, about 3.1×10 10 gc / ml CSF, about 3.2×10 10 gc / ml CSF, about 3.3×10 10 gc / ml CSF, about 3.4×10 10 gc / ml CSF, about 3.5×10 10 gc / ml CSF, about 3.6×10 10 gc / ml CSF, about 3.7×10 10 gc / ml CSF, about 3.8×10 10 gc / ml CSF, about 3.9×1010 gc / ml CSF, about 4.0×10 10 gc / ml CSF, about 4.1×10 10 gc / mlCSF, about 4.2×10 10 gc / ml CSF, about 4.3×10 10 gc / ml CSF, about 4.4×10 10 gc / ml CSF, about 4.5×10 10 gc / ml CSF, about 4.6×10 10 gc / ml CSF, about 4.7×10 10 gc / ml CSF, about 4.8×10 10 gc / ml CSF, about 4.9×10 10 gc / ml CSF, about 5.0×10 10 gc / ml CSF, about 5.1×10 10 gc / ml CSF, about 5.2×10 10 gc / mlCSF, about 5.3×10 10 gc / ml CSF, about 5.4×10 10 gc / ml CSF, about 5.5×10 10 gc / ml CSF, about 5.6×10 10 gc / ml CSF, about 5.7×10 10 gc / ml CSF, about 5.8×10 10 gc / ml CSF, about 5.9×10 10 gc / ml CSF, about 6.0×10 10 gc / ml CSF, about 6.1×10 10 gc / ml CSF, about 6.2×10 10 gc / ml CSF, about 6.3×10 10 gc / mlCSF, about 6.4×10 10 gc / ml CSF, about 6.5×10 10 gc / ml CSF, about 6.6×10 10 gc / ml CSF, about 6.7×10 10 gc / ml CSF, about 6.8×10 10 gc / ml CSF, about 6.9×10 10 gc / ml CSF, about 7.0×10 10 gc / ml CSF, about 7.1×10 10 gc / ml CSF, about 7.2×10 10gc / ml CSF, about 7.3×10 10 gc / ml CSF, about 7.4×10 10 gc / mlCSF, about 7.5×10 10 gc / ml CSF, about 7.6×10 10 gc / ml CSF, about 7.7×10 10 gc / ml CSF, about 7.8×10 10 gc / ml CSF, about 7.9×10 10 gc / ml CSF, about 8.0×10 10 gc / ml CSF, about 8.1×10 10 gc / ml CSF, about 8.2×10 10 gc / ml CSF, about 8.3×10 10 gc / ml CSF, about 8.4×10 10 gc / ml CSF, about 8.5×10 10 gc / mlCSF, about 8.6×10 10 gc / ml CSF, about 8.7×10 10 gc / ml CSF, about 8.8×10 10 gc / ml CSF, about 8.9×10 10 gc / ml CSF, about 9.0×10 10 gc / ml CSF, about 9.1×10 10 gc / ml CSF, about 9.2×10 10 gc / ml CSF, about 9.3×10 10 gc / ml CSF, about 9.4×10 10 gc / ml CSF, about 9.5×10 10 gc / ml CSF, about 9.6×10 10 gc / mlCSF, about 9.7×10 10 gc / ml CSF, about 9.8×10 10 gc / ml CSF or about 9.9×10 10 gc / ml CSF or any dose in between.

[0194] In some aspects, about 1.0×10 11 gc / ml CSF, about 1.1×10 11 gc / ml CSF, about 1.2×10 11 gc / mlCSF, about 1.3×10 11 gc / ml CSF, about 1.4×10 11gc / ml CSF, about 1.5×10 11 gc / ml CSF, about 1.6×10 11 gc / ml CSF, about 1.7×10 11 gc / ml CSF, about 1.8×10 11 gc / ml CSF, about 1.9×10 11 gc / ml CSF, about 2.0×10 11 gc / ml CSF, about 2.1×10 11 gc / ml CSF, about 2.2×10 11 gc / ml CSF, about 2.3×10 11 gc / mlCSF, about 2.4×10 11 gc / ml CSF, about 2.5×10 11 gc / ml CSF, about 2.6×10 11 gc / ml CSF, about 2.7×10 11 gc / ml CSF, about 2.8×10 11 gc / ml CSF, about 2.9×10 11 gc / ml CSF, about 3.0×10 11 gc / ml CSF, about 3.1×10 11 gc / ml CSF, about 3.2×10 11 gc / ml CSF, about 3.3×10 11 gc / ml CSF, about 3.4×10 11 gc / mlCSF, about 3.5×10 11 gc / ml CSF, about 3.6×10 11 gc / ml CSF, about 3.7×10 11 gc / ml CSF, about 3.8×10 11 gc / ml CSF, about 3.9×10 11 gc / ml CSF, about 4.0×10 11 gc / ml CSF, about 4.1×10 11 gc / ml CSF, about 4.2×10 11 gc / ml CSF, about 4.3×10 11 gc / ml CSF, about 4.4×10 11 gc / ml CSF, about 4.5×10 11 gc / mlCSF, about 4.6×10 11 gc / ml CSF, about 4.7×10 11gc / ml CSF, about 4.8×10 11 gc / ml CSF, about 4.9×10 11 gc / ml CSF, about 5.0×10 11 gc / ml CSF, about 5.1×10 11 gc / ml CSF, about 5.2×10 11 gc / ml CSF, about 5.3×10 11 gc / ml CSF, about 5.4×10 11 gc / ml CSF, about 5.5×10 11 gc / ml CSF, about 5.6×10 11 gc / mlCSF, about 5.7×10 11 gc / ml CSF, about 5.8×10 11 gc / ml CSF, about 5.9×10 11 gc / ml CSF, about 6.0×10 11 gc / ml CSF, about 6.1×10 11 gc / ml CSF, about 6.2×10 11 gc / ml CSF, about 6.3×10 11 gc / ml CSF, about 6.4×10 11 gc / ml CSF, about 6.5×10 11 gc / ml CSF, about 6.6×10 11 gc / ml CSF, about 6.7×10 11 gc / mlCSF, about 6.8×10 11 gc / ml CSF, about 6.9×10 11 gc / ml CSF, about 7.0×10 11 gc / ml CSF, about 7.1×10 11 gc / ml CSF, about 7.2×10 11 gc / ml CSF, about 7.3×10 11 gc / ml CSF, about 7.4×10 11 gc / ml CSF, about 7.5×10 11 gc / ml CSF, about 7.6×10 11 gc / ml CSF, about 7.7×10 11 gc / ml CSF, about 7.8×10 11 gc / mlCSF, about 7.9×10 11 gc / ml CSF, about 8.0×10 11gc / ml CSF, about 8.1×10 11 gc / ml CSF, about 8.2×10 11 gc / ml CSF, about 8.3×10 11 gc / ml CSF, about 8.4×10 11 gc / ml CSF, about 8.5×10 11 gc / ml CSF, about 8.6×10 11 gc / ml CSF, about 8.7×10 11 gc / ml CSF, about 8.8×10 11 gc / ml CSF, about 8.9×10 11 gc / mlCSF, about 9.0×10 11 gc / ml CSF, about 9.1×10 11 gc / ml CSF, about 9.2×10 11 gc / ml CSF, about 9.3×10 11 gc / ml CSF, about 9.4×10 11 gc / ml CSF, about 9.5×10 11 gc / ml CSF, about 9.6×10 11 gc / ml CSF, about 9.7×10 11 gc / ml CSF, about 9.8×10 11 gc / ml CSF or about 9.9×10 11 gc / ml CSF or any dose in between.

[0195] In some aspects, the dose is about 1.0×10 12 gc / ml CSF, about 1.1×10 12 gc / ml CSF, about 1.2×10 12 gc / ml CSF, about 1.3×10 12 gc / ml CSF, about 1.4×10 12 gc / ml CSF, about 1.5×10 12 gc / ml CSF, about 1.6×10 12 gc / ml CSF, about 1.7×10 12 gc / ml CSF, about 1.8×10 12 gc / ml CSF, about 1.9×10 12 gc / mlCSF, about 2.0×10 12 gc / ml CSF, about 2.1×10 12 gc / ml CSF, about 2.2×10 12gc / ml CSF, about 2.3×10 12 gc / ml CSF, about 2.4×10 12 gc / ml CSF, about 2.5×10 12 gc / ml CSF, about 2.6×10 12 gc / ml CSF, about 2.7×10 12 gc / ml CSF, about 2.8×10 12 gc / ml CSF, about 2.9×10 12 gc / ml CSF, about 3.0×10 12 gc / mlCSF, about 3.1×10 12 gc / ml CSF, about 3.2×10 12 gc / ml CSF, about 3.3×10 12 gc / ml CSF, about 3.4×10 12 gc / ml CSF, about 3.5×10 12 gc / ml CSF, about 3.6×10 12 gc / ml CSF, about 3.7×10 12 gc / ml CSF, about 3.8×10 12 gc / ml CSF, about 3.9×10 12 gc / ml CSF, about 4.0×10 12 gc / ml CSF, about 4.1×10 12 gc / mlCSF, about 4.2×10 12 gc / ml CSF, about 4.3×10 12 gc / ml CSF, about 4.4×10 12 gc / ml CSF, about 4.5×10 12 gc / ml CSF, about 4.6×10 12 gc / ml CSF, about 4.7×10 12 gc / ml CSF, about 4.8×10 12 gc / ml CSF, about 4.9×10 12 gc / ml CSF, about 5.0×10 12 gc / ml CSF, about 5.1×10 12 gc / ml CSF, about 5.2×10 12 gc / mlCSF, about 5.3×10 12 gc / ml CSF, about 5.4×10 12 gc / ml CSF, about 5.5×10 12gc / ml CSF, about 5.6×10 12 gc / ml CSF, about 5.7×10 12 gc / ml CSF, about 5.8×10 12 gc / ml CSF, about 5.9×10 12 gc / ml CSF, about 6.0×10 12 gc / ml CSF, about 6.1×10 12 gc / ml CSF, about 6.2×10 12 gc / ml CSF, about 6.3×10 12 gc / mlCSF, about 6.4×10 12 gc / ml CSF, about 6.5×10 12 gc / ml CSF, about 6.6×10 12 gc / ml CSF, about 6.7×10 12 gc / ml CSF, about 6.8×10 12 gc / ml CSF, about 6.9×10 12 gc / ml CSF, about 7.0×10 12 gc / ml CSF, about 7.1×10 12 gc / ml CSF, about 7.2×10 12 gc / ml CSF, about 7.3×10 12 gc / ml CSF, about 7.4×10 12 gc / mlCSF, about 7.5×10 12 gc / ml CSF, about 7.6×10 12 gc / ml CSF, about 7.7×10 12 gc / ml CSF, about 7.8×10 12 gc / ml CSF, about 7.9×10 12 gc / ml CSF, about 8.0×10 12 gc / ml CSF, about 8.1×10 12 gc / ml CSF, about 8.2×10 12 gc / ml CSF, about 8.3×10 12 gc / ml CSF, about 8.4×10 12 gc / ml CSF, about 8.5×10 12 gc / mlCSF, about 8.6×10 12 gc / ml CSF, about 8.7×10 12 gc / ml CSF, about 8.8×10 12gc / ml CSF, about 8.9×10 12 gc / ml CSF, about 9.0×10 12 gc / ml CSF, about 9.1×10 12 gc / ml CSF, about 9.2×10 12 gc / ml CSF, about 9.3×10 12 gc / ml CSF, about 9.4×10 12 gc / ml CSF, about 9.5×10 12 gc / ml CSF, about 9.6×10 12 gc / mlCSF, about 9.7×10 12 gc / ml CSF, about 9.8×10 12 gc / ml CSF or about 9.9×10 12 gc / ml CSF or any dose in between.

[0196] In some aspects, 1.4×10 10 gc / mL CSF, ii) 4.4×10 10 gc / mL CSF and 1.4×10 11 The dose of gc / mL CSF was quantified by ddPCR.

[0197] In some aspects, i) 5.0×10 10 gc / mL CSF, ii) 1.6×10 11 gc / mL CSF, or iii) 5.0×10 11 The dose of gc / mL CSF was quantified by qPCR.

[0198] In some aspects, quantitative PCR (qPCR) doses can be converted to digital droplet PCR (ddPCR) doses. Digital droplet PCR (ddPCR) assays can be used to ensure the precision and accuracy of titration methods for administering rAAV vectors, AAV vectors, or pharmaceutical compositions disclosed herein. After evaluating an initial batch of APOE2 AAV viral capsids, a conversion factor of 3.6 was specified for qPCR to ddPCR, enabling conversion of qPCR doses to ddPCR doses. Those skilled in the art will recognize that the conversion factor can be adjusted based on changes in the AAV viral capsid batches.

[0199] In some aspects, the AAV vectors of the present disclosure are expressed at about 1.0×10 10 gc is about 1.0×10 16 In some aspects, the AAV vectors of the present disclosure are administered at a total dose of about 1.0×10 11 gc is about 1.0×1015 In some aspects, the AAV vectors of the present disclosure are administered at a total dose of about 1.0×10 12 gc is about 9.9×10 14 In some aspects, the AAV vectors of the present disclosure are administered at a total dose of about 1.0×10 12 gc, about 2.0×10 12 gc, about 3.0×10 12 gc, about 4.0×10 12 gc, about 5.0×10 12 gc, about 6.0×10 12 gc, about 7.0×10 12 gc, about 8.0×10 12 gc, about 9.0×10 12 gc, about 1.0×10 13 gc, about 2.0×10 13 gc, about 3.0×10 13 gc, about 4.0×10 13 gc, about 5.0×10 13 gc, about 6.0×10 13 gc, about 7.0×10 13 gc, about 8.0×10 13 gc, about 9.0×10 13 gc, about 1.0×10 14 , about 2.0×10 14 gc, about 3.0×10 14 gc, about 4.0×10 14 gc, about 5.0×10 14 gc, about 6.0×10 14 gc, about 7.0×10 14 gc, about 8.0×10 14 gc, about 9.0×10 14 gc or about 1.0×10 15 or any dose therebetween.

[0200] In some aspects, the AAV vectors of the present disclosure are expressed at about 1.0×10 14 gc, about 1.1×10 14 gc, about 1.2×10 14 gc, about 1.3×10 14 gc, about 1.4×10 14 gc, about 1.5×10 14 gc, about 1.6×10 14 gc, about 1.7×10 14 gc, about 1.8×1014 gc, about 1.9×10 14 gc or about 2.0×10 14 gc or any dose therebetween. In some aspects, the AAV vectors of the present disclosure are administered at a total dose of about 1.4×10 14 The total dose of gc administered.

[0201] In some aspects, the therapeutically effective dose of the AAV vector of the present disclosure is administered as a fixed dose. In some aspects, the fixed dose is expressed as the total number of genome copies (gc).

[0202] In some aspects, the AAV vectors of the present disclosure are expressed at about 1.0×10 10 gc is about 1.0×10 16 In some aspects, the AAV vectors of the present disclosure are administered at a fixed dose of about 1.0×10 13 gc is about 1.0×10 15 In some aspects, the AAV vectors of the present disclosure are administered at a fixed dose of about 1.0×10 14 gc is about 9.0×10 14 In some aspects, the AAV vectors of the present disclosure are administered at a fixed dose of about 1.0×10 14 gc, 2.0×10 14 gc, 2.1×10 14 gc, 2.2×10 14 gc, 2.3×10 14 gc, 2.4×10 14 gc, 2.5×10 14 gc, 2.6×10 14 gc, 2.7×10 14 gc, 2.8×10 14 gc, 2.9×10 14 gc, 3.0×10 14 gc, 4.0×10 14 gc, 5.0×10 14 gc, 6.0×10 14 gc, 7.0×10 14 gc, 8.0×10 14 gc or about 9.0×10 14 gc or any dose in between for a fixed dose administration.

[0203] In some aspects, the AAV vectors of the present disclosure are expressed at about 1.0×10 14 gc, 1.1×10 14 gc, 1.2×10 14 gc, 1.3×10 14 gc, 1.4×1014 gc, 1.5×10 14 gc, 1.6×10 14 gc, 1.7×10 14 gc, 1.8×10 14 gc, 1.9×10 14 gc or about 2.0×10 14 gc or any dose therebetween. In some aspects, the AAV vectors of the present disclosure are administered at a fixed dose of about 2.5×10 14 In some aspects, the AAV vectors of the present disclosure are administered at a fixed dose of about 1.4×10 14 Fixed dose administration of gc.

[0204] Any dosage form of the AAV vector disclosed herein or a method of calculating the dosage form is contemplated herein. In some aspects, the dosage is based on the mass and / or volume of the brain. In some aspects, the dosage is based on the weight of the subject. In some aspects, the dosage is calculated using a qPCR titer method. In some aspects, the dosage is calculated using a ddPCR titer method.

[0205] In some aspects, a therapeutically effective dose can be customized for each AAV capsid serotype. In some aspects, a therapeutically effective dose is customized based on the CNS tropism differences of different AAV capsid serotypes. In some aspects, a therapeutically effective dose is customized based on the liver tropism differences of different AAV capsid serotypes. In some aspects, a therapeutically effective dose is customized based on the cardiac tropism differences of different AAV capsid serotypes.

[0206] In some aspects, a single dose of the AAV vector is administered to the subject. In some aspects, the subject is also administered a second, third, fourth, or fifth dose of the AAV vector. In some aspects, the second and subsequent administrations of the AAV vector can be different doses from the first dose.

[0207] In some aspects, the dose is measured by quantitative polymerase chain reaction (qPCR) titer. In some aspects, the dose is measured by droplet digital polymerase chain reaction (ddPCR) titer.

[0208] In some aspects, the subject is also administered an immunosuppressant while the AAV vector is administered. The immunosuppressant can be any immunosuppressant and / or corticosteroid known in the art. The immunosuppressant can be administered in any amount at any schedule or interval. Immunosuppressants can be administered to improve patient safety, minimize host immune responses to AAV-based therapies, and / or enhance the therapeutic efficacy of AAV-based therapies. In some aspects, the subject is also administered prednisone while the AAV vector is administered.

[0209] In some aspects, prednisone is administered in the following doses:

[0210] About 40 mg once a day for 1 week before AAV viral vector administration;

[0211] 40 mg once daily in week 1 to 2 after AAV viral vector administration;

[0212] 30 mg once daily in the third week after AAV viral vector administration;

[0213] 20 mg once daily in the 4th week after AAV viral vector administration;

[0214] 10 mg once daily in the 5th week after AAV viral vector administration;

[0215] 5 mg once daily in the 6th week after AAV viral vector administration;

[0216] 2.5 mg once daily in week 7 after AAV viral vector administration; and

[0217] 2.5 mg every other day in week 8 after AAV viral vector administration.

[0218] In some aspects, if evidence of a host immune response is observed after administration of the AAV vector, the dose of the immunosuppressant can be kept constant (i.e., not gradually reduced as described above) or increased. In some aspects, the maximum dose of prednisone is about 60 mg. Once evidence of a host immune response subsides or decreases, a gradual reduction of the immunosuppressant can be initiated.

[0219] In some aspects, during the period of prednisone use, the subject is further administered a proton pump inhibitor.

[0220] In some aspects, the invention relates to a vector comprising a nucleic acid encoding APOE2 for use in treating or preventing Alzheimer's disease in a subject, wherein the AAV vector is delivered to a subject in need thereof, and wherein APOE2 is expressed by the transduced cells at therapeutically effective levels.

[0221] In a specific embodiment, the invention relates to a vector comprising a nucleic acid encoding APOE2 for reversing symptoms of Alzheimer's disease in a subject in need thereof, wherein the AAV vector is delivered to the subject in need thereof, and wherein APOE2 is expressed by the transduced cells at therapeutically effective levels.

[0222] Non-viral vectors

[0223] In a specific embodiment, the vector used according to the present invention is a non-viral vector. Generally, the non-viral vector can be a plasmid comprising a nucleic acid sequence encoding the APOE2 gene or a variant thereof as described above.

[0224] Pharmaceutical composition

[0225] In some aspects, the present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease in a subject in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of an AAV vector comprising a nucleic acid encoding APOE2.

[0226] The present disclosure provides a pharmaceutical composition comprising an APOE2 rAAV viral vector; wherein the rAAV viral vector comprises an AAVrh10 capsid protein and an APOE2 rAAV vector.

[0227] In some aspects, the pharmaceutical composition comprises at least about 1.0×10 11 gc / mL to about 1.0×10 15 gc / mL of APOE2rAAV vector. In some aspects, the pharmaceutical composition comprises at least about 1.0×10 11 gc / mL to about 1.0×10 14 gc / mL of APOE2 rAAV vector. In some aspects, the pharmaceutical composition comprises at least about 1.0×10 12 gc / mL, about 1.5×10 12 gc / mL, about 2.0×10 12 gc / mL, about 2.5×10 12 gc / mL, about 3.0×10 12 gc / mL, about 3.5×10 12 gc / mL, about 4.0×10 12 gc / mL, about 4.5×10 12 gc / mL, about 5.0×10 12 gc / mL, about 5.5×10 12 gc / mL, about 6.0×10 12 gc / mL, about 6.5×10 12 gc / mL, about 7.0×10 12 gc / mL, about 7.5×10 12 gc / mL, about 8.0×10 12 gc / mL, about 8.5×10 12 gc / mL, about 9.0×10 12 gc / mL, about 9.5×10 12 gc / mL, about 1.0×10 13 gc / mL, about 1.5×10 13 gc / mL, about 2.0×10 13 gc / mL, about 2.5×10 13 gc / mL, about 3.0×10 13 gc / mL, about 4.0×1013 gc / mL, about 4.5×10 13 gc / mL, about 5.0×10 13 gc / mL, about 5.5×10 13 gc / mL, about 6.0×10 13 gc / mL, about 6.5×10 13 gc / mL, about 7.0×10 13 gc / mL, about 7.5×10 13 gc / mL, about 8.0×10 13 gc / mL, about 8.5×10 13 gc / mL, about 9.0×10 13 gc / mL, about 9.5×10 13 gc / mL or any number therebetween of APOE2 rAAV vectors. In some aspects, the pharmaceutical composition comprises at least about 1.5×10 13 gc / mL of APOE2 rAAV vector.

[0228] In some aspects, the APOE2 rAAV pharmaceutical composition comprises less than about 50% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises less than about 40% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises less than about 15% empty rAAV capsids.

[0229] In some aspects, the APOE2 rAAV pharmaceutical compositions disclosed herein comprise less than about 15% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical compositions comprise less than about 50% empty rAAV capsids, less than about 45% empty rAAV capsids, less than about 40% empty rAAV capsids, less than about 39% empty rAAV capsids, less than about 38% empty rAAV capsids, less than about 37% empty rAAV capsids, less than about 36% empty rAAV capsids, less than about 35% empty rAAV capsids, less than about 34% empty rAAV capsids, less than about 33% empty rAAV capsids, less than about 32% empty rAAV capsids, less than about 31% empty rAAV capsids, less than about 30% empty rAAV capsids, less than about 29% empty rAAV capsids, less than about 28% empty rAAV capsids, less than about 27% empty rAAV capsids, less than about 26% empty rAAV capsids, less than about 25% empty rAAV capsids, less than about 24% empty rAAV capsids, less than about 23% empty rAAV capsids, less than about 22 % empty rAAV capsids, less than about 21% empty rAAV capsids, less than about 20% empty rAAV capsids, less than about 19% empty rAAV capsids, less than about 18% empty rAAV capsids, less than about 17% empty rAAV capsids, less than about 16% empty rAAV capsids, less than about 15% empty rAAV capsids, less than about 14% empty rAAV capsids, less than about 13% empty rAAV capsids, less than about 12% empty rAAV capsids, In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 10% empty rAAV capsids.

[0230] In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 40% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 35% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 30% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 25% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 20% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 15% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 10% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises about 2% to about 8% empty rAAV capsids. In some aspects, the APOE2 rAAV pharmaceutical composition comprises less than or equal to about 6% empty rAAV capsids, about 5% empty rAAV capsids, about 4% empty rAAV capsids, about 3% empty rAAV capsids, about 2% empty rAAV capsids, or about 1% empty rAAV capsids. In some aspects, the number of empty rAAV capsids is below the limit of detection. In some aspects, the percentage of empty rAAV capsids is determined, for example, by electron microscopy as a percentage of total rAAV capsids.

[0231] In some aspects, the percentage of empty rAAV capsids is determined as the percentage of total rAAV capsids, for example, using analytical ultracentrifugation (AUC). In some aspects, AUC is sedimentation velocity AUV (SV-AUC). In some aspects, for example, compared with the administration of a composition with a higher percentage of empty rAAV particles, these low percentages of empty rAAV particles improve therapeutic efficacy and / or reduce adverse events (e.g., inflammatory response, liver damage) after administration to a subject. In some aspects, compared with the level of empty rAAV particles produced in other methods (e.g., those methods that do not use the production and / or purification methods described herein), the method for preparing the rAAV composition disclosed herein provides these low percentages of empty rAAV particles.

[0232] In some aspects, the APOE2 rAAV pharmaceutical compositions disclosed herein contain at least 50% complete rAAV particles. In some aspects, the APOE2 rAAV pharmaceutical compositions disclosed herein contain at least 60% complete rAAV particles. In some aspects, the APOE2 rAAV pharmaceutical compositions disclosed herein contain at least 70% complete rAAV particles. In some aspects, the APOE2 rAAV pharmaceutical compositions disclosed herein contain at least 80% complete rAAV particles. In some aspects, the APOE2 rAAV pharmaceutical compositions disclosed herein contain at least 85% complete rAAV particles, at least 90% complete rAAV particles, at least 95% complete rAAV particles, or at least about 99% complete rAAV particles. In some aspects, the APOE2 rAAV pharmaceutical compositions contain 100% complete rAAV particles.

[0233] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual host cell DNA is removed. In some aspects, the amount of residual host cell DNA is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0234] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual host cell proteins are removed. In some aspects, the amount of residual host cell proteins is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0235] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual adenoviral early region 1A (E1a DNA) is removed. In some aspects, the amount of E1a DNA is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0236] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual SV40 large T antigen DNA (SV40 LTA) DNA is removed. In some aspects, the amount of SV40 LTA DNA is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0237] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared so that residual unencapsulated rAAV vector plasmid DNA is removed. In some aspects, the amount of unencapsulated rAAV vector plasmid DNA is below a predetermined value, which is determined to be necessary for safe and effective treatment suitable for administration to a subject. In some aspects, the rAAV vector plasmid DNA is a plasmid DNA that does not encode between the ITR regions.

[0238] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual polyethyleneimine (PEI) is removed. In some aspects, the amount of PEI is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0239] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual benzonase is removed. In some aspects, the amount of benzonase is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0240] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual bovine serum albumin (BSA) is removed. In some aspects, the amount of BSA is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0241] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that residual iodixanol is removed. In some aspects, the amount of iodixanol is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0242] The APOE2 rAAV pharmaceutical compositions of the present disclosure are prepared such that replication-competent AAV (rcAAV) is removed. In some aspects, the amount of rcAAV is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject.

[0243] By "therapeutically effective amount" is meant an amount of an AAV vector of the invention sufficient to treat Alzheimer's disease at a reasonable benefit / risk ratio applicable to any medical treatment.

[0244] It should be understood that the single dose or total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound employed; the specific composition employed; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination or concomitantly with the specific polypeptide employed; and similar factors well known in the medical field. For example, it is entirely within the skill of the art to start using the compound at a dosage level lower than the dosage level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product can vary widely per adult per day. The therapeutically effective amount of the vector according to the present invention that should be administered and the dosage of the pathological condition treated with the virus or non-viral particles and / or pharmaceutical composition of the present invention will depend on a variety of factors, including the age and condition of the patient, the severity of the disorder or condition, the method and frequency of administration, and the specific peptide to be used.

[0245] The presentation form of the pharmaceutical composition containing the AAV vector according to the present invention can be any form suitable for the selected mode of administration (eg, C1-C2 administration or ICM).

[0246] In the pharmaceutical composition of the present invention for administration to the CNS, the active ingredient (alone or in combination with another active ingredient) can be administered to animals and humans as a mixture with a conventional pharmaceutical support in a unit administration form.

[0247] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for a formulation capable of being injected. These may in particular be isotonic sterile saline solutions (mono- or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts) or dried, especially lyophilized, compositions which, after adding, as appropriate, for example, sterile water or physiological saline, allow the constitution of injectable solutions.

[0248] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. The composition must be stable under the conditions of manufacture and storage and must be preserved in storage against the contaminating action of microorganisms such as bacteria and fungi.

[0249] Solutions containing the compounds of the invention in the form of free alkali or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactants such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under common storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0250] The AAV vector according to the present invention can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), and pharmaceutically acceptable salts are formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0251] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant. Preventing the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents, for example, sugar or sodium chloride. The absorption of the injectable composition can be extended by using an agent that delays absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0252] Sterile injectable solutions are prepared by incorporating the active polypeptide in the desired amount in an appropriate solvent, as necessary, along with several other ingredients listed above, followed by filter sterilization. Typically, dispersions are prepared by incorporating a variety of sterilized active ingredients into a sterile vehicle containing an alkaline dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying techniques and freeze drying techniques, thereby producing a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof.

[0253] After formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is readily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like may also be employed.

[0254] Multiple doses may also be administered.

[0255] In some aspects, pharmaceutical compositions comprising the rAAV viral vectors of the present disclosure are formulated with more or more excipients suitable for administration to a subject in need thereof by any suitable method of administration. In some aspects, one or more excipients include phosphate buffer and salts.

[0256] In some aspects, phosphate buffer comprises potassium dihydrogen phosphate and disodium hydrogen phosphate. In some aspects, potassium dihydrogen phosphate is used with a concentration of about 0.01mM to about 100mM. In some aspects, the concentration of potassium dihydrogen phosphate is about 0.1mM, about 0.25mM, about 0.5mM, about 0.75mM, about 1mM, about 1.25mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 15mM, about 20mM or about 30mM or any concentration therebetween. In some aspects, potassium dihydrogen phosphate is used with a concentration of about 1mM. In some aspects, disodium hydrogen phosphate is used with a concentration of about 0.01mM to about 100mM. In some aspects, the concentration of sodium phosphate dibasic is about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 15mM, about 20mM or about 30mM or any concentration therebetween. In some aspects, sodium phosphate dibasic is used at a concentration of about 3mM.

[0257] In some aspects, the salt is sodium chloride. In some aspects, the concentration of sodium chloride is about 0.01mM to 1M. In some aspects, the concentration of sodium chloride is about 25mM to 300mM. In some aspects, the concentration of sodium chloride is about 100mM, about 110mM, about 120mM, about 130mM, about 140mM, about 150mM, about 155mM, about 160mM, about 170mM, about 180mM, about 190mM or about 200mM. In some aspects, the concentration of sodium chloride is about 155mM.

[0258] In some aspects, the pharmaceutical composition is formulated to a pH suitable for administration to a subject. In some aspects, the pH of the pharmaceutical composition is about 6.0 to about 9.0. In some aspects, the pH of the pharmaceutical composition is about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some aspects, the pH of the pharmaceutical composition is about 7.4.

[0259] The pharmaceutical composition of the present disclosure can be applied to the subject in any volume deemed suitable by the attending physician, and can be varied according to the specific needs of each individual subject. In some aspects, the pharmaceutical composition of the present disclosure (including prepackaged pharmaceutical compositions) can be diluted in a fluid of appropriate volume before application. In some aspects, if the pharmaceutical composition of the present disclosure is diluted, the volume of the diluted pharmaceutical composition applied to the subject is equal to the dosage of the rAAV viral particles provided herein. In other words, if the volume of the pharmaceutical composition is increased, the concentration of the rAAV viral particles is diluted, and the patient will still apply the same total number of rAAV vector genome copies or the number of rAAV viral capsids specified by the dosage disclosed herein.

[0260] In some aspects, the volume of the subject's CSF determines the final volume of the pharmaceutical composition.In some aspects, CSF volume is measured by magnetic resonance imaging (MRI).

[0261] In some aspects, the pharmaceutical composition is administered in a total volume of about 5 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, or about 50 mL, or any volume therebetween.

[0262] Manufacturing of APOE2 AAV viral vector

[0263] The present disclosure provides methods for producing rAAV vectors and rAAV viral vectors encoding APOE2. In some aspects, the rAAV viral vector is derived from a cell lysate produced by the cell culture and purification methods described herein.

[0264] The present disclosure provides a method for producing a cell lysate containing a rAAV viral vector, the method comprising: (i) obtaining a culture container containing HEK293T cells in a culture medium; (ii) transfecting the HEK293T cells with a first plasmid encoding an APOE2 AAV vector and a second plasmid encoding an AAV Rep protein and an AAV Cap protein in a transfection medium, wherein the ratio of the second plasmid to the first plasmid is 2:1; (iii) culturing the transfected HEK293T cells in a culture medium under conditions in which the transfected HEK293T cells produce a recombinant adeno-associated virus (rAAV) viral vector encoding APOE2; (iv) harvesting the transfected HEK293T cells; and (v) lysing the transfected HEK293T cells to produce a cell lysate containing a rAAV viral vector.

[0265] The culture medium of the present disclosure can be any culture medium capable of culturing mammalian cells (including but not limited to HEK293T cells). In some aspects, the culture medium comprises Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Cells can be expanded to produce any length of time required for the desired number of cells. In some aspects, HEK293T cells are obtained after amplification culture for about two days to about five days.

[0266] Transfection can be carried out in any suitable culture medium. In some aspects, the transfection medium comprises serum-free DMEM and polyethyleneimine (PEI).

[0267] The transfection of the two plasmids can occur in any order. In some aspects, the transfection of the first plasmid and the second plasmid occurs simultaneously.

[0268] In some aspects, prior to transfection, HEK293T cells were grown at a density of about 2.0×10 4 About 2.0×10 6 cells / cm 2 In some aspects, the HEK293T cells are present in the culture vessel at a density of about 2.0×10 5 cells / cm 2 The density exists.

[0269] After transfection, the cells can be cultured for any suitable length of time to produce the desired amount of APOE2 rAAV viral vector. In some aspects, the transfected cells are cultured for about 1 day to about 7 days. In some aspects, the transfected cells are cultured for about 3 days.

[0270] After harvesting the transfected cells, the cells containing the APOE2 rAAV viral vector are cracked to produce a cell lysate containing the APOE2 rAAV viral vector. Cell lysis can be performed by any suitable method, including freeze-thaw, mechanical lysis, and lysis by chemical reagents (such as detergents). In some aspects, the HEK293T cells are lysed by at least about 4 continuous freeze-thaw cycles to produce a cell lysate.

[0271] The cell lysate of the present disclosure can be treated to remove any residual unencapsulated viral or cellular DNA. In some aspects, the cell lysate is further treated with a recombinant nuclease to digest any viral DNA or cellular DNA. In some aspects, the recombinant nuclease is benzonase. In some aspects, the DNA digestion is carried out in the presence of magnesium chloride.

[0272] After DNA digestion, the lysate containing the APOE2 rAAV viral vector is clarified. Clarification can be performed according to any method known in the art. In some aspects, clarification is performed by centrifugation or ultracentrifugation. Clarification of the cell lysate removes cell debris and unbroken cells.

[0273] After clarification, the number of rAAV vectors or rAAV viral vectors in the cell lysate can be quantified. In some aspects, the cell lysate contains about 1.0×10 9 to about 5.0×10 14 genome copies (gc) / ml.

[0274] The present disclosure also provides a method for producing an APOE2 rAAV pharmaceutical composition, the method comprising: (i) obtaining a cell lysate containing an rAAV viral vector encoding APOE2; (ii) contacting a density gradient with the cell lysate containing an rAAV viral vector encoding APOE2, and subjecting the density gradient to centrifugation; (iii) contacting a chromatographic column with the cell lysate containing an rAAV viral vector encoding APOE2; (iv) eluting the rAAV viral vector from the column; and (v) concentrating the eluted rAAV viral vector into a formulation buffer by ultrafiltration, thereby producing an APOE2 rAAV pharmaceutical composition.

[0275] The density gradient of the present disclosure can be any suitable density gradient. Density gradient provides a means to separate components in a mixture by their size and / or molecular weight. Density gradient purification can be used to separate empty AAV capsids from AAV capsids comprising APOE2 rAAV vectors. In some aspects, the density gradient is an iodixanol density gradient.

[0276] In some aspects, the iodixanol gradient comprises a stepwise density gradient comprising: (i) about 10% to about 20% iodixanol solution; (ii) about 20% to about 30% iodixanol solution; (iii) about 40% to about 50% iodixanol solution; and (iv) about 50% to about 60% iodixanol solution. In some aspects, the iodixanol gradient comprises a stepwise density gradient comprising: (i) about 15% iodixanol solution; (ii) about 25% iodixanol solution; (iii) about 40% iodixanol solution; and (iv) about 54% iodixanol solution.

[0277] In some aspects, alternative purification means can be used instead of density gradient purification. In some aspects, chromatography can be used. In some aspects, ion exchange chromatography can be used.

[0278] After density gradient purification, chromatography can be used to purify rAAV viral vectors. In some aspects, the chromatography is anion exchange chromatography. In some aspects, the anion exchange column is a Q agarose high efficiency strong quaternary ammonium anion exchange resin column.

[0279] After chromatographic purification, the APOE2 rAAV viral vector buffer is exchanged into a formulation buffer by ultrafiltration, thereby producing a pharmaceutical composition of the present disclosure. In some aspects, the formulation buffer comprises phosphate buffered saline (PBS).

[0280] In some aspects, after ultracentrifugation, the pharmaceutical composition comprises about 1.0×10 10 to about 5.0×10 13 In some aspects, after ultracentrifugation, the pharmaceutical composition comprises about 1.5×10 13 Genome copies (viral genomes) / ml.

[0281] Example

[0282] The present invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way.

[0283] Example 1: Administration of APOE2 rAAV vector to AD patients results in increased APOE2 expression and altered expression of AD biomarkers.

[0284] As part of a Phase 1 / 2 trial, an AAV vector encoding human APOE2 (LX1001) was administered to APOE4 homozygous patients with Alzheimer's disease. Administration was performed by C1-C2 injection under CT guidance. C1-C2 administration is preferred, but ICM administration can be used when C1-C2 administration is impractical or impossible. Patients were administered a dose of 5.0×10 10 gc / ml CSF (determined by qPCR) or 1.4×10 10 LX1001 (as measured by ddPCR). Total CSF volumes in administered patients ranged from 313 mL to 490 mL. No serious adverse events were observed following administration. Adverse events of transient headache occurred in 2 / 5 subjects and were considered to be related to the administration procedure. No other adverse events occurred in more than one subject.

[0285] Patient population

[0286] Eligibility criteria for participation in the trial included: a) age greater than or equal to 50 years, homozygous for APOE4 expression, mild cognitive impairment, mild to moderate dementia with a positive amyloid PET and biomarkers consistent with Alzheimer's disease.

[0287] end

[0288] Primary End Point

[0289] Security

[0290] Secondary End Points

[0291] Conversion of APOE4 to APOE2 in CSF

[0292] Other secondary endpoints

[0293] CSF biomarker levels / expression: Aβ42, T Tau, and P Tau

[0294] Amyloid PET scan

[0295] Quantitative MRI

[0296] Cognitive testing

[0297] APOE2 expression after LX1001 administration was evaluated at fixed time points and compared with the baseline of APOE4 expression before administration. APOE2 / APOE4 expression was evaluated in the CSF of patients at 3, 6, and 12 months ( Figure 1 and Figure 2 APOE2 / APOE4 expression was calculated as total APOE2 expression divided by total APOE4 expression multiplied by 100 to give the percent increase in APOE2 expression relative to APOE4 expression ( Figure 1 Patients all experienced an increase in APOE2 expression following treatment with LX1001. APOE2 expression was confirmed in all patients with follow-up data and persisted through 12 months ( Figure 2 ).

[0298] AD biomarkers were also evaluated after LX1001 administration. Aβ42, T-tau, and P-tau expression were measured at 2 months, 6 months, and 12 months after LX1001 administration and compared to pre-treatment baseline (Figure 3). Biomarker expression was measured in CSF. After treatment with LX1001, treated patients generally experienced Aβ42 ( Figure 3A )、T-tau( Figure 3B ) and P-tau( Figure 3C ) expression reduction.

[0299] Example 2: In vivo studies of AAV vectors encoding APOE2 polypeptides

[0300] The hypothesis that AAVrh.10-mediated expression of combined human APOE2 would effectively protect against the development of Alzheimer's disease was evaluated. AAVrh.10hAPOE2C was tested in the APP.PSEN1 / TRE4 amyloid plaque mouse model, which has a humanized APOE 4 gene, amyloid plaques that promote presenilin mutations, and mutant amyloid precursor protein (Neurobiology of Aging 2016 44:159). AAVrh.10APOE2 and AAHrh.10 null vectors were administered to the hippocampus of APP.PSEN / APOE4 mice (2×10 10 genomic copies) and were evaluated at 5.5 months after administration. APP.PSEN / APOE4 mice administered PBS were used as controls. Compared with PBS, the hippocampal human APOE2 protein in the AAVrh.10hAPOE2-treated group was significantly increased ( Figure 4C ). AAVrh.10hAPOE2 administration reduced the levels of soluble and insoluble amyloid peptide β42 and soluble and insoluble amyloid peptide β40, respectively, compared with the PBS group ( Figure 5A and Figure 5B The amount of viral genome copies in the mouse brain after administration was evaluated ( Figure 4A The amount of APOE mRNA was also measured ( Figure 4B ).

[0301] Immunohistochemical staining of β-amyloid in the hippocampus showed a reduction in the number and size of amyloid plaques after AAVrh.10hAPOE2 administration compared to PBS controls ( Figure 6 ). Fluoro-Jade staining of neuronal degeneration was reduced from PBS mice to AAVrh.10hAPOE2 treated mice ( Figure 7 GFAP ( Figure 8 ) and Iba1( Fig. 9 )-positive cells. The marker of amyloid aggregates and structure, X-34, depicts a reduction in amyloid levels, as measured by a decrease in X-34 staining intensity ( Fig.10 ).

[0302] Before sacrifice, mice were evaluated in three behavioral sensorimotor assays. Compared with PBS, AAVrh.10hAPOE2 treatment: (1) increased Y-maze ( Fig.12 , Fig.13 ), Fig.14 ; (2) Improved new target discrimination index ( Fig.15); (3) decreased escape latency in the Barn maze test ( Fig.16 ); and (4) observed changes in nesting behavior ( Fig.11 ).

[0303] In conclusion, intrahippocampal delivery of AAVrh.10 expressing human APOE2 improved morphological, biochemical, and behavioral disease parameters in amyloid-driven Alzheimer's disease mice.

[0304] Example 3: Preparation of APOE2 rAAV viral vector

[0305] The preparation of APOE2 rAAV vector pharmaceutical compositions can be carried out according to the methods outlined below.

[0306] The APOE2 rAAV vector upstream production method consists of a series of cell culture amplification steps until the transfection of cells. The production of AAVrh.10hAPOE2 involves the co-transfection of HEK293T cells with two plasmids: the AAV vector plasmid (pAAV-hAPOE2) and the helper plasmid (pPAK-MArh.10). After transfection, the cells are incubated until the cell harvest. The cell harvest can be stored frozen before further processing. The crude viral lysate is recovered from the transfected cells by multiple freeze-thaw cycles and is cultured with Incubate with to digest residual plasmid DNA and host nucleic acid. Clarification of upstream process material is performed by ultracentrifugation to remove cell debris and unbroken cells. Purification of process streams is performed by iodixanol gradient and anion exchange chromatography.

[0307] The final processing step is to concentrate the processing pool and exchange the buffer into the formulation buffer to form the AAVrh.10hAPOE2 drug substance. The subbatches can be combined and then diluted to the target bulk drug substance (BDS) concentration prior to drug product manufacturing to form a pharmaceutical composition comprising the APOE2 rAAV vector.

[0308] HEK293T WCB Thawing and Amplification:

[0309] To start a batch of AAVrh.10hAPOE2, a vial of HEK293T WCB was thawed and propagated by serial culture in DMEM and 10% qualified, gamma-irradiated fetal bovine serum (FBS) without antibiotics or phenol red indicator. The 10% FBS and DMEM media used for the cell expansion phases were pre-warmed before use at each phase.

[0310] Frozen HEK293T WCB vials were thawed by suspending the frozen vials in a beaker containing warm water for injection (WFI). After visual confirmation that the material was thawed, the WCB cells were transferred to a 50 mL conical tube containing preheated culture medium and mixed manually. HEK293T cells were grown as adherent cultures on the original polystyrene surface of Corning 175 cm2 cell culture flasks. The cell suspension from the 50 mL conical tube was transferred to a T75 flask. The T75 flask was cultured in a humidity controlled incubator at 37 ° C and 5% CO2 for up to 48 hours.

[0311] After the initial expansion phase, the cells were washed with phosphate buffered saline and detached from the flask walls using TrypLE select. Warm 10% FBSDMEM medium was added to the flask to wash the cells from the culture vessel surface and to break up the cell clusters. The detached cells suspended in the medium were transferred to a sterile container and the cell density and viability were determined. The cell density was used to calculate the inoculation volume for the next expansion phase. After adding the target inoculation density in the T175 flask, the flask was incubated at 37°C, 90% relative humidity and 5% CO2 for 2 to 4 days. The cells were expanded until there were enough cells for transfection.

[0312] Cell monolayers and ten (10) cell stacks were seeded from cells recovered from T175 flasks at a target cell density of ≥5E3 cells / cm2. During recovery from the flask, the cell monolayers were washed with PBS, detached from the surface with TrypLE select, and then resuspended in warm 10% FBSDMEM. Each cell stack was incubated at 37°C, 90% relative humidity, and 5% CO2 for up to 4 days prior to transfection.

[0313] PEI-mediated transfection

[0314] When the cells seeded in the cell stack have grown to a density of approximately 2E5 viable cells / cm2, the AAV vector and auxiliary plasmid are added to the cells in the presence of PEI (a synthetic transfection reagent that is not derived from any animal product). The molar ratio of the auxiliary plasmid to the transgenic plasmid used in the transfection is approximately 1:2. The transfection mixture is prepared by adding the calculated volume of DNA to a dilution tube containing PEI. For each 10-layer cell stack, 1272μg of auxiliary plasmid and 636μg of transgenic plasmid are added to SF-DMEM to dilute the plasmid, and then they are combined with 1908μg PEI pro in a dilution tube. The solution is mixed and incubated at room temperature for 15 minutes, and then the prepared transfection mixture is divided into two bottles, each containing 750mL of 2.5% DMEM, and the total complex volume of each cell stack is 36.7mL. Before adding the transfection mixture, the residual culture medium from the expansion stage is removed from the cell stack.

[0315] The transfected cells were cultured in an incubator at 37° C., 90% humidity and 5% CO 2 for the production phase.

[0316] Cell harvesting

[0317] After transfection for three days, the cells floating in the culture medium were harvested from the culture medium. The culture medium in the cell pile was collected and added to a centrifuge bottle. The remaining cells were released from the cell pile using sterile PBS / EDTA solution (pH 7.5). The floating cells in the cells and culture medium in the PBS / EDTA solution were precipitated by centrifugation. The supernatant was discarded, and the cell pellet was resuspended in a hypotonic resuspension buffer (20mM Tris pH 8.0 and 2mM MgCl ) and merged. The sample of the cell harvest material (cell and supernatant) was collected for determination of vector genome copy (GC) titer, bioburden and the presence of external factors evaluated before further processing. Before further processing, at ≤-65°C, the cell harvest was stored in a 50mL sterile and pyrogen-free polypropylene conical tube with a screw top cap for up to 3 months.

[0318] Dissolving / clarifying

[0319] The cell harvest was subjected to multiple freeze / thaw cycles to release the target vector from the cells producing the crude viral lysate (CVL). For each cycle, the frozen cell harvest was thawed on a heating block, mixed, and then placed on dry ice for refrozen. Five freeze / thaw cycles were completed before collecting in-process samples for vector GC titer.

[0320] CVL was treated with a target concentration of 200 U / mL of recombinant nuclease (Benzonase) in a resuspension buffer containing 2 mM magnesium chloride (MgCl2) to digest any additional viral DNA and host cell-derived nucleic acids. After the addition of benzonase, the CVL was incubated at 37°C for 60 minutes. After benzonase treatment, the CVL was clarified by centrifugation to remove cell debris and unbroken cells. After the clarified CVL was combined, in-process samples were collected and analyzed for vector GC titer.

[0321] Iodixanol Gradient Purification

[0322] Clarified CVL was purified using a differential iodixanol density gradient. In this step, 15% / 25% / 40% / 54% iodixanol solution prepared in a sealed tube was used to separate AAVrh.10hAPOE2 from cellular components and empty capsids. The test tubes were centrifuged with a fixed angle rotor to perform gradient separation and collect gradient fractions containing complete capsids. After collection, the fractions were diluted with QHP binding buffer (50mM Tris, 50mM NaCl, pH 9.0) and stored overnight at 2-8°C. QC samples of the purified AAVrh.10hAPOE2 were taken and the vector GC titer was analyzed.

[0323] Anion exchange chromatography

[0324] The fractions containing the desired AAVrh.10hAPOE2 vector product were combined and loaded onto Hi-Trap QHP anion exchange columns. These columns are disposable and filled with Q agarose high efficiency strong quaternary ammonium anion exchange resin. The AAVrh.10hAPOE2 vector was loaded onto the equilibrated column and collected in the flow-through. The column was washed with 1 column volume of binding buffer and combined with the AAVrh.10hAPOE2 flow-through to recover the residual product from the column. Up to two columns were used to purify the iodixanol fractions collected from a single 10-layer cell stack. In-process QC samples of purified AAVrh.10hAPOE2 were collected and analyzed for vector GC titer.

[0325] Ultrafiltration

[0326] The AAVrh.10hAPOE2 vector was concentrated and buffer exchanged using a disposable ultrafiltration device with a 100 kDa MWCO polysulfone (PES) filter to produce the drug substance in its final formulation buffer, phosphate buffered saline. The filter membrane was configured to work with a swing bucket rotor in a centrifuge. Prior to the initial concentration step, the filter membrane was equilibrated with the final formulation buffer. The concentration and buffer exchange operations were controlled by a volume gradient marker on the side of the ultrafiltration unit. The dead stop concentration ratio was limited by filling the collection bottom to the total volume minus the reduction volume in the sample.

[0327] In-process QC samples of drug substance were taken and analyzed for vector GC titer, SDS PAGE purity, empty capsids, and endotoxins.Drug substance interim batches were stored at ≤ -65 °C in 5 mL cryogenic vials (USP VI) for up to 1 year prior to further processing.

[0328] Bulk drug substances

[0329] To account for 10% losses during sterile filtration in preparing the drug product, frozen drug substance subbatches are thawed, combined and adjusted with PBS to a target concentration 10% higher than the target drug product concentration (in some aspects, 1.0E13 gc / mL, but other target drug product concentrations are contemplated and described herein). Multiple batches of drug substance can be combined to form the BDS. The target BDS volume is based on the amount of AAVrh.10hAPOE2 vector drug product (DP) required to provide the ongoing clinical trial. The combination and dilution of drug substance batches are performed in sterile disposable containers immediately prior to sterile filtration and filling of the AAVrh.10hAPOE2 vector DP.

[0330] Analytical evaluation of AAVrh.10hAPOE2 vector-drug compositions

[0331] Analytical evaluation of the AAVrh.10hAPOE2 vector pharmaceutical composition (LX1001) can be performed using the following techniques and methods.

[0332] Transgenic identity

[0333] Sanger sequencing of AAV was performed to confirm the nucleotide sequence of the encapsulated payload. The sample was first treated with protease to release the digested AAV capsid protein, and then overlapping PCR was performed to completely cover the promoter, enhancer, transgene and poly A region of the expression cassette. After PCR, individual bands were purified from agarose gels and sequenced using a Sanger-based method. Bases were assigned using a prevalence-based score, and any changes detected were individually reviewed using manual inspection.

[0334] Capsid purity

[0335] Each sample was loaded into an SDS-PAGE gel along with a reference control based on the total viral genome calculated by qPCR. Proteins that migrated into the gel were stained with Oriole fluorescent dye. All protein bands in each lane were analyzed for densitometry and individual impurity bands were quantified as a percentage of the sample. Overall purity was reported as total VP1, VP2, VP3 quantification as a percentage of all quantified bands.

[0336] Vector genome titer (qPCR)

[0337] The qPCR assay based on Taqman targeting the CMV portion of the promoter region was used to quantify the genome copy titer. Before qPCR analysis, the sample was first treated with proteinase K to digest the viral capsid protein shell. In each assay, a positive control AAVrh.10 was included to ensure consistent assay performance, and a reference plasmid containing CMV (CMV PNY1160) was used to generate a standard curve to quantify the AAV samples tested. The reported DS batch titer is the average of triplicates at two dilution levels. The titer of the stability DS sample reported is the average of two independent analyses of triplicate samples at two dilution levels.

[0338] Vector genome titer (ddPCR)

[0339] A ddPCR assay was developed to target a region spanning the 5' end of the transgene coding region in LX1001 to ensure specificity. Each execution of this method included an AAVrh.10 hAPOE-specific reference control to ensure assay performance.

[0340] Infectious titer

[0341] An infectious titer assay will be used to determine the productive uptake and replication of the AAVrh.10hAPOE vector in HeLaRC 32 cells (HeLa expressing rep2). This assay measures the ability of vector particles to infect a reporter cell line and replicate in this cell line to estimate the infectious ability of the vector. Serial vector dilutions (10× dilutions) were co-infected in 12 replicates and then infected with type 5 adenovirus. Seventy-two hours later, the cells were lysed and qPCR was performed to detect the input AAV vector amplification. Endpoint dilution TCID50 calculations (Spearman-Karber) were performed to determine the replication titer expressed as TCID50 / mL. Since the "infectivity" values ​​depend on the particles in contact with the cells, they are affected by the assay geometry and are not an absolute measure of the number of "infectious" particles present. However, the ratio of vector genomes to "infectious units" (described as gc: infectious titer ratio) can be used as a measure of product consistency between batches.

[0342] Empty Capsid

[0343] In order to quantify the amount of AAV capsid species present in the sample, analytical ultracentrifugation (SV-AUC) using sedimentation velocity was used. The AAV sample was diluted to an A230nm of 0.8 ± 0.1 and run at 12,000RPM on a Beckman XLI AUC instrument. The AUC cells were scanned at A230nm every 90 seconds for a total of 200 scans, and then the scans were fitted to sedfit using the c(s) distribution model at a sedimentation range of 0-200S and a resolution of 200. At sedimentation coefficient values ​​consistent with empty, partial, complete or higher-order particle species, the integral of the resulting peak and the calculation of the area under the c(s) fitting curve allow the relative abundance of the particle population to be measured. In order to evaluate whether each peak is reproducible or a possible modeling artifact due to model fitting, a reproducible threshold standard was implemented. The reproducible threshold is defined as the percentage of the absorbance signal for a given species, which is <0.5% and / or <0.002OD ​​of the total absorbance. The result of this analysis is a set of peaks that can be interpreted based on literature precedent and / or additional sample analysis (e.g., by analyzing preparations containing only empty particles).Electron microscopy can also be used to evaluate empty and intact AAV capsids.

[0344] Residual host cell DNA

[0345] qPCR assay was used to detect residual human DNA from the HEK293 process. DNA was extracted from the test samples and three separate amplicons were targeted in the 18S ribosomal (r) DNA gene using quantitative PCR (qPCR). A standard curve was generated using HEK293 derived genomic DNA to convert the copy number results of each amplicon to ng / mL.

[0346] Residual host cell proteins

[0347] ELISA is performed to measure the level of contaminated host HEK293 cell proteins. Cygnus Technologies HEK 293 host cell protein ELISA kit is used for this analysis. Sample and pre-diluted HEK 293HCP standards are added to the pre-coated microtiter wells of the anti-HEK 293HCP capture antibody and the polyclonal anti-HEK 293HCP detection antibody conjugated with affinity purification. After incubation, the wells are washed to remove unbound reactants, and TMB (a peroxidase substrate) is added. After development, sulfuric acid solution is used to stop the reaction. The absorbance of the obtained colored product is measured using a microplate reader, and the amount of HEK 293HCP in each sample is calculated according to the standard curve.

[0348] Residual SV40 large T antigen DNA

[0349] Quantitative PCR (qPCR) was used to measure DNA sequences containing SV40. Quantification was performed relative to a reference standard plasmid containing a single copy of the SV40 LTA sequence. Test samples were run with and without spiking to report the assay LOD and assess assay performance.

[0350] Residual plasmid DNA

[0351] Plasmid DNA that is not encoded between the ITR regions was determined in AAV samples by quantitative PCR (qPCR) targeting the kanamycin resistance gene portion of the plasmid sequence. Prior to qPCR analysis, samples were first treated with proteinase K to digest the viral capsid protein coat. Samples were tested with DNase (treated before proteinase K) and without DNase (performed in quadruplicate) to remove any residual unencapsulated DNA and reported relative to known standards.

[0352] Residual E1a DNA

[0353] DNA sequences containing adenoviral E1a genetic elements were determined in AAV samples by quantitative PCR (qPCR) using primers and probes specific for adenoviral E1a sequences. A standard curve was prepared using genomic DNA extracted from HEK293T cells (which express E1a). Prior to qPCR analysis, samples were first treated with proteinase K to digest the viral capsid protein coat. Test samples were run with and without spiking to report the assay LOD and evaluate assay performance.

[0354] Residual Benzonase

[0355] Benzonase is used to degrade nucleic acids during production to facilitate vector purification and therefore represents a process impurity. A commercial ELISA was used to measure the concentration of residual Benzonase (EMD Millipore No. 1.01681.0002). Samples were analyzed in triplicate wells.

[0356] Replication-competent AAV

[0357] The replication capacity determination based on cells is composed of inoculated monolayer HEK293 cells, cell lines transformed by adenovirus (Ad5) and test products. Due to the presence and co-infection of adenovirus, the replication-competent AAV will be replicated (if present) and amplified in cell culture. After a few days, the virus will induce cytopathic effect and harvest cells. Cell lysate is used to re-infect the fresh monolayer of HEK293 cells twice, a total of 3 rounds of amplification. Total cell DNA is then separated from the third generation culture and quantitative PCR (qPCR) analysis is performed. The target for analysis is derived from the AAV2 Rep sequence, which is required for AAV replication in the presence of adenovirus. The limit of detection (LOD) of the assay is 1rcAAV / 1E9 rAAV, and the spike-in control ensures that there is no inhibition.

[0358] Bioburden

[0359] The method is based on modified membrane filtration for quantitative analysis. The sample is divided into two equal parts, filtered onto a 0.2 μm membrane, and incubated on TSA and SAB media at 30-35°C and 20-25°C for five to seven days, respectively. At the end of incubation, the total number of colonies is counted and divided by the sample volume to generate CFU / mL sample.

Claims

1. A pharmaceutical composition comprising an APOE2 rAAV viral vector; wherein the rAAV viral vector comprises an AAVrh10 capsid protein and an APOE2 rAAV vector; wherein the pharmaceutical composition comprises at least about 1.0×10 11 genome copies (gc) / mL to about 1.0×10 14 gc / mL, and wherein the pharmaceutical composition comprises less than about 40% empty rAAV capsids.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises less than about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10% or about 5% empty rAAV capsids.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises at least about 1.5×10 13 gc / mL.

4. A pharmaceutical composition as described in any of the preceding claims, wherein the APOE2 rAAV viral vector is formulated in about 1 mM potassium dihydrogen phosphate, 3 mM sodium dihydrogen phosphate, and about 155 mM sodium chloride (NaCl) at a pH of about 7.

4.

5. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the rAAV vector comprises in the 5' to 3' direction: The first AAV ITR sequence; enhancer sequences; Promoter sequence; chimeric intron; A nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide; polyA sequence; and Second ITR sequence.

6. The pharmaceutical composition of any of the preceding claims, wherein the nucleic acid sequence encoding the APOE2 polypeptide comprises SEQ ID NO:

5.

7. The pharmaceutical composition according to any one of the preceding claims, wherein the first ITR sequence comprises the nucleic acid sequence shown in SEQ ID NO:

1.

8. The pharmaceutical composition of any one of the preceding claims, wherein the second ITR sequence comprises the nucleic acid sequence shown in SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:

12.

9. The pharmaceutical composition of any one of the preceding claims, wherein the enhancer sequence comprises the nucleic acid sequence shown in SEQ ID NO:

2.

10. The pharmaceutical composition of any one of the preceding claims, wherein the promoter sequence comprises the nucleic acid sequence shown in SEQ ID NO:

3.

11. The pharmaceutical composition of any one of the preceding claims, wherein the polyA sequence comprises the nucleic acid sequence shown in SEQ ID NO:

6.

12. A pharmaceutical composition as described in any of the preceding claims, wherein the rAAV vector comprises the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:

11.

13. A pharmaceutical composition as described in any of the preceding claims, wherein the rAAV vector is packaged as a rAAV viral vector comprising AAV capsid protein.

14. A pharmaceutical composition as described in any of the preceding 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.

15. The pharmaceutical composition of any one of the preceding claims, wherein the AAV capsid protein is an AAVrh10 capsid protein.

16. A pharmaceutical composition comprising an APOE2 rAAV viral vector; wherein the rAAV viral vector comprises an AAVrh10 capsid protein and an APOE2 rAAV vector; wherein the APOE2 rAAV vector comprises the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:

11.

17. A method of treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 1 or 16.

18. The method of claim 17, wherein following administration, the subject experiences at least about a 5% increase in APOE2 expression relative to a pre-administration baseline.

19. The method of claim 17, wherein the patient is homozygous for APOE4.

20. The method of claim 17, wherein the pharmaceutical composition is administered by C1-C2 administration or intracisternal (ICM) administration.

21. The method of claim 17, wherein the pharmaceutical composition is present in an amount of about 5.0×10 9 gc / mL CSF to approximately 5.0×10 12 A dose of gc / mL CSF was administered.

22. The method of claim 17, wherein the pharmaceutical composition is administered at a dose of about: i)1.4×10 10 gc / mL CSF, ii)4.4×10 10 gc / mL CSF, iii)5.0×10 10 gc / mL CSF, iv)1.4×10 11 gc / mL CSF, v)1.6×10 11 gc / mL CSF, or vi)5.0×10 11 gc / mL CSF.

23. The method of claim 17, wherein the pharmaceutical composition is administered in a total volume of about 5 mL, about 10 mL, about 15 mL, or about 20 mL.

24. The method of claim 17, wherein the subject experiences 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% increase in APOE2 expression.

25. The method of claim 17, wherein the APOE2 expression occurs in the central nervous system.

26. The method of claim 17, wherein the APOE2 expression is measured in cerebrospinal fluid (CSF).

27. The method of claim 17, 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 relative to a pre-administration baseline.

28. The method of claim 27, wherein the expression level of T-tau and / or P-tau is 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%.

29. The method of claim 17, wherein after administration of the pharmaceutical composition, amyloid β42 / amyloid β40 (Aβ 42 / 40 ) ratio increased.

30. The method of claim 29, wherein Aβ 42 / 40 The ratio is increased 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%.

31. The method of claim 17, wherein an immunosuppressant is administered to the subject prior to treatment with the pharmaceutical composition.

32. The method of claim 31, wherein the immunosuppressant is prednisone.

33. The method of claim 31, wherein the prednisone is administered at a dose of: 40 mg once daily for 1 week before AAV viral vector administration; 40 mg once daily in week 1 to 2 after AAV viral vector administration; 30 mg once daily in the third week after AAV viral vector administration; 20 mg once daily in the 4th week after AAV viral vector administration; 10 mg once daily in the 5th week after AAV viral vector administration; 5 mg once daily in the 6th week after AAV viral vector administration; 2.5 mg once daily in week 7 after AAV viral vector administration; and 2.5 mg every other day in week 8 after AAV viral vector administration.

34. A method for producing a cell lysate containing a rAAV viral vector, the method comprising: (i) transfecting a cell culture comprising HEK293T cells with a first plasmid encoding an APOE2 AAV vector and a second plasmid encoding an AAV Rep protein and an AAV Cap protein in a transfection medium, wherein the ratio of the second plasmid to the first plasmid is 2:1; (ii) culturing the transfected HEK293T cells in the culture medium under conditions where the transfected HEK293T cells produce a recombinant adeno-associated virus (rAAV) viral vector encoding APOE2; (iii) harvesting the transfected HEK293T cells; as well as (iv) lysing the transfected HEK293T cells to produce cell lysate containing the rAAV viral vector.

35. The method of claim 34, wherein the culture medium comprises Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).

36. The method of claim 34, wherein the transfection medium comprises serum-free DMEM and polyethyleneimine (PEI).

37. The method of claim 34, wherein the HEK293T cells are obtained after about two days to about five days of expansion culture.

38. The method of claim 34, wherein transfection of the first plasmid and the second plasmid occurs simultaneously.

39. The method of claim 34, wherein the HEK293T cells are expressed at about 2.0×10 4 About 2.0×10 6 cells / cm 2 The density of is present in the culture container.

40. The method of claim 34, wherein the transfected cells are cultured for about 3 days.

41. The method of claim 34, wherein the HEK293T cells are lysed by at least about 4 consecutive freeze-thaw cycles to produce the cell lysate.

42. The method of claim 34, wherein the cell lysate is further treated with a recombinant nuclease to digest any non-encapsulated DNA.

43. The method of claim 34, wherein after DNA digestion, the cell lysate is clarified by ultracentrifugation.

44. The method of any one of claims 34 to 43, wherein the cell lysate comprises about 1.0×10 9 to about 5.0×10 14 genome copies (gc) / ml.

45. A method for producing an APOE2 rAAV pharmaceutical composition, the method comprising: (i) obtaining a cell lysate containing an rAAV viral vector encoding APOE2; (ii) contacting a density gradient with the cell lysate containing the rAAV viral vector encoding APOE2, and subjecting the density gradient to centrifugation; (iii) contacting an anion exchange column with the cell lysate containing the rAAV viral vector encoding APOE2; (iv) eluting the rAAV viral vector from the column; and (v) concentrating the eluted rAAV viral vector into a formulation buffer by ultrafiltration, thereby producing an APOE2 rAAV pharmaceutical composition.

46. ​​The method of claim 45, wherein the density gradient is an iodixanol density gradient.

47. The method of claim 45, wherein the iodixanol gradient comprises a stepwise density gradient comprising: (i) about 10% to about 20% iodixanol solution; (ii) about 20% to about 30% iodixanol solution; (iii) about 40% to about 50% iodixanol solution; and (iv) about 50% to about 60% iodixanol solution.

48. The method of claim 45, wherein the anion exchange column is a Q Sepharose high efficiency strong quaternary ammonium anion exchange resin column.

49. The method of claim 45, wherein the formulation buffer comprises phosphate buffered saline (PBS).

50. The method of claim 45, wherein after ultracentrifugation, the pharmaceutical composition comprises about 1.0×10 10 to about 5.0×10 13 viral genomes / ml.

51. An APOE2 rAAV pharmaceutical composition produced by the method of claim 45.

52. A cell lysate comprising a rAAV viral vector produced by the method of claim 34.

53. An rAAV vector comprising the nucleic acid sequence shown in SEQ ID NO:8 or SEQ ID NO:

11.

54. A rAAV vector comprising, in the 5' to 3' direction: The first AAV ITR sequence; enhancer sequences; Promoter sequence; chimeric intron; A nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide; polyA sequence; and Second ITR sequence.

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