Methods and pharmaceutical compositions for the treatment and prevention of 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 include AAV vectors and AAV viral vectors that contain a transgene nucleic acid molecule comprising a nucleic acid sequence encoding an APOE2 polypeptide.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority and the benefit of U.S. Provisional Patent Application No. 63 / 367,995, filed on July 8, 2022; U.S. Provisional Patent Application No. 63 / 385,889, filed on December 2, 2022; and U.S. Provisional Patent Application No. 63 / 496,792, filed on April 18, 2023. The entire contents of each of these are incorporated herein by reference in their entirety.

[0002] Submission of Sequence Listing in XML Format The contents of the electronic sequence listing (LEXE_010_001WO_SeqList_ST26.xml, size: 28,886, creation date: July 7, 2023) are incorporated herein by reference in their entirety.

[0003] 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 an apolipoprotein 2 (APOE2) - encoding nucleic acid. A method for producing an AAV viral vector encoding APOE2 is also provided.

Background Art

[0004] There are long-standing but still unmet needs for therapies effective in the treatment or prevention of Alzheimer's disease (AD). An AAV vector encoding the human APOE2 gene is disclosed. The AAV vector is formulated for administration to an individual having Alzheimer's disease or at risk of developing Alzheimer's disease. Methods for producing the APOE2 AAV vector are also provided. The common APOE alleles (APOE4, APOE3, APOE2) are, by far, the most major genetic risk modifiers for AD, with the APOE4 allele increasing risk and decreasing age of onset, and the APOE2 allele decreasing risk and significantly delaying 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, instead of having a 4-fold higher risk of AD than APOE3 / APOE4 heterozygotes. APOE4 homozygotes have a significantly higher risk of developing AD (14.5-fold compared to APOE3 homozygotes) and an earlier age of onset of the disease (about 5 years earlier compared to APOE3 homozygotes for each APOE4 allele). 45% - 50% of AD patients carry at least one APOE4 allele, compared to only 15% of age-matched healthy controls who carry at least one APOE4 allele. In contrast, APOE2 is a protective allele, reducing the risk of AD by about 50% (a 1.8-fold reduction in risk) even in the presence of the APOE4 allele and significantly delaying the age of onset. Thus, nearly equivalent expression of APOE2 counteracts the deleterious effects of the E4 allele in humans.

[0005] Amyloid burden is associated with the neuropathology of AD. Both experimental animals and clinical trials have shown that the timing and amount of cerebral Aβ peptide deposition, as well as amyloid burden, can be predicted by APOE genotype (APOE4 > APOE3 > APOE2). Intracerebroventricular administration of APOE4 and APOE2 using gene transfer vectors increases and decreases cerebral Aβ / amyloid burden, respectively, in a model of AD-related amyloidosis. Amyloid burden is one mechanism by which APOE isoforms exert their effects. Another mechanism is the tau pathway.

[0006] Therefore, patients with homozygous APOE4 (APOE4 homozygotes) have the highest risk of developing AD, and APOE3 / APOE4 heterozygotes also have a high risk of developing AD. By using the methods and compositions of the present disclosure to increase APOE2 expression in the CNS of APOE4 homozygous or APOE3 / APOE4 heterozygous subjects and convert their brains into brains similar to APOE2 / APOE4 brains (i.e., with a low risk of developing AD), the risk of developing AD can be reduced, the symptoms of AD can be treated, and / or the symptoms of AD can be restored. SUMMARY OF THE INVENTION

[0007] The present disclosure provides a pharmaceutical composition comprising an APOE2 rAAV viral vector, the rAAV viral vector comprising an AAVrh10 capsid protein and an APOE2 rAAV vector, the pharmaceutical composition comprising at least about 1.0×10 11 genome copies (gc) / mL to about 1.0×10 14 gc / mL, and the pharmaceutical composition comprising less than about 40% empty rAAV capsids.

[0008] In some embodiments, 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.

[0009] In some embodiments, the pharmaceutical composition comprises at least about 1.5×1013 It contains gc / mL.

[0010] In some embodiments, the APOE2 rAAV viral vector is formulated at a pH of about 7.4 in about 1 mM potassium monobasic phosphate, 3 mM sodium dibasic phosphate, and about 155 mM sodium chloride (NaCl).

[0011] In some embodiments, the rAAV vector contains, 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 apolipoprotein 2 (APOE2) polypeptide, a polyA sequence, and a second ITR sequence.

[0012] In some embodiments, the nucleic acid sequence encoding the APOE2 polypeptide contains SEQ ID NO: 5.

[0013] In some embodiments, the first ITR sequence contains the nucleic acid sequence set forth in SEQ ID NO: 1.

[0014] In some embodiments, the second ITR sequence contains the nucleic acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12.

[0015] In some embodiments, the enhancer sequence contains the nucleic acid sequence set forth in SEQ ID NO: 2.

[0016] In some embodiments, the promoter sequence contains the nucleic acid sequence set forth in SEQ ID NO: 3.

[0017] In some embodiments, the polyA sequence contains the nucleic acid sequence set forth in SEQ ID NO: 6.

[0018] In some embodiments, the rAAV vector contains the nucleic acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 11.

[0019] In some embodiments, the rAAV vector is packaged as an rAAV viral vector containing an AAV capsid protein.

[0020] In some embodiments, 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 embodiments, the AAV capsid protein is an AAVrh10 capsid protein.

[0021] 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, and the APOE2 rAAV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 11.

[0022] The present disclosure provides a method of treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition according to an embodiment of the present disclosure.

[0023] In some embodiments, after administration, the subject experiences at least about a 5% increase in APOE2 expression compared to the baseline prior to administration.

[0024] In some embodiments, the patient is an APOE4 homozygote.

[0025] In some embodiments, the pharmaceutical composition is administered via C1-C2 administration or intracisternal (ICM) administration.

[0026] In some embodiments, the pharmaceutical composition is about 5.0×10 9 gc / mL CSF~about 5.0×10 12It is administered at a dose of gc / mL CSF. In some embodiments, the pharmaceutical composition is 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.

[0027] In some embodiments, the pharmaceutical composition is administered at a total volume of about 5 mL, about 10 mL, about 15 mL, or about 20 mL.

[0028] In some embodiments, the subject experiences an increase in APOE2 expression of at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0029] In some embodiments, APOE2 expression occurs in the central nervous system. In some embodiments, APOE2 expression is measured in cerebrospinal fluid (CSF).

[0030] In some embodiments, after administration of the pharmaceutical composition, the expression level of at least one of T-tau and P-tau in the subject decreases compared to the baseline before administration. In some embodiments, the expression level of T-tau and / or P-tau decreases 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%.

[0031] In some embodiments, after administration of the pharmaceutical composition, the amyloid beta 42 / amyloid beta 40 (Aβ 42 / 40 ) ratio increases. In some embodiments, the Aβ 42 / 40 ratio increases by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0032] In some embodiments, prior to treatment with the pharmaceutical composition, the subject is administered an immunosuppressant. In some embodiments, the immunosuppressant is prednisone. In some embodiments, prednisone is administered once daily at 40 mg one week prior to administration of the AAV viral vector; once daily at 40 mg from week 1 to week 2 after AAV viral vector administration; once daily at 30 mg in week 3 after AAV viral vector administration; once daily at 20 mg in week 4 after AAV viral vector administration; once daily at 10 mg in week 5 after AAV viral vector administration; once daily at 5 mg in week 6 after AAV viral vector administration; once daily at 2.5 mg in week 7 after AAV viral vector administration; and every other day at a dose of 2.5 mg in week 8 after AAV viral vector administration.

[0033] The present disclosure provides a method for producing a cell lysate comprising an rAAV viral vector, comprising: (i) transfecting a cell culture comprising HEK293T cells in a transfection medium with a first plasmid encoding an APOE2 AAV vector and a second plasmid encoding an AAV Rep protein and an AAV Cap protein, 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 that produce a recombinant adeno-associated virus (rAAV) viral vector encoding APOE2, collecting the transfected HEK293T cells; and (iv) lysing the transfected HEK293T cells to produce a cell lysate comprising the rAAV viral vector.

[0034] In some embodiments, the culture medium comprises Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). In some embodiments, the transfection medium comprises serum-free DMEM and polyethyleneimine (PEI).

[0035] In some embodiments, the HEK293T cells are obtained after a growth culture over a period of about 2 to about 5 days.

[0036] In some embodiments, transfection of the first plasmid and the second plasmid occurs simultaneously.

[0037] In some embodiments, the HEK293T cells are present in a culture vessel at a density of about 2.0×10 4 ~about 2.0×10 6 cells / cm 2 . In some embodiments, the transfected cells are cultured for about 3 days.

[0038] In some embodiments, the HEK293T cells are lysed through at least about 4 consecutive freeze-thaw cycles to produce a cell lysate.

[0039] In some embodiments, the cell lysate is further treated with a recombinant nuclease to digest all of the non-capsidated DNA.

[0040] In some embodiments, following DNA digestion, the cell lysate is clarified via ultracentrifugation.

[0041] In some embodiments, the cell lysate contains from about 1.0×10 9 to about 5.0×10 14 genomic copies (gc) per milliliter.

[0042] The present disclosure provides a method for producing an APOE2 rAAV pharmaceutical composition, 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 via ultrafiltration to produce an APOE2 rAAV pharmaceutical composition.

[0043] In some embodiments, the density gradient is an iodixanol density gradient.

[0044] In some embodiments, the iodixanol gradient comprises a stepwise density gradient comprising: (i) a solution of about 10% to about 20% iodixanol; (ii) a solution of about 20% to about 30% iodixanol; (iii) a solution of about 40% to about 50% iodixanol; and (iv) a solution of about 50% to about 60% iodixanol.

[0045] In some embodiments, the anion exchange column is a Q Sepharose High Performance strong anion exchange resin column.

[0046] In some embodiments, the formulation buffer comprises phosphate buffered saline (PBS).

[0047] In some embodiments, the pharmaceutical composition comprises from about 1.0×10 10 to about 5.0×10 13 viral genomes per milliliter after ultracentrifugation.

[0048] The present disclosure provides an APOE2 rAAV pharmaceutical composition produced by the method of any embodiment of the present disclosure.

[0049] The present disclosure provides a cell lysate comprising an rAAV viral vector produced by the method of any embodiment of the present disclosure.

[0050] The present disclosure provides an rAAV vector comprising the nucleic acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 11.

[0051] The present disclosure provides an rAAV vector comprising, 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.

[0052] Any of the above embodiments, or any other embodiment described herein, can be combined with any other embodiment.

[0053] Unless defined otherwise, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification, unless the context clearly dictates otherwise, the singular forms also include the plural forms, and terms such as "a", "an", and "the" are to be understood as being singular or plural, and the term "or" is to be understood as being inclusive. By way of example, "an element" means one or more elements.

[0054] When carrying out the practice or testing of the present disclosure, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted as prior art with respect to the claimed invention. In case of conflict, this specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description of the invention and the claims.

Brief Description of the Drawings

[0055]

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[0056] The present disclosure provides a method of treating or preventing Alzheimer's disease in a subject in need thereof. In some embodiments, the subject having Alzheimer's disease is homozygous for APOE4 expression. In some embodiments, 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 apolipoprotein 2 (APOE2) polypeptide or a fragment thereof. In some embodiments, the vector is administered at a therapeutically effective amount at a dose in the range of 5.0×10 9 genome copies (gc) / mL CSF to about 5.0×10 12 gc / mL CSF. In some embodiments, the vector is administered via C1-C2 administration or intracisternal (ICM) administration. In some embodiments, the rAAV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 11.

[0057] AAV vector In some embodiments, the isolated nucleic acid sequence comprising a nucleic acid sequence encoding apolipoprotein 2 (APOE2) can be a recombinant AAV vector (rAAV vector).

[0058] 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 repeat sequences (ITRs). In some embodiments, the rAAV vector comprises one or more of an enhancer, a promoter, at least one nucleic acid capable of encoding at least one protein, an intron sequence, and a polyA sequence.

[0059] In some embodiments, the invention relates to an rAAV vector comprising an APOE2-encoding nucleic acid for use in treating or preventing Alzheimer's disease in a subject in need thereof.

[0060] In some aspects, the present invention relates to an rAAV vector comprising an apolipoprotein 2 (APOE2) - encoding nucleic acid for use in treating cardiomyopathy associated with Alzheimer's disease in a subject in need thereof.

[0061] In some aspects, the present invention relates to an rAAV vector comprising an apolipoprotein 2 (APOE2) - encoding nucleic acid for use in restoring or stabilizing symptoms of Alzheimer's disease in a subject in need thereof.

[0062] In some aspects, the present invention relates to an rAAV vector comprising an apolipoprotein 2 (APOE2) - encoding nucleic acid for use in ameliorating symptoms of Alzheimer's disease in a subject in need thereof.

[0063] 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.

[0064] In some aspects, the AAV vector encoding APOE2 of the present disclosure 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% (or any percentage therebetween) identical to SEQ ID NO: 8. In some aspects, the AAV vector encoding APOE2 of the present disclosure 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% (or any percentage therebetween) identical to SEQ ID NO: 11.

[0065] In some embodiments, the enhancer sequence can comprise, consist essentially of, or consist of the human cytomegalovirus (CMV) enhancer sequence. The CMV enhancer 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% (or any percentage therebetween) identical to SEQ ID NO: 2.

[0066] In some embodiments, the promoter sequence can comprise, consist essentially of, or consist of the chicken β-actin promoter sequence. The chicken β-actin promoter 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% (or any percentage therebetween) identical to SEQ ID NO: 3.

[0067] In some embodiments, the chimeric intron sequence can consist essentially 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: 4.

[0068] In some embodiments, the polyA sequence can comprise, consist essentially of, or consist of the 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% (or any percentage therebetween) identical to SEQ ID NO: 6.

[0069] The nucleic acid sequence encoding APOE2 Apolipoprotein (APOE) is a protein involved in the metabolism of fats in the body. It is a family of proteins that bind to fats and interact with low-density lipoprotein receptors (LDLR), which are important for the normal processing of triglyceride-rich lipoproteins. In peripheral tissues, APOE is produced by the liver and macrophages and mediates cholesterol metabolism. In the central nervous system, APOE is produced by astrocytes and transports cholesterol to neurons via APO receptors, which are members of the LDLR family.

[0070] APOE is synthesized as a 317-amino acid protein that is processed intracellularly to cleave the N-terminal 18-amino acid signal peptide (SEQ ID NO: 13) to obtain a 299-amino acid long mature protein. There are three major variants of APOE: APOE2, APOE3, and APOE4. The three variants differ from each other at two positions: residue 112 (residue 130 including the signal peptide) and residue 158 (residue 176 including the signal peptide). The amino acid differences among the three APOE variants are shown in Table 1. Table 1 also shows the prevalence in the world population and the relative risk of developing AD.

[0071] APOE2 is characterized by having cysteine at position 112 (residue 130 including the signal peptide) and cysteine at position 158 (residue 176 including the signal peptide). APOE3 is characterized by having cysteine at position 112 (residue 130 including the signal peptide) and arginine at position 158 (residue 176 including the signal peptide). APOE4 is characterized by having arginine at position 112 (residue 130 including the signal peptide) and arginine at position 158 (residue 176 including the signal peptide).

[0072]

Table 1

[0073] In some embodiments, a nucleic acid sequence encoding an apolipoprotein 2 (APOE2) polypeptide 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% (or any percentage therebetween) identical to SEQ ID NO: 5.

[0074] In some embodiments, an 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% (or any percentage therebetween) identical to SEQ ID NO: 9.

[0075] In some embodiments, a mature APOE2 polypeptide without 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% (or any percentage therebetween) identical to SEQ ID NO: 14.

[0076] In some embodiments, an APOE3 polypeptide with 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% (or any percentage therebetween) identical to SEQ ID NO: 17.

[0077] In some embodiments, a mature APOE3 polypeptide without 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% (or any percentage therebetween) identical to SEQ ID NO: 15.

[0078] In some embodiments, an APOE4 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% (or any percentage therebetween) identical to SEQ ID NO: 18.

[0079] In some embodiments, a mature APOE4 polypeptide without 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% (or any percentage therebetween) identical to SEQ ID NO: 16.

[0080] In some embodiments, the invention provides a nucleic acid sequence comprising SEQ ID NO: 8 or SEQ ID NO: 11, or a variant thereof, for treating Alzheimer's disease.

[0081] Inverted terminal repeat In some embodiments, the first ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 1 or its complement. In some embodiments, the first ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 7 or its complement. In some embodiments, the first ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 10 or its complement. In some embodiments, the first ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 12 or its complement.

[0082] In some embodiments, the second ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 7 or its complement. In some embodiments, the second ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 1 or its complement. In some embodiments, the second ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 10 or its complement. In some embodiments, the second ITR 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% (or any percentage therebetween) identical to SEQ ID NO: 12 or its complement.

[0083] AAV vector As used herein, the terms "adeno-associated virus" or "AAV" refer to members of a class of viruses related to this name and belonging to the genus Dependoparvovirus of the family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows in cells provided with certain functions by a co-infecting helper virus. General information and reviews on AAV are described, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Since it is well known that various serotypes are closely related both structurally and functionally, and even at the genetic level, it is fully anticipated that the same principles described in these reviews are applicable to additional AAV serotypes characterized since the publication dates of these reviews (see, for example, Blacklowe, 1988 (pp. 165-174 in Parvoviruses and Human Disease, J.R. Pattison, ed.); and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes and all have three related capsid proteins as expressed in AAV2. Furthermore, extensive cross-hybridization between serotypes along the length of the genome and heteroduplex analysis revealing the presence of similar self-annealing segments corresponding to "inverted terminal repeats" (ITRs) at the termini further suggest the degree of relatedness. Similarities in infectious patterns also suggest that the replication functions in each serotype are under similar regulatory controls. Multiple serotypes of this virus are known to be suitable for gene delivery and all known serotypes can infect cells of various tissue types. At least 11 consecutively numbered AAV serotypes are known in the art.Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, such as, for example, AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. The AAV particles comprise, consist essentially of, or consist of the three major viral proteins VPl, VP2, and VP3. In some embodiments, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, or AAVrh.10.

[0084] 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), as well as AAV hybrids that contain the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (details are described in Kerun et al. JCI Insight, 2019;4(22):e131610), and AAV-NP59 (details are described in Paulk et al. Molecular Therapy, 2018;26(1):289-303).

[0085] AAV is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb in length and containing two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided by GenBank accession number NC_002077, the complete genome of AAV-2 is provided by GenBank accession numbers NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided by GenBank accession number NC_l829, the complete genome of AAV-4 is provided by GenBank accession number NC_001829, the AAV-5 genome is provided by GenBank accession number AF085716, the complete genome of AAV-6 is provided by GenBank accession number NC_001862, at least a portion of the AAV-7 and AAV-8 genomes are provided by GenBank accession numbers AX753246 and AX753249, respectively, the AAV-9 genome is provided by Gao et al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided by Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided by Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is described 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 integration into the host cell chromosome are contained within the AAV ITRs. Three AAV promoters (referred to as p5, p19, and p40 from their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) result in the generation of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene, with alternative splicing of one AAV intron (nucleotides 2107 and 2227). The rep proteins have multiple enzymatic properties and ultimately are involved in the replication of the viral genome.

[0086] The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are involved in the production of the three related capsid proteins. More specifically, after a single mRNA from which each of the VP1, VP2, and VP3 proteins is translated is transcribed, it is spliced in two different ways. That is, either a long intron or a short intron can be excised, resulting in the formation of two mRNA pools of 2.3 kb and 2.6 kb in length. Since the longer intron is often preferred, the 2.3 kb-long mRNA is sometimes called the major splice variant. This form lacks the first AUG codon that initiates the synthesis of the VP1 protein, resulting in an overall reduction in the synthesis level of the VP1 protein. The first AUG codon remaining in the major splice variant is the start codon of the VP3 protein. However, upstream of this codon within the same open reading frame, there is an ACG sequence (encoding threonine) surrounded by an optimal Kozak (translation initiation) context.This contributes to the synthesis of the VP2 protein (actually, like VP1, with an N-terminal residue added to the VP3 protein) at low levels, which is described in Becerra SP et al., (December 1985) “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 hereby incorporated by reference herein. A single consensus polyA site is present at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0087] 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 that is unique to the VP1 protein. The VP2 portion is the amino acid sequence present within the VP1 protein, which is also found 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 that is shared with the VP1 and VP2 proteins.

[0088] The VP3 protein can be further classified into discrete variable surface regions I to IX (VR-I to IX). Each of the variable surface regions (VRs) can confer a unique infectious phenotype (e.g., reduced antigenicity compared to other AAV serotypes, improved transduction, and / or tissue-specific tropism) to a particular serotype, either alone or in combination with the specific amino acid sequences of each of the other VRs, by containing or being capable of containing specific amino acid sequences (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 (this content is incorporated herein by reference)).

[0089] AAV has unique features that make it an attractive vector for delivering foreign DNA into cells, for example, in gene therapy. Infection of cells in culture with AAV is non-cytopathic, and natural infection in humans and other animals is asymptomatic. Furthermore, since AAV infects many mammalian cells, it has the potential to target many different tissues in vivo. Additionally, AAV can transduce both slowly dividing and non-dividing cells and persist essentially throughout the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome can be inserted into a plasmid as cloned DNA, enabling the construction of recombinant genomes. Moreover, since the signals that induce AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, it is possible to generate AAV vectors by replacing some or all of approximately 4.3 kb of the interior of the genome (encoding the replication and structural capsid proteins rep-cap) with foreign DNA. The rep and cap proteins may also be provided in trans. Another important feature of AAV is that it is a very stable and robust virus. This makes cryopreservation of AAV less important because it can easily withstand the conditions used for adenovirus inactivation (several hours at 56 °C to 65 °C). AAV can even be lyophilized. Finally, cells infected with AAV are resistant to superinfection.

[0090] AAV viral vector The AAV vectors of the present disclosure can be packaged as AAV viral vectors.

[0091] The term "rAAV viral vector" refers to a viral particle composed of at least one rAAV capsid protein and a capsidated polynucleotide AAV vector. Therefore, the production of an rAAV viral vector necessarily involves the production of an rAAV vector. The term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into a host cell. Generally, a capsid is composed of oligomeric structural subunits of protein ("capsid proteins"). As used herein, the term "capsidated" means enclosed within a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.

[0092] The rAAV viral vectors useful for the practice of the present invention can be constructed using methodologies well known in the art of molecular biology. Typically, an AAV viral vector having a transgene is assembled from a polynucleotide encoding the transgene, suitable regulatory elements, and elements necessary for the production of viral proteins that mediate cell transduction.

[0093] The term "gene transfer" or "gene delivery" refers to a method or system for reliably inserting foreign DNA into a host cell. By such methods, it is also possible to transiently express the non-integrated transferred DNA, replicate and express the transferred replicon (e.g., episome) extrachromosomally, or integrate the transferred genetic material into the genomic DNA of the host cell.

[0094] Examples of viral vectors include, but are not limited to, adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV) vectors.

[0095] Such recombinant viruses can be generated by techniques known in the art (e.g., transfecting packaging cells, or transient transfection with a helper plasmid or virus). Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Patent No. 5,882,877, No. 6,013,516, No. 4,861,719, No. 5,278,056, and WO 94 / 19478.

[0096] In one embodiment, an adeno-associated virus (AAV) vector is used.

[0097] 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 may infect humans, monkeys, or other species.

[0098] In an exemplary embodiment, the rAAV vector is an AAVrh10 vector.

[0099] The term "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 can have one or more of the AAV wild-type genes, preferably the rep gene and / or the cap gene, deleted in whole or in part, but can retain the functional flanking ITR sequences. The functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion. Thus, as used herein, an AAV vector is defined as including at least the sequences (e.g., functional ITRs) that are cis-required for viral replication and packaging. The ITRs need not be wild-type nucleotide sequences and may be modified by nucleotide insertions, deletions, or substitutions so long as the sequences provide functional rescue, replication, and packaging. An AAV expression vector is constructed using known techniques to provide, as at least components operably linked in the transcriptional direction, a control element including a transcription initiation region, the DNA of interest (i.e., the APOE2 gene), and a transcription termination region.

[0100] The control element is selected to function in mammalian cells. The resulting construct containing the operably linked components is linked (at the 5' and 3' ends) to a functional AAV ITR sequence. "Adeno-associated virus inverted terminal repeat" or "AAV ITR" means the region recognized in the art found at both ends of the AAV genome, which functions cis together as an origin of DNA replication and as a viral packaging signal. The AAV ITR, together with the AAV rep coding region, efficiently excises, rescues, and integrates the nucleotide sequence intervening between the two flanking ITRs into the mammalian cell genome. In some embodiments, the ITR sequences of the present disclosure may contain deletions of one or more nucleotides at one or more positions of the ITR sequence. In some embodiments, the nucleotides deleted within the ITR sequence may be repaired in vivo or during vector replication. The nucleotide sequence of the AAV ITR region is known. For example, for the AAV-2 sequence, see Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M. Knipe, eds.). As used herein, "AAV ITR" does not necessarily include the wild-type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution. In addition, the AAV ITR 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, etc. Furthermore, the 5' and 3' ITRs flanking the selected nucleotide sequence within the AAV vector do not necessarily have to be identical and do not have to be derived from the same AAV serotype or isolate, as long as they function as intended, i.e., are able to excise and rescue the sequence of interest from the host cell genome or vector and integrate the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell.In addition, the AAV ITR may be derived from any of several AAV serotypes including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV5, AAV-6, etc. Further, the 5' and 3' ITRs adjacent to the selected nucleotide sequence within the AAV expression vector need not necessarily be identical, nor need they be derived from the same AAV serotype or isolate, so long as they function as intended, i.e., enable excision and rescue of the sequence of interest from the host cell genome or vector, and function to enable integration of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0101] Particularly preferred are vectors derived from AAV serotypes having tropism and high transduction efficiency for mammalian cardiomyocytes, particularly cardiomyocytes and cardiomyocyte precursors. An overview and comparison of the transduction efficiencies of different serotypes are provided by 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.

[0102] The selected nucleotide sequence is operably linked to control elements that induce its transcription or expression in the subject in vivo. Such control elements can include control sequences normally associated with the selected gene.

[0103] Alternatively, heterologous control sequences may be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG, MCK (muscle creatine kinase), SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoters such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, etc. The promoter may be of human origin or derived from other species including mice. In addition, sequences derived from non-viral genes such as the mouse metallothionein gene also find use herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA).

[0104] Examples of heterologous promoters include the CMV promoter.

[0105] Examples of inducible promoters include DNA response elements to ecdysone, tetracycline, hypoxia, and aufin.

[0106] An AAV expression vector having a DNA molecule of interest joined by AAV ITRs can be constructed by directly inserting a selected sequence into an AAV genome having the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome may also be deleted so long as portions of the ITRs sufficient to allow replication and packaging functions remain. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publications WO92 / 01070 (published Jan. 23, 1992) and WO93 / 03769 (published Mar. 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 ITRs can be excised from the viral genome or from an AAV vector containing the same using standard ligation techniques and fused to the 5' and 3' of a selected nucleic acid construct present in another vector. AAV vectors containing ITRs are described, for example, in the specification of U.S. Pat. No. 5,139,941. Specifically, this document describes several AAV vectors available from the American Type Culture Collection (“ATCC”) under accession numbers 53222, 53223, 53224, 53225, and 53226. Further, chimeric genes can be synthetically generated to include AAV ITR sequences disposed 5' and 3' of one or more selected nucleic acid sequences. Codons preferred for expressing chimeric gene sequences in mammalian CNS cells can be used. The complete chimeric sequence can be assembled from overlapping oligonucleotides prepared by standard methods. See, for example, Edge, 1981; Nambair et al., 1984; Jay et al., 1984.To generate AAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques such as transfection. Many transfection techniques are generally known in the art. See, for example, Graham et al., 1973, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981. Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome-mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Felgner et al., 1987), and nucleic acid delivery using a high-velocity microprojectile (Klein et al., 1987).

[0107] The AAV viral vector of the present disclosure comprises i) the AAV vector described herein, and ii) an AAV capsid protein.

[0108] In some embodiments, the AAV capsid protein can be any AAV capsid protein. In some embodiments, 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 embodiments, the AAV capsid protein is an AAVrh10 capsid protein.

[0109] Inverted terminal repeat "Inverted terminal repeat" or "ITR" means a region recognized in the art found at both ends of the AAV genome that functions cis - together as an origin of DNA replication and as a viral packaging signal. The AAV ITR, together with the AAV rep coding region, efficiently provides for excision and rescue from the mammalian cell genome and integration of a nucleotide sequence inserted between two adjacent ITRs into the mammalian cell genome. The nucleotide sequence of the AAV ITR region is known. For example, for the AAV - 2 sequence, see Kotin, 1994; Berns, KI “Parvoviridae and their Replication” in Fundamental Virology, 2nd Edition, (B.N.Fields and D.M.Knipe, eds.). As used herein, "AAV ITR" does not necessarily include the wild - type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution. In addition, the AAV ITR may be derived from any of several AAV serotypes including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6. Further, the 5' and 3' ITRs flanking a selected nucleotide sequence within an AAV vector do not necessarily have to be identical and do not have to be derived from the same AAV serotype or isolate, as long as they function as intended, i.e., enable excision and rescue of the sequence of interest from the host cell genome or vector and enable integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell. In addition, the AAV ITR may be derived from any of several AAV serotypes including, but not limited to, AAV1, AA2, AAV3, AAV4, AAV5, AAV6.Furthermore, the 5' and 3' ITRs adjacent to the selected nucleotide sequence within the AAV expression vector do not necessarily have to be identical, nor do they have to be derived from the same AAV serotype or isolate, as long as they function as intended, i.e., enable excision and rescue of the sequence of interest from the host cell genome or vector, and enable integration of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present intracellularly.

[0110] In some embodiments, the AAV ITR sequence can include any AAV ITR sequence known in the art. In some embodiments, 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.

[0111] Thus, in some embodiments, the AAV ITR sequence can include, 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.

[0112] In some embodiments, the rAAV vector of the disclosure can include, consist essentially of, or consist of an AAV2 ITR sequence. In some embodiments, the rAAV vector of the disclosure can include, consist essentially of, or consist of an AAV2 ITR sequence or a modified AAV2 ITR sequence. Promoter and enhancer sequences

[0113] As used herein, the terms "promoter" and "promoter sequence" mean a control sequence that is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or transgene. A promoter can be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter can include gene elements to which regulatory proteins and molecules, such as RNA polymerase and transcription factors, can bind.

[0114] A selected nucleotide sequence, such as a frataxin coding nucleotide sequence, is operably linked to a control element that directs its transcription or expression in a subject in vivo. Such control elements can include control sequences normally associated with the selected gene.

[0115] Alternatively, heterologous control sequences may be used. Useful heterologous control sequences generally include sequences derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG, MCK (muscle creatine kinase), SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoters such as the CMV immediate early promoter region (CMVIE), chicken β-actin (CBA) promoter, Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, etc. The promoter can be of human origin or derived from other species including mice. Furthermore, sequences derived from non-viral genes such as the mouse metallothionein gene are also used herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA).

[0116] An example of a heterologous promoter is the CMV promoter.

[0117] Examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and andaufin.

[0118] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence capable of providing both promoter and enhancer functions. For example, the long terminal repeat of a retrovirus contains both promoter and enhancer functions. Enhancer / promoters can be "endogenous", "exogenous", or "heterologous". An "endogenous" enhancer / promoter is one that is naturally associated with a given gene within the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is placed in parallel with a gene by genetic manipulation (i.e., molecular biology techniques) or synthetic techniques, such that the transcription of the gene is directed by the attached enhancer / promoter. Non-limiting examples of attached enhancer / promoters for use in the methods, compositions, and constructs provided herein include the CMV enhancer attached to the CBA promoter. In the art, it is understood that an enhancer can act at a distance and regardless of the orientation relative to the position of an endogenous or heterologous promoter. Thus, an enhancer that acts at a distance from a promoter is further understood to be "operably linked" to that promoter, regardless of its position within the vector or its orientation relative to the position of the promoter.

[0119] 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 spatially connected promoter. The promoter can be located either 5' (upstream) or 3' (downstream) of the gene under its control. The promoter can be located 5' (upstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls within the gene from which the promoter is derived. Variations in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0120] In some embodiments, the enhancer sequence can comprise, consist essentially of, or consist of a human cytomegalovirus (CMV) enhancer sequence. The CMV enhancer 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% (or any percentage therebetween) identical to SEQ ID NO: 2.

[0121] In some embodiments, the promoter sequence can comprise, consist essentially of, or consist of a chicken β-actin promoter sequence. The chicken β-actin promoter 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% (or any percentage therebetween) identical to SEQ ID NO: 3.

[0122] Methods of treatment The present disclosure provides a method for treating or preventing Alzheimer's disease (AD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an rAAV vector comprising a nucleic acid sequence encoding an APOE2 polypeptide or a fragment thereof.

[0123] 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.

[0124] 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 after administration, the subject experiences an increase in APOE2 expression.

[0125] Subjects of the present disclosure can express any combination of APOE variants, including being APOE2 homozygotes, APOE3 homozygotes, APOE4 homozygotes, APOE2 / APOE4 heterozygotes, APOE2 / APOE3 heterozygotes, or APOE3 / APOE4 heterozygotes. In some embodiments, the subject of the present disclosure is an APOE4 homozygote.

[0126] Subjects of the present disclosure can have a range of cognitive impairments associated with AD, including no impairment, mild cognitive impairment (CI), or mild, moderate, or severe dementia. In some embodiments, the subject of the present disclosure is at least 50 years old. In some embodiments, the subject of the present disclosure can be of any age.

[0127] In some embodiments, the subject of the present disclosure has CSF biomarkers consistent with Alzheimer's disease. In some embodiments, the subject of the present disclosure is determined to be positive via amyloid-targeted positron emission tomography (PET).

[0128] In some embodiments, the subject experiences an increase in APOE2 expression as compared to the baseline prior to administration. Quantification of APOE2 expression can be performed according to any method known in the art. In some embodiments, the subject experiences an increase in APOE2 expression of at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% as compared to the baseline prior to administration.

[0129] Expression of APOE2 can occur anywhere in the central nervous system, including any region of the brain and cerebrospinal fluid (CSF).

[0130] Expression of APOE2, APOE3, and APOE4 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 coupled with mass spectrometry (i.e., LC-MS).

[0131] In some embodiments, APOE2 expression is reflected as a percentage of APOE4 expression and / or total APOE expression. In some embodiments, APOE2 expression is calculated as the total APOE2 expression divided by the APOE4 expression. This ratio can be multiplied by 100 to obtain a percentage. In some embodiments, APOE2 expression is calculated as the total APOE2 expression divided by the total APOE expression (total expression of APOE2, APOE3, and / or APOE4). This ratio can be multiplied by 100 to obtain a percentage. Evaluation of APOE expression can be performed according to any suitable protein quantification method.

[0132] Hippocampal volume decreases over time in individuals with prodromal and subsequent AD. Since hippocampal volume is typically correlated with cognitive and functional changes over time, it serves as a useful marker of disease progression. In some embodiments, after treatment with the APOE2 pharmaceutical compositions of the present disclosure, the subject experiences an increase in hippocampal volume. In some embodiments, after treatment with the APOE2 pharmaceutical compositions of the present disclosure, the subject's hippocampal volume remains the same. In some embodiments, after treatment with the APOE2 pharmaceutical compositions of the present disclosure, the subject experiences a decrease in the rate of decrease of hippocampal volume. In some embodiments, the decrease in hippocampal volume is evaluated by brain MRI.

[0133] Cognitive assessment Improvement in cognition after administration of the APOE2 pharmaceutical composition can be evaluated according to any method known in the art. Such evaluations may include, but are not limited to, Clinical Dementia Rating (CDR), Alzheimer's Disease Assessment Scale - Cognitive Assessment (ADAS-Cog 13), or Mini-Mental State Examination.

[0134] The Clinical Dementia Rating (CDR) scale is a clinician-administered dementia staging system that tracks the progression of cognitive impairment in six categories (memory, orientation, judgment and problem-solving, community affairs, home and hobbies, and personal care). Each category is scored on a 5-point scale: none = 0, questionable = 0.5, mild = 1, moderate = 2, and severe = 3. The global CDR score is established by clinical scoring rules and has values of 0 (no dementia), 0.5 (dementia suspected), 1 (mild dementia), 2 (moderate dementia), and 3 (severe dementia). CDR-SB is obtained by adding the evaluations in each of the six categories and ranges from 0 to 18, with higher scores indicating greater impairment.

[0135] CDR-SB is performed at screening / baseline visit before administration of the APOE2 pharmaceutical composition of the present disclosure. These evaluations are used as a measure of clinical efficacy.

[0136] The Alzheimer's Disease Assessment Scale - Cognitive Subscale (13 items) (ADAS-Cog13) is a structured scale that evaluates memory, orientation, attention, reasoning, language, and constructional praxis. The higher the score, the greater the impairment.

[0137] The MMSE is a simple 30-item questionnaire used to assess cognitive impairment, with lower scores indicating greater impairment. The MMSE evaluates 11 cognitive categories including orientation to time, memory, attention, concentration, naming, repetition, comprehension, as well as writing ability and the ability to copy two intersecting polygons. Biomarker testing

[0138] APOE2 expression after administration of the APOE2 pharmaceutical composition of the present disclosure can be evaluated in any region of the CNS. In some embodiments, APOE2 expression is evaluated in the brain. In some embodiments, APOE2 expression is evaluated in the CSF. Amyloid beta (Aβ)42, Aβ42 / 40, T-tau, and P-tau are considered core biomarkers for AD. T-tau and P-tau are thought to increase after amyloid accumulates in the brain (amyloid cascade hypothesis). Both amyloid and tau accumulation are associated with inflammation in the brain at various time points during disease progression, with amyloid driving inflammation during initial accumulation (MCI cases) and tau driving inflammation after the amyloid load in the brain approaches AD levels (preclinical AD). In the CSF of AD participants, Aβ42 is decreased and T-tau and P-tau are increased. Amyloid beta 40, although not a core CSF biomarker, is also evaluated.

[0139] In some embodiments, after administration of the pharmaceutical composition, the expression level of at least one of amyloid beta 42 (Aβ 42 )), amyloid beta 40 (Aβ 40 )), T-tau, and P-tau in the subject decreases compared to the baseline before administration. In some embodiments, after administration of the pharmaceutical composition, amyloid beta 42 (Aβ 42)、Amyloid beta 40 (Aβ 40 )、T-tau, and the expression level of at least one of P-tau increases as compared to the baseline before administration. In some embodiments, after administration of the pharmaceutical composition, amyloid beta 42 (Aβ 42 )、Amyloid beta 40 (Aβ 40 )、T-tau, and the expression level of at least one of P-tau remains constant as compared to the baseline before administration.

[0140] In some embodiments, the expression level of Aβ 42 、Aβ 40 、T-tau, and / or P-tau decreases 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%.

[0141] In some embodiments, after administration of the pharmaceutical composition, the expression level of Aβ 42 changes. In some embodiments, after administration of the pharmaceutical composition, the expression level of Aβ 42 increases. In some embodiments, after administration of the pharmaceutical composition, the expression level of Aβ 42 decreases. In some embodiments, the change in the expression level of Aβ 42 after administration of the pharmaceutical composition is subject-specific.

[0142] In some embodiments, after administration of the pharmaceutical composition, the amyloid beta 42 / amyloid beta 40 (Aβ 42 / 40 ) ratio changes. In some embodiments, after administration of the pharmaceutical composition, the Aβ 42 / 40 ratio increases. In some embodiments, after administration of the pharmaceutical composition, the Aβ 42 / 40 ratio decreases. In some embodiments, the change in the Aβ 42 / 40 ratio after administration of the pharmaceutical composition is subject-specific.

[0143] In some embodiments, Aβ 42 / 40 ratio increases by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0144] In certain embodiments, the gene encoded by the nucleic acid sequence in the AAV vector is the APOE2 gene.

[0145] As used herein in the broadest sense, the terms "preventing" or "prevention" refer to preventing a disease or condition from occurring in a subject who has not yet been diagnosed as having it or who has no clinical symptoms.

[0146] As used herein, the terms "treating" or "treatment" mean restoring, alleviating, or inhibiting the progression of such a disorder or condition, or one or more symptoms of such a disorder or condition. A "therapeutically effective amount" is intended to be 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, alleviates, stabilizes, slows the progression of, or otherwise causes an improvement in a pathological symptom, disease progression, or physiological condition associated with or resistant to succumbing to a disorder.

[0147] As used herein, the term "subject" means a mammal (e.g., rodents, cats, dogs, and primates). In some embodiments, the subject according to the invention is a human. In the context of the present invention, "a subject in need thereof" refers to a subject, preferably a human, more specifically a subject having Alzheimer's disease.

[0148] As used herein, the term "gene" refers to a polynucleotide that contains at least one open reading frame capable of encoding a specific polypeptide or protein after transcription and translation.

[0149] As used herein, the terms "coding sequence", "sequence encoding a specific protein", or "coding nucleic acid" refer to a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and furthermore synthetic DNA sequences.

[0150] In certain embodiments, the present invention relates to a method for preventing or treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV vector containing APOE2 coding nucleic acid, an AAV viral vector, or a pharmaceutical composition to the subject.

[0151] In certain embodiments, the present invention relates to a method for treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV vector containing APOE2 coding nucleic acid, an AAV viral vector, or a pharmaceutical composition to the subject.

[0152] In certain embodiments, the present invention relates to a method for restoring or stabilizing the symptoms of Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV vector containing APOE2 coding nucleic acid, an AAV viral vector, or a pharmaceutical composition to the subject.

[0153] As used herein, the terms "asymptomatic" or "pre-symptomatic" refer to a subject who has a disease (Alzheimer's disease) defined by genetic diagnosis but has no detectable clinical symptoms.

[0154] As used herein, the term "symptomatic" refers to a subject who has a disease (Alzheimer's disease) defined by genetic diagnosis and has a cognitive impairment including MCI, or mild, moderate, or severe dementia.

[0155] Delivery of the vector Provided herein is a method for treating Alzheimer's disease in a subject in need thereof, comprising: (a) providing an AAV vector comprising a nucleic acid sequence encoding an APOE2 polypeptide or a fragment thereof as defined above; and (b) delivering the AAV vector to a subject in need thereof such that APOE2 is expressed at a therapeutically effective level by the transduced cells.

[0156] Preferred dosages and regimens can be determined by a physician and may vary depending on the age, sex, weight, and disease stage of the subject.

[0157] In some embodiments, the AAV vector is administered to the central nervous system (CNS) of the subject. In some embodiments, the administration is a C1-C2 administration. C1-C2 administration refers to administering the AAV vector between the first two vertebrae of the cervical spine. In some embodiments, the C1-C2 administration is CT-guided. In some embodiments, the administration is intracisternal (ICM). In some embodiments, ICM administration is performed when C1-C2 administration cannot be performed. In some embodiments, factors limiting C1-C2 administration include the vagus artery along the needle trajectory and / or a restricted dorsal subarachnoid space (i.e., less than 2 mm).

[0158] In some embodiments, administration of the AAV vector is carried out over 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 embodiments, IV infusion is carried out over 60 minutes. In some embodiments, C1-C2 administration is carried out at a rate of about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, or about 10 mL / min.

[0159] In some embodiments, the volume of the AAV vector administered is about 5 mL, about 10 mL, about 15 mL, about 20 mL, or about 25 mL.

[0160] In some embodiments, the subject is administered the AAV vector of the present disclosure in a therapeutically effective dose. In some embodiments, the dose is about 5.0×10 9 genome copies (gc) (gc / mL) per 1 milliliter (mL) of cerebrospinal fluid (CSF) ~ about 5.0×10 12 gc / mL CSF. In some embodiments, the dose is about 1.4×10 10 gc / mL CSF. In some embodiments, the dose is about 4.4×10 10 gc / mL CSF. In some embodiments, the dose is about 5.0×10 10 gc / mL CSF. In some embodiments, the dose is about 1.4×10 11 gc / mL CSF. In some embodiments, the dose is about 1.6×10 11 gc / mL CSF. In some embodiments, the dose is about 5.0×10 11 gc / mL CSF.

[0161] In some embodiments, 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 10gc / ml CSF, approximately 1.4×10 10 gc / ml CSF, approximately 1.5×10 10 gc / ml CSF, approximately 1.6×10 10 gc / ml CSF, approximately 1.7×10 10 gc / ml CSF, approximately 1.8×10 10 gc / ml CSF, approximately 1.9×10 10 gc / ml CSF, approximately 2.0×10 10 gc / ml CSF, approximately 2.1×10 10 gc / ml CSF, approximately 2.2×10 10 gc / ml CSF, approximately 2.3×10 10 gc / ml CSF, approximately 2.4×10 10 gc / ml CSF, approximately 2.5×10 10 gc / ml CSF, approximately 2.6×10 10 gc / ml CSF, approximately 2.7×10 10 gc / ml CSF, approximately 2.8×10 10 gc / ml CSF, approximately 2.9×10 10 gc / ml CSF, approximately 3.0×10 10 gc / ml CSF, approximately 3.1×10 10 gc / ml CSF, approximately 3.2×10 10 gc / ml CSF, approximately 3.3×10 10 gc / ml CSF, approximately 3.4×10 10 gc / ml CSF, approximately 3.5×10 10 gc / ml CSF, approximately 3.6×10 10 gc / ml CSF, approximately 3.7×10 10 gc / ml CSF, approximately 3.8×10 10 gc / ml CSF, approximately 3.9×10 10 gc / ml CSF, approximately 4.0×10 10 gc / ml CSF, approximately 4.1×10 10 gc / ml CSF, approximately 4.2×10 10 gc / ml CSF, approximately 4.3×10 10 gc / ml CSF, approximately 4.4×10 10 gc / ml CSF, approximately 4.5×10 10 gc / ml CSF, approximately 4.6×10 10gc / ml CSF, approximately 4.7×10 10 gc / ml CSF, approximately 4.8×10 10 gc / ml CSF, approximately 4.9×10 10 gc / ml CSF, approximately 5.0×10 10 gc / ml CSF, approximately 5.1×10 10 gc / ml CSF, approximately 5.2×10 10 gc / ml CSF, approximately 5.3×10 10 gc / ml CSF, approximately 5.4×10 10 gc / ml CSF, approximately 5.5×10 10 gc / ml CSF, approximately 5.6×10 10 gc / ml CSF, approximately 5.7×10 10 gc / ml CSF, approximately 5.8×10 10 gc / ml CSF, approximately 5.9×10 10 gc / ml CSF, approximately 6.0×10 10 gc / ml CSF, approximately 6.1×10 10 gc / ml CSF, approximately 6.2×10 10 gc / ml CSF, approximately 6.3×10 10 gc / ml CSF, approximately 6.4×10 10 gc / ml CSF, approximately 6.5×10 10 gc / ml CSF, approximately 6.6×10 10 gc / ml CSF, approximately 6.7×10 10 gc / ml CSF, approximately 6.8×10 10 gc / ml CSF, approximately 6.9×10 10 gc / ml CSF, approximately 7.0×10 10 gc / ml CSF, approximately 7.1×10 10 gc / ml CSF, approximately 7.2×10 10 gc / ml CSF, approximately 7.3×10 10 gc / ml CSF, approximately 7.4×10 10 gc / ml CSF, approximately 7.5×10 10 gc / ml CSF, approximately 7.6×10 10 gc / ml CSF, approximately 7.7×10 10 gc / ml CSF, approximately 7.8×10 10 gc / ml CSF, approximately 7.9×10 10gc / ml CSF, approximately 8.0×10 10 gc / ml CSF, approximately 8.1×10 10 gc / ml CSF, approximately 8.2×10 10 gc / ml CSF, approximately 8.3×10 10 gc / ml CSF, approximately 8.4×10 10 gc / ml CSF, approximately 8.5×10 10 gc / ml CSF, approximately 8.6×10 10 gc / ml CSF, approximately 8.7×10 10 gc / ml CSF, approximately 8.8×10 10 gc / ml CSF, approximately 8.9×10 10 gc / ml CSF, approximately 9.0×10 10 gc / ml CSF, approximately 9.1×10 10 gc / ml CSF, approximately 9.2×10 10 gc / ml CSF, approximately 9.3×10 10 gc / ml CSF, approximately 9.4×10 10 gc / ml CSF, approximately 9.5×10 10 gc / ml CSF, approximately 9.6×10 10 gc / ml CSF, approximately 9.7×10 10 gc / ml CSF, approximately 9.8×10 10 gc / ml CSF, or approximately 9.9×10 10 gc / ml CSF, or any dosage in between.

[0162] In some embodiments, approximately 1.0×10 11 gc / ml CSF, approximately 1.1×10 11 gc / ml CSF, approximately 1.2×10 11 gc / ml CSF, approximately 1.3×10 11 gc / ml CSF, approximately 1.4×10 11 gc / ml CSF, approximately 1.5×10 11 gc / ml CSF, approximately 1.6×10 11 gc / ml CSF, approximately 1.7×10 11 gc / ml CSF, approximately 1.8×10 11 gc / ml CSF, approximately 1.9×10 11 gc / ml CSF, approximately 2.0×10 11gc / ml CSF, approximately 2.1×10 11 gc / ml CSF, approximately 2.2×10 11 gc / ml CSF, approximately 2.3×10 11 gc / ml CSF, approximately 2.4×10 11 gc / ml CSF, approximately 2.5×10 11 gc / ml CSF, approximately 2.6×10 11 gc / ml CSF, approximately 2.7×10 11 gc / ml CSF, approximately 2.8×10 11 gc / ml CSF, approximately 2.9×10 11 gc / ml CSF, approximately 3.0×10 11 gc / ml CSF, approximately 3.1×10 11 gc / ml CSF, approximately 3.2×10 11 gc / ml CSF, approximately 3.3×10 11 gc / ml CSF, approximately 3.4×10 11 gc / ml CSF, approximately 3.5×10 11 gc / ml CSF, approximately 3.6×10 11 gc / ml CSF, approximately 3.7×10 11 gc / ml CSF, approximately 3.8×10 11 gc / ml CSF, approximately 3.9×10 11 gc / ml CSF, approximately 4.0×10 11 gc / ml CSF, approximately 4.1×10 11 gc / ml CSF, approximately 4.2×10 11 gc / ml CSF, approximately 4.3×10 11 gc / ml CSF, approximately 4.4×10 11 gc / ml CSF, approximately 4.5×10 11 gc / ml CSF, approximately 4.6×10 11 gc / ml CSF, approximately 4.7×10 11 gc / ml CSF, approximately 4.8×10 11 gc / ml CSF, approximately 4.9×10 11 gc / ml CSF, approximately 5.0×10 11 gc / ml CSF, approximately 5.1×10 11 gc / ml CSF, approximately 5.2×10 11 gc / ml CSF, approximately 5.3×10 11gc / ml CSF, approximately 5.4×10 11 gc / ml CSF, approximately 5.5×10 11 gc / ml CSF, approximately 5.6×10 11 gc / ml CSF, approximately 5.7×10 11 gc / ml CSF, approximately 5.8×10 11 gc / ml CSF, approximately 5.9×10 11 gc / ml CSF, approximately 6.0×10 11 gc / ml CSF, approximately 6.1×10 11 gc / ml CSF, approximately 6.2×10 11 gc / ml CSF, approximately 6.3×10 11 gc / ml CSF, approximately 6.4×10 11 gc / ml CSF, approximately 6.5×10 11 gc / ml CSF, approximately 6.6×10 11 gc / ml CSF, approximately 6.7×10 11 gc / ml CSF, approximately 6.8×10 11 gc / ml CSF, approximately 6.9×10 11 gc / ml CSF, approximately 7.0×10 11 gc / ml CSF, approximately 7.1×10 11 gc / ml CSF, approximately 7.2×10 11 gc / ml CSF, approximately 7.3×10 11 gc / ml CSF, approximately 7.4×10 11 gc / ml CSF, approximately 7.5×10 11 gc / ml CSF, approximately 7.6×10 11 gc / ml CSF, approximately 7.7×10 11 gc / ml CSF, approximately 7.8×10 11 gc / ml CSF, approximately 7.9×10 11 gc / ml CSF, approximately 8.0×10 11 gc / ml CSF, approximately 8.1×10 11 gc / ml CSF, approximately 8.2×10 11 gc / ml CSF, approximately 8.3×10 11 gc / ml CSF, approximately 8.4×10 11 gc / ml CSF, approximately 8.5×10 11 gc / ml CSF, approximately 8.6×10 11gc / ml CSF, approximately 8.7×10 11 gc / ml CSF, approximately 8.8×10 11 gc / ml CSF, approximately 8.9×10 11 gc / ml CSF, approximately 9.0×10 11 gc / ml CSF, approximately 9.1×10 11 gc / ml CSF, approximately 9.2×10 11 gc / ml CSF, approximately 9.3×10 11 gc / ml CSF, approximately 9.4×10 11 gc / ml CSF, approximately 9.5×10 11 gc / ml CSF, approximately 9.6×10 11 gc / ml CSF, approximately 9.7×10 11 gc / ml CSF, approximately 9.8×10 11 gc / ml CSF, or approximately 9.9×10 11 gc / ml CSF, or any dosage in between.

[0163] In some embodiments, the dosage is approximately 1.0×10 12 gc / ml CSF, approximately 1.1×10 12 gc / ml CSF, approximately 1.2×10 12 gc / ml CSF, approximately 1.3×10 12 gc / ml CSF, approximately 1.4×10 12 gc / ml CSF, approximately 1.5×10 12 gc / ml CSF, approximately 1.6×10 12 gc / ml CSF, approximately 1.7×10 12 gc / ml CSF, approximately 1.8×10 12 gc / ml CSF, approximately 1.9×10 12 gc / ml CSF, approximately 2.0×10 12 gc / ml CSF, approximately 2.1×10 12 gc / ml CSF, approximately 2.2×10 12 gc / ml CSF, approximately 2.3×10 12 gc / ml CSF, approximately 2.4×10 12 gc / ml CSF, approximately 2.5×10 12 gc / ml CSF, approximately 2.6×10 12 gc / ml CSF, approximately 2.7×1012 gc / ml CSF, approximately 2.8×10 12 gc / ml CSF, approximately 2.9×10 12 gc / ml CSF, approximately 3.0×10 12 gc / ml CSF, approximately 3.1×10 12 gc / ml CSF, approximately 3.2×10 12 gc / ml CSF, approximately 3.3×10 12 gc / ml CSF, approximately 3.4×10 12 gc / ml CSF, approximately 3.5×10 12 gc / ml CSF, approximately 3.6×10 12 gc / ml CSF, approximately 3.7×10 12 gc / ml CSF, approximately 3.8×10 12 gc / ml CSF, approximately 3.9×10 12 gc / ml CSF, approximately 4.0×10 12 gc / ml CSF, approximately 4.1×10 12 gc / ml CSF, approximately 4.2×10 12 gc / ml CSF, approximately 4.3×10 12 gc / ml CSF, approximately 4.4×10 12 gc / ml CSF, approximately 4.5×10 12 gc / ml CSF, approximately 4.6×10 12 gc / ml CSF, approximately 4.7×10 12 gc / ml CSF, approximately 4.8×10 12 gc / ml CSF, approximately 4.9×10 12 gc / ml CSF, approximately 5.0×10 12 gc / ml CSF, approximately 5.1×10 12 gc / ml CSF, approximately 5.2×10 12 gc / ml CSF, approximately 5.3×10 12 gc / ml CSF, approximately 5.4×10 12 gc / ml CSF, approximately 5.5×10 12 gc / ml CSF, approximately 5.6×10 12 gc / ml CSF, approximately 5.7×10 12 gc / ml CSF, approximately 5.8×10 12 gc / ml CSF, approximately 5.9×10 12 gc / ml CSF, approximately 6.0×10 12gc / ml CSF, approximately 6.1×10 12 gc / ml CSF, approximately 6.2×10 12 gc / ml CSF, approximately 6.3×10 12 gc / ml CSF, approximately 6.4×10 12 gc / ml CSF, approximately 6.5×10 12 gc / ml CSF, approximately 6.6×10 12 gc / ml CSF, approximately 6.7×10 12 gc / ml CSF, approximately 6.8×10 12 gc / ml CSF, approximately 6.9×10 12 gc / ml CSF, approximately 7.0×10 12 gc / ml CSF, approximately 7.1×10 12 gc / ml CSF, approximately 7.2×10 12 gc / ml CSF, approximately 7.3×10 12 gc / ml CSF, approximately 7.4×10 12 gc / ml CSF, approximately 7.5×10 12 gc / ml CSF, approximately 7.6×10 12 gc / ml CSF, approximately 7.7×10 12 gc / ml CSF, approximately 7.8×10 12 gc / ml CSF, approximately 7.9×10 12 gc / ml CSF, approximately 8.0×10 12 gc / ml CSF, approximately 8.1×10 12 gc / ml CSF, approximately 8.2×10 12 gc / ml CSF, approximately 8.3×10 12 gc / ml CSF, approximately 8.4×10 12 gc / ml CSF, approximately 8.5×10 12 gc / ml CSF, approximately 8.6×10 12 gc / ml CSF, approximately 8.7×10 12 gc / ml CSF, approximately 8.8×10 12 gc / ml CSF, approximately 8.9×10 12 gc / ml CSF, approximately 9.0×10 12 gc / ml CSF, approximately 9.1×10 12 gc / ml CSF, approximately 9.2×10 12 gc / ml CSF, approximately 9.3×10 12gc / ml CSF, approximately 9.4×10 12 gc / ml CSF, approximately 9.5×10 12 gc / ml CSF, approximately 9.6×10 12 gc / ml CSF, approximately 9.7×10 12 gc / ml CSF, approximately 9.8×10 12 gc / ml CSF, or approximately 9.9×10 12 gc / ml CSF, or any dosage therebetween.

[0164] In some embodiments, 1.4×10 10 gc / mL CSF, ii) 4.4x10 10 gc / mL CSF, and the dosage of 1.4x10 11 gc / mL CSF is quantified by ddPCR.

[0165] In some embodiments, i) 5.0x10 10 gc / mL CSF, ii) 1.6x10 11 gc / mL CSF, or iii) 5.0x10 11 gc / mL CSF dosage is quantified by qPCR.

[0166] In some embodiments, the dosage of quantitative PCR (qPCR) can be converted to the dosage of digital droplet PCR (ddPCR). The digital droplet PCR (ddPCR) assay can be used to ensure the accuracy and precision of the titration method used for the administration of the rAAV vectors, AAV vectors, or pharmaceutical compositions of the present disclosure. After evaluating the initial lot of the APOE2 AAV viral capsid, a conversion factor of 3.6 from qPCR to ddPCR was assigned to enable the conversion of qPCR dosages to ddPCR dosages. Those skilled in the art will understand that the conversion factor can be adjusted according to the variation of the AAV viral capsid lot.

[0167] In some embodiments, the AAV vectors of the present disclosure are from about 1.0×10 10 gc to about 1.0×10 16It is administered at a total dose of gc. In some embodiments, the AAV vectors of the present disclosure are about 1.0×10 11 gc to about 1.0×10 15 gc. It is administered at a total dose of gc. In some embodiments, the AAV vectors of the present disclosure are about 1.0×10 12 gc to about 9.9×10 14 gc. It is administered at a total dose of gc. In some embodiments, the AAV vectors of the present disclosure are 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 at any dose therebetween.

[0168] In some embodiments, the AAV vectors of the present disclosure are about 1.0×10 14 gc, about 1.1×10 14 gc, about 1.2×10 14 gc, about 1.3×1014 gc, approximately 1.4×10 14 gc, approximately 1.5×10 14 gc, approximately 1.6×10 14 gc, approximately 1.7×10 14 gc, approximately 1.8×10 14 gc, approximately 1.9×10 14 gc, or approximately 2.0×10 14 gc, or at any dosage between these is administered. In some embodiments, the AAV vectors of the present disclosure are administered at a total dosage of approximately 1.4×10 14 gc.

[0169] In some embodiments, a therapeutically effective dosage of the AAV vectors of the present disclosure is administered as a fixed dosage. In some embodiments, the fixed dosage is expressed as the total number of genomic copies (gc).

[0170] In some embodiments, the AAV vectors of the present disclosure are administered at a fixed dosage of approximately 1.0×10 10 gc to approximately 1.0×10 16 gc. In some embodiments, the AAV vectors of the present disclosure are administered at a fixed dosage of approximately 1.0×10 13 gc to approximately 1.0×10 15 gc. In some embodiments, the AAV vectors of the present disclosure are administered at a fixed dosage of approximately 1.0×10 14 gc to approximately 9.0×10 14 gc. In some embodiments, the AAV vectors of the present disclosure are administered at a fixed dosage of approximately 1.0×10 14 gc, approximately 2.0×10 14 gc, approximately 2.1×10 14 gc, approximately 2.2×10 14 gc, approximately 2.3×10 14 gc, approximately 2.4×10 14 gc, approximately 2.5×10 14 gc, approximately 2.6×10 14 gc, approximately 2.7×10 14 gc, approximately 2.8×10 14 gc, approximately 2.9×10 14 gc, approximately 3.0×10 14 gc, approximately 4.0×10 14 gc, approximately 5.0×1014 gc, about 6.0×10 14 gc, about 7.0×10 14 gc, about 8.0×10 14 gc, or about 9.0×10 14 gc, or at a fixed dose of any dose in between is administered.

[0171] In some embodiments, the AAV vector of the present disclosure is 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 1.8×10 14 gc, about 1.9×10 14 gc, or about 2.0×10 14 gc, or at a fixed dose of any dose in between is administered. In some embodiments, the AAV vector of the present disclosure is about 2.5×10 14 gc at a fixed dose is administered. In some embodiments, the AAV vector of the present disclosure is about 1.4×10 14 gc at a fixed dose is administered.

[0172] Any dosage form of the AAV vector of the present disclosure, or a method for calculating the dosage form, is contemplated herein. In some embodiments, the dose is based on the mass and / or volume of the brain. In some embodiments, the dose is based on the body weight of the subject. In some embodiments, the dose is calculated using the qPCR titer method. In some embodiments, the dose is calculated using the ddPCR titer method.

[0173] In some embodiments, the therapeutically effective dosage can be adjusted for each AAV capsid serotype. In some embodiments, the therapeutically effective dosage is adjusted taking into account differences in CNS tropism of different AAV capsid serotypes. In some embodiments, the therapeutically effective dosage is adjusted taking into account differences in liver tropism of different AAV capsid serotypes. In some embodiments, the therapeutically effective dosage is adjusted taking into account differences in heart tropism of different AAV capsid serotypes.

[0174] In some embodiments, the subject is administered a single dose of the AAV vector. In some embodiments, the subject is further administered a second, third, fourth, or fifth dose of the AAV vector. In some embodiments, subsequent administrations of the AAV vector can be at a dosage different from the first dosage.

[0175] In some embodiments, the dosage is measured by quantitative polymerase chain reaction (qPCR) titer. In some embodiments, the dosage is measured by droplet digital polymerase chain reaction (ddPCR) titer.

[0176] In some embodiments, the subject is further administered an immunosuppressant in combination with administration of the AAV vector. 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. The immunosuppressant can be administered to improve patient safety, minimize the host immune response to AAV-based therapy, and / or enhance the therapeutic effect of AAV-based therapy. In some embodiments, the subject is further administered prednisone in combination with administration of the AAV vector.

[0177] In some embodiments, prednisone is 40 mg once daily starting approximately one week before administration of the AAV viral vector; 40 mg once daily from week 1 to week 2 after administration of the AAV viral vector; 30 mg once daily in week 3 after administration of the AAV viral vector. Once daily at 20 mg on the 4th week after AAV virus vector administration; Once daily at 10 mg on the 5th week after AAV virus vector administration; Once daily at 5 mg on the 6th week after AAV virus vector administration; Once daily at 2.5 mg on the 7th week after AAV virus vector administration; and Administered at a dose of 2.5 mg every other day on the 8th week after AAV virus vector administration.

[0178] In some embodiments, if evidence of a host immune response is observed after AAV vector administration, the dose of the immunosuppressant can be kept constant (i.e., not tapered as described above) or increased. In some embodiments, the maximum dose of prednisone is about 60 mg. Once the evidence of the host immune response has subsided or decreased, tapering of the immunosuppressant can be initiated.

[0179] In some embodiments, the subject may be further administered a proton pump inhibitor during the use of prednisone.

[0180] In some embodiments, the invention relates to a vector comprising an APOE2-encoding nucleic acid for use in the treatment or prevention of Alzheimer's disease in a subject, wherein the AAV vector is delivered to a subject in need thereof and APOE2 is expressed at a therapeutically effective level by the transduced cells.

[0181] In certain embodiments, the invention relates to a vector comprising an APOE2-encoding nucleic acid for effecting recovery of symptoms of Alzheimer's disease in a subject in need thereof, wherein the AAV vector is delivered to a subject in need thereof and APOE2 is expressed at a therapeutically effective level by the transduced cells.

[0182] Non-viral vector In certain embodiments, the use of the vector according to the invention is a non-viral vector. Typically, the non-viral vector can be a plasmid containing a nucleic acid sequence encoding the APOE2 gene or a variant thereof as described above. Pharmaceutical composition

[0183] 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 an APOE2-encoding nucleic acid.

[0184] The present disclosure provides a pharmaceutical composition comprising an APOE2 rAAV viral vector, the rAAV viral vector comprising an AAVrh10 capsid protein and an APOE2 rAAV vector.

[0185] 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 the APOE2 rAAV 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 the 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 12gc / mL, approximately 8.0×10 12 gc / mL, approximately 8.5×10 12 gc / mL, approximately 9.0×10 12 gc / mL, approximately 9.5×10 12 gc / mL, approximately 1.0×10 13 gc / mL, approximately 1.5×10 13 gc / mL, approximately 2.0×10 13 gc / mL, approximately 2.5×10 13 gc / mL, approximately 3.0×10 13 gc / mL, approximately 4.0×10 13 gc / mL, approximately 4.5×10 13 gc / mL, approximately 5.0×10 13 gc / mL, approximately 5.5×10 13 gc / mL, approximately 6.0×10 13 gc / mL, approximately 6.5×10 13 gc / mL, approximately 7.0×10 13 gc / mL, approximately 7.5×10 13 gc / mL, approximately 8.0×10 13 gc / mL, approximately 8.5×10 13 gc / mL, approximately 9.0×10 13 gc / mL, approximately 9.5×10 13 gc / mL, or any number of APOE2 rAAV vectors in between. In some embodiments, the pharmaceutical composition comprises at least about 1.5×10 13 gc / mL of the APOE2 rAAV vector.

[0186] In some embodiments, the APOE2 rAAV pharmaceutical composition comprises less than about 50% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises less than about 40% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises less than about 15% empty rAAV capsids.

[0187] In some embodiments, the APOE2 rAAV pharmaceutical composition disclosed herein comprises less than about 15% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises 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, less than about 11% empty rAAV capsids, less than about 10% empty rAAV capsids, less than about 9% empty rAAV capsids, less than about 8% empty rAAV capsids, less than about 7% empty rAAV capsids, less than about 6% empty rAAV capsids, less than about 5% empty rAAV capsids, less than about 4% empty rAAV capsids, less than about 3% empty rAAV capsids, less than about 2% empty rAAV capsids, or less than about 1% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises about 1% to about 10% empty rAAV capsids.

[0188] In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 40% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 35% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 30% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 25% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 20% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 15% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 1% to about 10% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises from about 2% to about 8% empty rAAV capsids. In some embodiments, the APOE2 rAAV pharmaceutical composition comprises no more than 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 embodiments, the number of empty rAAV capsids is below the limit of detection. In some embodiments, the percentage of empty rAAV capsids is specified as a percentage of the total rAAV capsids (e.g., by electron microscopy).

[0189] In some embodiments, the percentage of empty rAAV capsids is determined as a percentage of total rAAV capsids, for example, using analytical ultracentrifugation (AUC). In some embodiments, the AUC is sedimentation velocity AUC (SV-AUC). In some embodiments, the low percentage of these empty rAAV particles results in an improved therapeutic effect and / or a reduction in adverse events (e.g., inflammatory response, liver damage) after administration to a subject, as compared to, for example, administering a composition having a high percentage of empty rAAV particles. In some embodiments, the methods for preparing the rAAV compositions disclosed herein provide a low percentage of empty rAAV particles as compared to the levels of empty rAAV particles produced by other methods, e.g., empty rAAV particles that do not use the production and / or purification methods described herein.

[0190] In some embodiments, the APOE2 rAAV pharmaceutical compositions disclosed herein comprise at least 50% full rAAV particles. In some embodiments, the APOE2 rAAV pharmaceutical compositions disclosed herein comprise at least 60% full rAAV particles. In some embodiments, the APOE2 rAAV pharmaceutical compositions disclosed herein comprise at least 70% full rAAV particles. In some embodiments, the APOE2 rAAV pharmaceutical compositions disclosed herein comprise at least 80% full rAAV particles. In some embodiments, the APOE2 rAAV pharmaceutical compositions comprise at least 85% full rAAV particles, at least 90% full rAAV particles, at least 95% full rAAV particles, or at least about 99% full rAAV particles. In some embodiments, the APOE2 rAAV pharmaceutical compositions comprise 100% full rAAV particles.

[0191] The APOE2 rAAV pharmaceutical compositions of the disclosure are prepared such that residual host cell DNA is removed. In some embodiments, 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.

[0192] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual host cell proteins are removed. In some embodiments, 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.

[0193] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual adenovirus early region 1A (E1a DNA) is removed. In some embodiments, 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.

[0194] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual SV40 large T antigen DNA (SV40 LTA) DNA is removed. In some embodiments, 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.

[0195] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual non-encapsidated rAAV vector plasmid DNA is removed. In some embodiments, the amount of non-encapsidated rAAV vector plasmid DNA is below a predetermined value determined to be necessary for a safe and effective treatment suitable for administration to a subject. In some embodiments, the rAAV vector plasmid DNA is plasmid DNA that does not encode between the ITR regions.

[0196] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual polyethyleneimine (PEI) is removed. In some embodiments, 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.

[0197] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual benzonase is removed. In some embodiments, 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.

[0198] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual bovine serum albumin (BSA) is removed. In some embodiments, 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.

[0199] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual iodixanol is removed. In some embodiments, 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.

[0200] The APOE2 rAAV pharmaceutical composition of the present disclosure is prepared such that residual replication-competent AAV (rcAAV) is removed. In some embodiments, 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.

[0201] "Therapeutically effective amount" means an amount of the AAV vector of the present invention sufficient to treat Alzheimer's disease with a reasonable benefit / risk ratio applicable to any medical treatment.

[0202] It will be understood that the single dose or total daily dose of the compounds and compositions of the present invention is determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound being used, the specific composition being used, the age, weight, general health, sex, and diet of the patient, the time of administration, route of administration, and excretion rate of the specific compound being used, the duration of the treatment, drugs used in combination with or concurrently with the specific polypeptide being used, and like factors well known in the medical arts. For example, it is within the skill of the art to start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the total daily dosage of the product can vary widely for a single adult per day. The therapeutically effective amount of the vector according to the present invention to be administered, as well as the dosage for the treatment of a pathological condition by the number of viral or non-viral particles and / or pharmaceutical compositions of the present invention, will vary depending on a number of factors (including the age and condition of the patient, the severity of the affliction or disorder, the method and frequency of administration, and the specific peptide to be used).

[0203] The pharmaceutical composition comprising an AAV vector according to the present invention can be presented in any form suitable for the chosen mode of administration, for example, C1-C2 administration or ICM administration.

[0204] In the pharmaceutical compositions of the present invention for administration to the CNS, the active ingredient can be administered to animals and humans in unit dosage form, alone or in combination with another active ingredient, as a mixture with conventional pharmaceutical carriers.

[0205] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle as an injectable preparation. These may be, in particular, isotonic solutions, sterile solutions, saline (such as monosodium or disodium phosphate, sodium chloride, potassium, calcium, or magnesium, or mixtures of such salts), or may be dry, especially lyophilized compositions, which may optionally be made into an injectable solution by addition of sterile water or physiological saline.

[0206] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid. The form must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0207] Solutions containing the compounds of the present invention as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal storage and use conditions, such preparations contain preservatives to prevent the growth of microorganisms.

[0208] The AAV vectors according to the present invention can be formulated into the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may be derived from inorganic bases (such as sodium, potassium, ammonium, calcium, or ferric hydroxide), and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.).

[0209] The carrier may also be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size when dispersing, and the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to contain an isotonic agent (such as sugar or sodium chloride). Injectable compositions can be made to be absorbed sustainably by using them in compositions of agents that delay absorption (such as aluminum monostearate and gelatin).

[0210] Sterile injection solutions are prepared by incorporating the required amount of the active polypeptide, together with any of several other ingredients mentioned above as required, into a suitable solvent and then filtering and sterilizing. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required ingredients of the other ingredients mentioned above. In the case of sterile powders for preparing sterile injection solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, by using which powders containing the active ingredient and any additional desired ingredients can be obtained from a solution that has been previously sterilized by filtration.

[0211] When formulated, the solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulation can be easily administered in various dosage forms (such as the injection solution described above), but drug-release capsules, etc. may also be used.

[0212] Multiple doses can also be administered.

[0213] In some embodiments, the pharmaceutical composition comprising the rAAV viral vector of the present disclosure is formulated with more excipients suitable for administration to a subject in need thereof by any suitable method of administration. In some embodiments, the one or more excipients include a phosphate buffer and a salt.

[0214] In some embodiments, the phosphate buffer includes potassium monobasic phosphate and sodium dibasic phosphate. In some embodiments, potassium monobasic phosphate is used at a concentration of about 0.01 mM to about 100 mM. In some embodiments, potassium monobasic phosphate is about 0.1 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.25 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, or about 30 mM, or any concentration therebetween. In some embodiments, potassium monobasic phosphate is used at a concentration of about 1 mM. In some embodiments, sodium dibasic phosphate is used at a concentration of about 0.01 mM to about 100 mM. In some embodiments, sodium dibasic phosphate is about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, or about 30 mM, or any concentration therebetween. In some embodiments, sodium dibasic phosphate is used at a concentration of about 3 mM.

[0215] In some embodiments, the salt is sodium chloride. In some embodiments, sodium chloride is at a concentration of about 0.01 mM to 1 M. In some embodiments, sodium chloride is at a concentration of about 25 mM to 300 mM. In some embodiments, sodium chloride can be at a concentration of about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 155 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM. In some embodiments, sodium chloride is at a concentration of about 155 mM.

[0216] In some embodiments, the pharmaceutical composition is formulated at a pH suitable for administration to a subject. In some embodiments, the pH of the pharmaceutical composition is from about 6.0 to about 9.0. In some embodiments, 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 embodiments, the pH of the pharmaceutical composition is about 7.4.

[0217] The pharmaceutical compositions of the present disclosure can be administered to a subject in any amount deemed appropriate by the attending physician and may vary depending on the specific needs of the individual subject. In some embodiments, the pharmaceutical compositions of the present disclosure, which include pre-packaged pharmaceutical compositions, can be diluted with an appropriate volume of fluid prior to administration. In some embodiments, when the pharmaceutical compositions of the present disclosure are diluted, the volume of the diluted pharmaceutical composition administered to the subject is equal to the dosage of rAAV viral particles provided herein. In other words, even if the volume of the pharmaceutical composition increases and thereby dilutes the concentration of rAAV viral particles, the patient still receives the same total number of genomic copies of the rAAV vector or the number of rAAV viral capsids as specified by the dosage disclosed herein.

[0218] In some embodiments, the volume of the subject's CSF determines the final volume of the pharmaceutical composition. In some embodiments, the volume of CSF is measured by magnetic resonance imaging (MRI).

[0219] In some embodiments, 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 in between. Production of APOE2 AAV viral vector

[0220] The present disclosure provides an rAAV vector encoding APOE2 and a method for producing an rAAV viral vector. In some embodiments, the rAAV viral vector is derived from a cell lysate produced by the cell culture and purification methods described herein.

[0221] The present disclosure provides a method for producing a cell lysate containing an rAAV viral vector, comprising: (i) obtaining a culture vessel containing HEK293T cells in a culture medium; (ii) transfecting the HEK293T cells in the transfection medium with a first plasmid encoding an APOE2 AAV vector and a second plasmid encoding an AAV Rep protein and an AAV Cap protein, wherein the ratio of the second plasmid to the first plasmid is 2:1; (iii) culturing the transfected HEK293T cells in the culture medium under conditions such that 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 the rAAV viral vector.

[0222] The medium of the present disclosure can be any medium capable of culturing mammalian cells, including but not limited to HEK293T cells. In some embodiments, the culture medium comprises Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). The cells can be grown for any length of time necessary to produce the desired number of cells. In some embodiments, the HEK293T cells are obtained after a growth culture over a period of about 2 to about 5 days.

[0223] Transfection can be performed in any suitable medium. In some embodiments, the transfection medium comprises serum-free DMEM and polyethyleneimine (PEI).

[0224] The transfection of the two plasmids can be performed in any order. In some embodiments, the transfection of the first plasmid and the second plasmid occurs simultaneously.

[0225] In some embodiments, prior to transfection, HEK293T cells are present in a culture vessel at a density of about 2.0×10 4 ~ about 2.0×10 6 cells / cm 2 . In some embodiments, HEK293T cells are present at a density of about 2.0×10 5 cells / cm 2 .

[0226] 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 embodiments, the transfected cells are cultured for about 1 day to about 7 days. In some embodiments, the transfected cells are cultured for about 3 days.

[0227] Following collection of the transfected cells, the cells containing the APOE2 rAAV viral vector are lysed 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 with chemical agents such as detergents. In some embodiments, HEK293T cells are lysed via at least about 4 consecutive freeze-thaw cycles to produce a cell lysate.

[0228] The cell lysates of the present disclosure can be processed to remove all remaining non-capsidated virus or cellular DNA. In some embodiments, the cell lysates are further treated with a recombinant nuclease to digest all viral DNA or cellular DNA. In some embodiments, the recombinant nuclease is benzonase. In some embodiments, DNA digestion is performed in the presence of magnesium chloride.

[0229] Following 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 embodiments, clarification is performed by centrifugation or ultracentrifugation. Clarification of the cell lysate removes cell debris and unbroken cells.

[0230] Following clarification, the number of rAAV vectors or rAAV viral vectors in the cell lysate can be quantified. In some embodiments, the cell lysate contains from about 1.0×10 9 to about 5.0×10 14 genomic copies (gc) per milliliter.

[0231] The present disclosure further provides a method for producing an APOE2 rAAV pharmaceutical composition, 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 a chromatography 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 via ultrafiltration to produce an APOE2 rAAV pharmaceutical composition.

[0232] The density gradient of the present disclosure can be any suitable density gradient. The density gradient provides a means for separating the components in a mixture by size and / or molecular weight. The density gradient purification method can be used to separate empty AAV capsids from AAV capsids containing the APOE2 rAAV vector. In some embodiments, the density gradient is an iodixanol density gradient.

[0233] In some embodiments, the iodixanol gradient comprises a stepwise density gradient comprising (i) an iodixanol solution of about 10% to about 20%, (ii) an iodixanol solution of about 20% to about 30%, (iii) an iodixanol solution of about 40% to about 50%, and (iv) an iodixanol solution of about 50% to about 60%. In some embodiments, the iodixanol gradient comprises a stepwise density gradient comprising (i) an iodixanol solution of about 15%, (ii) an iodixanol solution of about 25%, (iii) an iodixanol solution of about 40%, and (iv) an iodixanol solution of about 54%.

[0234] In some embodiments, alternative purification means can be used instead of the density gradient purification method. In some embodiments, chromatography can be used. In some embodiments, ion exchange chromatography can be used.

[0235] Chromatography can be used to purify the rAAV viral vector following purification of the density gradient. In some embodiments, the chromatography is anion exchange chromatography. In some embodiments, the anion exchange column is a Q Sepharose High Performance high-strength quaternary ammonium anion exchange resin column.

[0236] Following chromatographic purification, the APOE2 rAAV viral vector is buffer-exchanged into a formulation buffer via ultrafiltration, thereby producing the pharmaceutical composition of the present disclosure. In some embodiments, the formulation buffer comprises phosphate buffered saline (PBS). In some embodiments, the pharmaceutical composition comprises about 1.0×10 10 to about 5.0×10 13 genome copies (viral genomes) per milliliter after ultracentrifugation. In some embodiments, the pharmaceutical composition comprises about 1.5×10 13 genome copies (vial genomes) per milliliter after ultracentrifugation.

Example

[0237] The present invention will be further illustrated by the following drawings and examples. However, these examples and drawings should not be construed as limiting the scope of the present invention in any way.

[0238] Example 1: Administration of APOE2 rAAV vector to AD patients results in increased expression of APOE2 and changes in the expression of AD biomarkers In APOE4 homozygous patients with Alzheimer's disease, as part of a Phase 1 / 2 trial, an AAV vector (LX1001) encoding human APOE2 was administered. The administration was performed by injection at C1-C2 under CT guidance. C1-C2 administration is preferred, but ICM administration can be used if C1-C2 administration is not practical or possible. Patients were administered LX1001 at a dose of 5.0×10 10 gc / ml CSF (determined by qPCR) or 1.4×10 10 (determined by ddPCR). The total CSF volume of the administered patients ranged from 313 to 490 mL. No serious adverse events were observed after administration. An adverse event of transient headache occurred in 2 out of 5 subjects, which was considered to be related to the administration procedure. No other adverse events occurred in multiple subjects.

[0239] Patient population The eligibility criteria for trial participation included a) aged 50 years or older, APOE4 expression being homozygous, mild cognitive impairment, amyloid PET positive mild to moderate dementia, and biomarkers consistent with Alzheimer's disease.

[0240] Evaluation items Primary evaluation items

[0241] Safety

[0242] Secondary evaluation items

[0243] Conversion of APOE4 to APOE2 in CSF Other secondary evaluation items

[0244] CSF Biomarker Levels / Expression: Aβ42, T Tau, and P Tau

[0245] Amyloid PET Scan

[0246] Quantitative MRI

[0247] Cognitive Tests

[0248] The expression of APOE2 was evaluated at the time point when it was fixed after LX1001 administration and compared with the pre - administration baseline of APOE4 expression. At 3 - month, 6 - month, and 12 - month time points, the APOE2 / APOE4 expression was evaluated in the patients' CSF (Figures 1 and 2). The APOE2 / APOE4 expression was calculated as 100 multiplied by the total APOE2 expression divided by the total APOE4 expression, and the increase in the ratio of APOE2 expression compared to APOE4 expression was obtained (Figure 1). All patients experienced an increase in APOE2 expression after treatment with LX1001. The increase in APOE2 expression was demonstrated in all patients with follow - up data and persisted up to 12 months (Figure 2).

[0249] AD biomarkers were also evaluated after LX1001 administration. The expressions of Aβ42, T - tau, and P - tau were measured 2, 6, and 12 months after LX1001 administration and compared with the pre - treatment baseline (Figure 3). The biomarker expressions were measured in CSF. After treatment with LX1001, the treated patients generally experienced a decrease in the expressions of Aβ42 (Figure 3A), T - Tau (Figure 3B), and P - Tau (Figure 3C).

[0250] Example 2: In Vivo Test of AAV Vector Encoding APOE2 Polypeptide The hypothesis that AAVrh10-mediated expression of combined human APOE2 effectively prevents the onset of Alzheimer's disease was evaluated. AAVrh.10hAPOE2C was tested in an APP.PSEN1 / TRE4 amyloid plaque mouse model with the humanized APOE4 gene, a presenilin mutation that promotes amyloid plaques, and a mutant amyloid precursor protein (Neurobiology of Aging 2016 44:159). AAVrh.10APOE2 and AAHrh.10 null vector were administered into the hippocampus (2×10 10 genome copies) of 2.5-month-old APP.PSEN / APOE4 mice and evaluated 5.5 months after administration. APP.PSEN / APOE4 mice administered with PBS were used as controls. The AAVrh.10hAPOE2 treatment cohort had a significant increase in hippocampal human APOE2 protein compared to PBS (Figure 4C). Administration of AAVrh.10hAPOE2 decreased the levels of both soluble and insoluble amyloid peptide β42 and soluble and insoluble amyloid peptide β40 compared to the PBS cohort (Figures 5A and 5B). The amount of viral genome copies in the mouse brain after administration was evaluated (Figure 4A). The amount of APOE mRNA was also determined (Figure 4B).

[0251] Immunohistochemical staining of the hippocampus for β-amyloid showed a decrease in the number and size of amyloid plaques after administration of AAVrh.10hAPOE2 compared to the PBS control (Figure 6). Fluorojade staining for neurodegeneration was decreased in AAVrh.10hAPOE2-treated mice compared to PBS mice (Figure 7). Markers of astrogliosis and microgliosis showed AAVrh.10hAPOE2-dependent decreases in GFAP (Figure 8) and Iba1 (Figure 9) positive cells. X-34, a marker of amyloid aggregates and structures, showed a decrease in amyloid levels measured by a decrease in X-34 staining intensity (Figure 10).

[0252] Before sacrifice, mice were evaluated by three behavioral sensorimotor assays. Compared with PBS, AAVrh.10hAPOE2 treatment increased (1) the number of alternations and entries in the Y-maze (Figures 12 and 13, Figure 14), (2) improved the novel object recognition index (Figure 15), (3) decreased the escape latency in the Barnes maze test (Figure 16), and (4) changes in nesting behavior were observed (Figure 11).

[0253] In conclusion, intracerebral delivery of AAVrh10 expressing human APOE2 improves morphological, biochemical, and behavioral disease parameters in mice with amyloid-driven Alzheimer's disease.

[0254] Example 3: Production of APOE2 rAAV viral vector The production of APOE2 rAAV vector pharmaceutical compositions can be carried out according to the process outlined below.

[0255] The APOE2 rAAV vector upstream manufacturing process consists of a series of cell culture expansion steps up to cell transfection. The production of AAVrh.10hAPOE2 involves co-transfection of HEK293T cells with two plasmids: the AAV vector plasmid (pAAVs-hAPOE2) and the helper plasmid (pPAK-MArh.10). After transfection, the cells are incubated until harvested. The cell harvest can be cryopreserved before further processing. The crude virus lysate is recovered from the transfected cells by multiple freeze-thaw cycles, and the remaining plasmid DNA and host cell nucleic acids are digested by incubation with Benzonase®. Clarification of the upstream process material to remove cell debris and unbroken cells is performed by ultracentrifugation. Purification of the process stream is performed by iodixanol gradient and anion exchange chromatography.

[0256] The final processing steps are concentration of the process pool and buffer exchange into formulation buffer, which forms the AAVrh.10hAPOE2 drug substance. Sub-lots may be pooled and then diluted to the target bulk drug substance (BDS) concentration prior to pharmaceutical manufacturing to form a pharmaceutical composition containing the APOE2 rAAV vector.

[0257] Thawing and Propagation of HEK293T WCB: To initiate a batch of AAVrh.10hAPOE2, thaw a vial of HEK293T WCB and propagate it by serial passage in DMEM and 10% qualified, gamma-irradiated fetal bovine serum (FBS) without antibiotics or phenol red indicator. The 10% FBS and DMEM medium used during the cell growth phase are preheated before use at each stage.

[0258] Thaw the frozen HEK293T WCB vial by suspending the cryovial in a beaker containing warmed water for injection (WFI). After visually confirming the thawing of the material, transfer the WCB cells to a 50 mL conical tube containing pre-warmed medium and mix manually. Grow the HEK293T cells as an adherent culture on the virgin polystyrene surface of a Corning 175 cm2 cell culture flask. Transfer the cell suspension from the 50 mL conical tube to a T75 flask. Incubate this T75 flask in a humidified incubator at 37 °C and 5% CO2 for up to 48 hours.

[0259] After the initial growth phase, wash the cells with phosphate-buffered saline and detach the cells from the flask walls with TrypLE select. Add warm 10% FBS DMEM medium to the flask to wash the cells off the culture vessel surface and break up cell clumps. Transfer the detached cells suspended in the medium to a sterile container and determine the cell density and viability. The cell density is used to calculate the seeding volume for the next growth phase. After adding the target seeding density to a T175 flask, incubate the flask at 37 °C, 90% relative humidity, and 5% CO2 for 2 - 4 days. Grow the cells until there are sufficient cells for transfection.

[0260] Seed monolayer cell stacks and 10-layer cell stacks from cells harvested from a T175 flask at a target cell density of 5E3 cells / cm2 or greater. During harvest from the flask, wash the cell monolayer with PBS, detach it from the surface with TrypLE select, and then resuspend it in warm 10% FBS DMEM. Prior to transfection, incubate each cell stack at 37 °C, 90% relative humidity, and 5% CO2 for up to 4 days.

[0261] PEI-mediated transfection Once the cells seeded in the cell stacks have grown to a density of approximately 2E5 viable cells / cm2, add the AAV vector and helper plasmid to the cells in the presence of PEI (a synthetic transfection reagent not derived from any animal product). The molar ratio of helper to transgene plasmid used in the transfection is approximately 1:2. The transfection mixture is prepared by adding the calculated amount of DNA to a dilution tube containing PEI. For each 10-layer cell stack, add 1272 μg of helper plasmid and 636 μg of transgene plasmid to SF-DMEM to dilute the plasmid, and then mix these with 1908 μg of PEI pro in the dilution tube. After mixing the solution and incubating it at room temperature for 15 minutes, divide the prepared transfection mixture into two bottles each containing 750 mL of 2.5% DMEM to give a total complex volume of 36.7 mL per cell stack. Remove the residual medium from the cell stack from the growth stage prior to adding the transfection mixture.

[0262] Culture the transfected cells in an incubator at 37 °C, 90% humidity, and 5% CO2 for the production stage.

[0263] Cell harvesting Three days after transfection, the cells floating in the medium are collected from the medium. The medium in the cell stack is collected and added to a centrifuge bottle. The remaining cells are detached from the cell stack using a sterile PBS / EDTA solution (pH 7.5). The cells in the PBS / EDTA solution and the floating cells in the medium are pelleted by centrifugation. The supernatant is discarded, and the cell pellet is resuspended and pooled in a hypotonic resuspension buffer (20 mM Tris pH 8.0 and 2 mM MgCl2). Samples of the cell harvest material (cells and supernatant) are collected for determination of vector genome copy (GC) titer, determination of bioburden, and assessment of the presence of adventitious contaminants prior to further processing. The cell harvest is stored at -65 °C or below for up to 3 months in a sterile, non-pyrogenic 50 mL polypropylene conical tube with a screw cap prior to further processing.

[0264] Lysis / Clarification The cell harvest is subjected to a plurality of freeze / thaw cycles to release the target vector from the cells producing the crude virus lysate (CVL). In each cycle, the harvested frozen cells are thawed on a heat block, mixed, and then placed on dry ice to refreeze. Five freeze / thaw cycles are completed before collecting an in-process sample for vector GC titer.

[0265] The CVL is treated with recombinant nuclease (Benzonase) at a target of 200 U / mL in a resuspension buffer containing 2 mM magnesium chloride (MgCl2) to digest any extra viral DNA and nucleic acids derived from the host cells. After addition of Benzonase, the CVL is incubated at 37 °C for 60 minutes. After Benzonase treatment, the CVL is clarified by centrifugation to remove cell debris and undestroyed cells. After pooling the clarified CVL, an in-process sample is collected and its vector GC titer is analyzed.

[0266] Iodixanol Gradient Purification The clarified CVL is purified using an iodixanol density gradient. In this step, a 15% / 25% / 40% / 54% iodixanol solution prepared in a sealed tube is used to separate AAVrh.10hAPOE2 from cellular components and empty capsids. These tubes are centrifuged in a fixed-angle rotor to achieve gradient separation, and the gradient fractions containing the complete capsids are collected. After collection, the fractions are diluted with QHP binding buffer (50 mM Tris, 50 mM NaCl, pH 9.0) and stored overnight at 2 - 8°C. In-process QC samples of the purified AAVrh.10hAPOE2 are taken and their vector GC titers are analyzed.

[0267] Anion exchange chromatography Fractions containing the desired AAVrh.10hAPOE2 vector product are pooled and loaded onto a Hi-Trap QHP anion exchange column. This column is disposable and is packed with Q Separose High Performance strong quaternary ammonium anion exchange resin. The AAVrh.10hAPOE2 vector is loaded onto the equilibrated column and collected in the flow-through. The column is washed with one column volume of binding buffer, and the AAVrh.10hAPOE2 flow-through is used to pool and recover residual product from the column. Up to two columns are used to purify the iodixanol fractions collected from a single 10-layer cell stack. In-process QC samples of the purified AAVrh.10hAPOE2 are collected and their vector GC titers are analyzed.

[0268] Ultrafiltration Using a single-use disposable ultrafiltration unit equipped with a 100 kDa MWCO polyethersulfone (PES) filter, the AAVrh.10hAPOE2 vector is concentrated and buffer-exchanged to generate the drug substance in phosphate-buffered saline, which is the final formulation buffer. The filter membrane is configured to be interlocked with a swing bucket rotor within a centrifuge. Prior to the first concentration step, the filter membrane is equilibrated with the final formulation buffer. The operations of concentration and buffer exchange are controlled by volume gradient markers on the side of the ultrafiltration unit. The dead stop concentration ratio is limited by filling the collection bottom up to the value obtained by subtracting the reduced volume of the sample from the total volume.

[0269] In-process QC samples of the drug substance are taken and analyzed for their vector GC titer, purity by SDS PAGE, empty capsids, and endotoxin. Intermediate lots of the drug substance are stored at -65°C or below for up to 1 year in 5 mL cryovials (USP VI) prior to further processing.

[0270] Bulk drug substance The frozen drug substance sublots are thawed, pooled, and adjusted to a target concentration 10% higher than the target pharmaceutical concentration with PBS, taking into account a 10% loss during sterile filtration for preparing the pharmaceutical (in some embodiments, it is 1.0E13 gc / mL, although other target pharmaceutical concentrations are also contemplated and described herein). Multiple lots of the drug substance may be pooled to form a BDS. The target BDS volume is based on the amount of AAVrh.10hAPOE2 vector pharmaceutical (DP) required to supply ongoing clinical trials. Pooling and dilution of the drug substance lots are performed in a sterile disposable container immediately prior to sterile filtration and filling of the AAVrh.10hAPOE2 vector DP. Analytical evaluation of the AAVrh.10hAPOE2 vector pharmaceutical composition

[0271] The analytical evaluation of the AAVrh.10hAPOE2 vector pharmaceutical composition (LX1001) can be performed using the following techniques and methods.

[0272] Identity of the transgene Perform Sanger sequencing of AAV to confirm the nucleotide sequence of the encapsulated payload. First, treat the sample with proteinase to release the digested AAV capsid protein, and then perform overlapping PCR to completely cover the promoter, enhancer, transgene, and polyA region of the expression cassette. After PCR, purify individual bands from the agarose gel and sequence them using a Sanger-based method. Bases are assigned using a score based on morbidity, and all detected mutations are individually examined using manual inspection.

[0273] Capsid purity Based on the total viral genome calculated by qPCR, load each sample into an SDS-PAGE gel along with a reference control. Stain the proteins migrating in the gel with Oriole fluorescent stain. Analyze all protein bands in each lane for densitometry and quantify individual impurity bands as a percentage of the sample. Report the overall purity as a percentage of all quantified bands with respect to the total quantification of VP1, VP2, and VP3.

[0274] Vector genome titer (qPCR) Use a Taqman-based qPCR assay targeting the CMV portion of the promoter region for quantification of the genomic copy titer. Prior to qPCR analysis, first treat the sample with proteinase K to digest the viral capsid protein membrane. Each assay includes a positive control AAVrh10 to ensure consistent assay performance and a CMV-containing reference plasmid (CMV PNY1160) is used to generate a standard curve for quantifying the tested AAV sample. The reported DS lot titer is the average of triplicates at two dilution levels. The titer reported for stable DS samples is the average of two independently performed analyses of triplicates of the sample at two dilution levels.

[0275] Vector genome titer (ddPCR) To ensure specificity, a ddPCR assay was developed that targets the region encompassing the 5’ end of the transgene coding region in LX1001. Each run of this method includes an AAVrh.10hAPOE-specific reference control to ensure assay performance.

[0276] Infectious titer An infectious titer assay is used to determine the productive uptake and replication of the AAVrh.10hAPOE vector in HeLaRC32 cells (HeLa expressing rep2). In this assay, the ability of vector particles to infect and replicate within a reporter cell line is measured to estimate the infectivity of the vector. Serial vector dilutions (10-fold dilutions) are co-infected in duplicate and then infected with adenovirus type 5. After 72 hours, the cells are lysed and qPCR is performed to detect amplification of the AAV vector relative to the input. Endpoint dilution TCID50 calculations (Spearman-Karber) are performed to determine the replication titer expressed as TCID50 / mL. The “infectious” value depends on the particles that contact the cells, so they are affected by the geometric shape of the assay, and this is not an absolute measure of the number of “infectious” particles present. However, the ratio of vector genome to “infectious unit” (expressed as the gc:infectious titer ratio) may be used as a measure of lot-to-lot product consistency.

[0277] Empty capsid To quantify the amount of AAV capsid species present in a sample, analytical ultracentrifugation using sedimentation velocity (SV-AUC) is used. The AAV sample is diluted to an A230nm of 0.8 ± 0.1 and run at 12,000 RPM on a Beckman XLI AUC instrument. The AUC cell is scanned every 90 seconds at A230nm for a total of 200 scans, and then the scans are fit with a resolution of 200 over a sedimentation range of 0 - 200 seconds using the c(s) distribution model in sedfit. Measurement of the relative abundance of the particle population is made possible by integration of the resulting peaks and calculation of the area under the c(s) fit profile at sedimentation coefficient values that match empty, partial, complete, or higher-order particle species. Reproducibility threshold criteria are implemented to evaluate whether each peak is reproducible or a modeling artifact that may be due to model fitting. The reproducibility threshold is defined as a percentage of the absorbance signal for a given species that is less than 0.5% of the total absorbance and / or less than 0.002 OD. The results of this analysis are a series of peaks that can be interpreted based on precedent literature and / or additional sample analysis (e.g., by analyzing a preparation containing only empty particles). Electron microscopy can also be used to evaluate empty and complete AAV capsids.

[0278] Residual host cell DNA The qPCR assay is used to detect residual human DNA from the HEK293 process. DNA is extracted from the test sample and tested using quantitative PCR (qPCR) targeting three separate amplicons in the 18S ribosomal (r)DNA gene. Genomic DNA from HEK293 is used to generate a standard curve for converting results from copy number to ng / mL for each amplicon.

[0279] Residual host cell protein Perform ELISA to measure the levels of contaminating host HEK293 cell proteins. Use the HEK293 Host Cell Proteins ELISA kit from Cygnus Technologies for the analysis. Add the samples, and the pre-diluted HEK293 HCP standards, to microtiter wells pre-coated with an affinity-purified anti-HEK293 HCP capture antibody together with a peroxidase-conjugated polyclonal anti-HEK293 HCP detection antibody. After incubation, wash the wells to remove unbound reactants and add TMB, a peroxidase substrate. After color development, stop the reaction using a sulfuric acid solution. Measure the absorbance of the resulting colored product using a microplate reader and calculate the amount of HEK293 HCP in each sample from the standard curve.

[0280] Residual SV40 large T antigen DNA Measure SV40 containing the DNA sequence using quantitative PCR (qPCR). Perform the quantification against a reference standard plasmid containing a single copy of the SV40 LTA sequence. Run the test samples with and without spiking to report the LOD of the assay and evaluate the assay performance.

[0281] Residual plasmid DNA Measure plasmid DNA not encoded between the ITR regions in the AAV sample by quantitative PCR (qPCR) targeting the kanamycin resistance gene portion of the plasmid sequence. Prior to qPCR analysis, first treat the samples with proteinase K to digest the viral capsid protein membrane. Perform the samples quadruplicate with and without DNase (treatment prior to proteinase K) to remove all remaining non-encapsidated DNA and test, reporting in comparison to known standards.

[0282] Residual E1a DNA A DNA sequence containing the adenovirus E1a gene element is determined in an AAV sample by quantitative PCR (qPCR) using primers and probes specific for the adenovirus E1a sequence. A standard curve is generated using genomic DNA extracted from HEK293T cells (which express E1a). Prior to qPCR analysis, the samples are first treated with proteinase K to digest the viral capsid protein membrane. Test samples are run with and without spiking to report the LOD of the assay and to evaluate assay performance.

[0283] Residual Benzonase Benzonase is used in the production process to degrade nucleic acids to facilitate vector purification and thus represents a process impurity. The concentration of residual Benzonase (EMD Millipore number 1.01681.0002) is measured using a commercially available ELISA. Samples are analyzed in triplicate wells.

[0284] Replication-competent AAV The cell-based assay for replication ability consists of a monolayer of HEK293 cells, an adenovirus (Ad5) transformed cell line, and inoculation with the test article. Due to the presence and co-infection of adenovirus, replication-competent AAV, if present, replicates and amplifies in cell culture. After several days, when the virus induces a cytopathic effect, the cells are harvested. The cell lysate is used to re-infect a new monolayer of HEK293 cells two more times for a total of three amplifications. Total cellular DNA is then isolated from the three passages and subjected to quantitative PCR (qPCR) analysis. The target used for the analysis is derived from the AAV2 Rep sequence required for AAV replication in the presence of adenovirus. The limit of detection (LOD) of this assay is 1 rcAAV / 1E9 rAAV, and it is confirmed that there is no inhibition by the spike control.

[0285] Bioburden This method is based on membrane filtration, including modifications for quantitative analysis. The sample is divided into two aliquots, filtered through a 0.2 μm membrane, and incubated in TSA and SAB media at 30 - 35 °C and 20 - 25 °C for 5 - 7 days, respectively. At the end of the incubation, the total number of colonies is counted and divided by the sample volume to obtain the CFU / mL of the 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, and the pharmaceutical composition comprises at least about 1.0×10 １１ genomic copies (gc) / mL to about 1.0×10 １４ gc / mL, and the pharmaceutical composition comprises less than about 40% empty rAAV capsids, said pharmaceutical composition.

2. The pharmaceutical composition according to 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 contains at least about 1.5×10 １３ g c / mL, and is the pharmaceutical composition according to claim 1.

4. The pharmaceutical composition according to any one of the preceding claims, wherein the APOE2 rAAV viral vector is formulated at a pH of about 7.4 in about 1 mM potassium monobasic phosphate, 3 mM sodium dibasic phosphate, and about 155 mM sodium chloride (NaCl).

5. The rAAV vector, 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 poly A sequence, and a second ITR sequence, and the pharmaceutical composition according to any one of the preceding claims.

6. The pharmaceutical composition according to any one 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 set forth in SEQ ID NO:

1.

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

12.

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

2.

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

3.

11. The pharmaceutical composition according to any one of the preceding claims, wherein the poly A sequence comprises the nucleic acid sequence set forth in SEQ ID NO:

6.

12. The pharmaceutical composition according to any one of the preceding claims, wherein the rAAV vector comprises the nucleic acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO:

11.

13. The pharmaceutical composition according to any one of the preceding claims, wherein the rAAV vector is packaged as an rAAV viral vector comprising an AAV capsid protein.

14. The pharmaceutical composition according to any one of the preceding claims, wherein 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.

15. The pharmaceutical composition according to 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, and the APOE2 rAAV vector comprises the nucleic acid sequence set forth 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 according to claim 1 or 16.

18. The method according to claim 17, wherein after administration, the subject experiences an increase in APOE2 expression of at least about 5% compared to the baseline before administration.

19. The method according to claim 17, wherein the patient is an APOE4 homozygote.

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

21. The pharmaceutical composition is administered at a dose of about 5.0 × 10 ９ gc / mL CSF to about 5.0 × 10 １２ gc / mL CSF, the method according to claim 17.

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

23. The method according to 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 according to claim 17, wherein the subject experiences an increase in APOE2 expression of at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

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

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

27. The method according to claim 17, wherein after administration of the pharmaceutical composition, the expression level of at least one of T-tau and P-tau in the subject decreases as compared to the baseline before administration.

28. The method according to claim 27, wherein the expression level of the T-tau and / or P-tau decreases 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. After administration of the pharmaceutical composition, the amyloid beta 42 / amyloid beta 40 (Aβ ４２／４０ ) ratio increases, the method according to claim 17.

30. Said Aβ ４２／４０ The method according to claim 29, wherein the ratio increases by at least about 5%, at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

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

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

33. The prednisone is 40 mg once a day, one week before administration of the AAV viral vector; 40 mg once a day, from the first week to the second week after administration of the AAV viral vector; 30 mg once a day, in the third week after administration of the AAV viral vector; 20 mg once a day, in the fourth week after administration of the AAV viral vector; 10 mg once a day, in the fifth week after administration of the AAV viral vector; 5 mg once a day, in the sixth week after administration of the AAV viral vector; 2.5 mg once a day, in the seventh week after administration of the AAV viral vector; and administered at a dose of 2.5 mg every other day, in the eighth week after administration of the AAV viral vector.

34. A method for producing a cell lysate containing an rAAV viral vector, comprising: (i) transfecting a cell culture containing HEK293T cells in transfection medium with a first plasmid encoding an APOE2 AAV vector and a second plasmid encoding an AAV Rep protein and an AAV Cap protein, 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; (iv) lysing the transfected HEK293T cells to produce a cell lysate containing the rAAV viral vector, the method comprising: [

35. ] The method according to claim 34, wherein the culture medium comprises Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). [

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

37. ] The method according to claim 34, wherein the HEK293T cells are obtained after growth culture for about 2 to about 5 days. [

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

39. ] The HEK293T cells are present in the culture vessel at a density of about 2.0×10 ４ to about 2.0×10 ６ cells / cm ２ according to claim 34, wherein the method is as described above. [

40. ] The method according to claim 34, wherein the transfected cells are cultured for about 3 days. [

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

42. ] The method according to claim 34, wherein the cell lysate is further treated with a recombinant nuclease to digest all non-capsidated DNA. [

43. ] The method according to claim 34, wherein following DNA digestion, the cell lysate is clarified via ultracentrifugation. [

44. ] The cell lysate contains from about 1.0×10 ９ to about 5.0×10 １４ genomic copies (gc) per milliliter, the method according to any one of claims 34 to 43. [

45. ] A method for producing an APOE2 rAAV pharmaceutical composition, 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. The method as described above, comprising: (v) producing an APOE2 rAAV pharmaceutical composition by concentrating the eluted rAAV viral vector into a formulation buffer via ultrafiltration.

46. The method according to claim 45, wherein the density gradient is an iodixanol density gradient.

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

48. The method according to claim 45, wherein the anion exchange column is a Q Sepharose High Performance high-strength quaternary ammonium anion exchange resin column.

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

50. The method according to claim 45, wherein the pharmaceutical composition contains from about 1.0×10 １０ to about 5.0×10 １３ virus genomes per milliliter after ultracentrifugation.

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

52. A cell lysate comprising an rAAV viral vector produced by the method according to claim 34.

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

11.

54. 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 an rAAV vector comprising a second ITR sequence.

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