Bifunctional molecules for targeted deamidation and methods of use

Abstract

Provided are bifunctional molecules for targeted deamidation. In some instances, the target for deamidation is amyloid-β (Aβ). For example, in certain embodiments, provided are bifunctional molecules comprising an Aβ targeting moiety stably associated with (1) a deamidation agent or (2) a deamidation agent-binding moiety. According to some embodiments, the target for deamidation is Aβ42 and the Aβ targeting moiety is an Aβ42 targeting moiety. Also provided are compositions and methods for deamidating Aβ in a subject in need thereof, e.g., to reduce or prevent Aβ neurotoxicity in the subject.

Description

[0001] BIFUNCTIONAL MOLECULES FOR TARGETED DEAMIDATION AND METHODS OF USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 551 ,477, filed February 8, 2024, which application is incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government support under contract AG074954 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE

[0007] LISTING XML FILE

[0008] A Sequence Listing is provided herewith as a Sequence Listing XML, UCSC- 405WO_SEQLIST, created on February 7, 2025 and having a size of 2,849 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0009] INTRODUCTION

[0010] Amyloid-p (Ap) is a 4 kDa fragment of the amyloid precursor protein (APP), a larger precursor molecule widely produced by brain neurons, vascular and blood cells (including platelets), and astrocytes. Two subsequent proteolytic cleavages of APP by p-secretase (p-APP- cleaving enzyme-1 (BACE1 )) at the ectodomain and y-secretase at intra-membranous sites generate A .

[0011] Alzheimer's disease is the most common type of dementia. It affects tens of millions of people worldwide, and this number is rising dramatically. The social and economic burden of Alzheimer's disease is high. The amyloid hypothesis proposes p-amyloid (AP) as the main cause of the disease and suggests that misfolding of the extracellular Ap protein accumulated in senile plaques and the intracellular deposition of misfolded tau protein in neurofibrillary tangles cause memory loss and confusion and result in personality and cognitive decline over time. Accumulated Ap peptide is the main component of senile plaques. Deposits of Ap peptides are mainly observed in the region of the hippocampus and the neocortex as well as in the cerebrovasculature.

[0012] Several prescription drugs are approved by the U.S. Food and Drug Administration (FDA) for Alzheimer’s disease to help either manage the symptoms of or to treat the disease. Most FDA- approved drugs work best for people in the early or middle stages of Alzheimer’s. There are currently no known interventions that will cure Alzheimer's. SUMMARY

[0013] Provided are bifunctional molecules for targeted deamidation. In some instances, the target for deamidation is amyloid-p (A|3). For example, in certain embodiments, provided are bifunctional molecules comprising an Ap targeting moiety stably associated with (1 ) a deamidation agent or (2) a deamidation agent-binding moiety. According to some embodiments, the target for deamidation is Ap4 and the Ap targeting moiety is an Ap42 targeting moiety. Also provided are compositions and methods for deamidating Ap in a subject in need thereof, e.g., to reduce or prevent Ap neurotoxicity in the subject.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 : Neurotoxicity of Ap42 and Ap42-N27D against SH-SY5Y cells at 20 pM and 50 M.

[0016] FIG. 2: Crystal structure of the KLVFFAE:klvffae rippled sheet.

[0017] DETAILED DESCRIPTION

[0018] Before the bifunctional molecules, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the bifunctional molecules, compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the bifunctional molecules, compositions and methods will be limited only by the appended claims.

[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the bifunctional molecules, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the bifunctional molecules, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the bifunctional molecules, compositions and methods.

[0020] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the bifunctional molecules, compositions and methods belong. Although any bifunctional molecules, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the bifunctional molecules, compositions and methods, representative illustrative bifunctional molecules, compositions and methods are now described.

[0021] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present bifunctional molecules, compositions and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0022] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0023] It is appreciated that certain features of the bifunctional molecules, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the bifunctional molecules, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present bifunctional molecules, compositions and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. BIFUNCTIONAL MOLECULES FOR TARGETED DEAMIDATION

[0025] The present disclosure provides bifunctional molecules for targeted deamidation. According to some embodiments, the bifunctional molecules comprise an Ap targeting moiety stably associated with (1 ) a deamidation agent or (2) a deamidation agent-binding moiety. Unlike existing therapeutic agents which do not chemically modify Ap such as Ap-targeting antibodies (e.g., Aducanumab, BAN2401 ) or small molecules used to remodel Ap aggregates to adopt nontoxic forms (e.g., ALZ801 , EGCG, RD2), the bifunctional molecules of the present disclosure detoxify Ap via chemical modification and therefore constitute a new class of therapeutic agents for treatment of diseases associated with protein or peptide aggregation (e.g., amyloid diseases) in view of the intrinsic sidechain fibrillogenicity of asparagine and glutamine. Details regarding embodiments of the bifunctional molecules of the present disclosure will now be provided.

[0026] The bifunctional molecules comprise an Ap targeting moiety. As used herein, “amyloid p” or “Ap” denotes a polypeptide of 36 to 49 amino acids (Ap36, Ap37, Ap38, Ap39, Ap40, Ap41 , Ap42, Ap43, Ap44, Ap45, Ap46, Ap47, Ap48, and Ap49) that are the main component of the amyloid plaques found in the brains of Alzheimer’s disease (AD) patients. The peptides result from the amyloid precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield Ap. The amino acid sequence of the wild-type Ap49 peptide is:

[0027] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITL (SEQ ID NO:1 )

[0028] The other wild-type Ap peptides are shorter versions of the Ap49 peptide. Ap42 peptides are more hydrophobic and fibrillogenic, have a higher aggregation potential and are the principal species deposited in the brain. The wild-type Ap42 peptide has the following amino acid sequence:

[0029] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:2)

[0030] The Ap targeting moiety is a moiety that binds an Ap polypeptide, e.g., Ap42. In some instances, the Ap targeting moiety is a peptide, polypeptide, a peptide or polypeptide multimer, an antibody, a ligand, an aptamer, a nanoparticle, and a small molecule.

[0031] The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof.

[0032] The term “amino acid” includes, but is not limited to, naturally-occurring amino acids and their stereoisomers. “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally- occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid (i.e., the D-amino acid). The Ap targeting moiety may comprise only of L-amino acids, may comprise only of D-amino acids, or may comprise a mixture of L-amino acids and D-amino acids. Propolypeptides comprised of D-amino acids (e.g., all-D amino acids) may be advantageous compared to their all-L counterparts in terms of PK, reduced immunogenicity, and / or the like. See Mandal et al. (2012) PNAS 109:14779-14784. Moreover, an Ap targeting moiety may comprise one or more beta amino acids, one or more gamma amino acids, and / or one or more of any other amino acid types that suitably replace alpha-amino acids. See, e.g., Sang et al. Acc. Chem. Res. 53, 10, 2425-2442. In certain embodiments, a targeting moiety comprises one or more amino acid analogs available from Bachem.

[0033] Naturally-occurring a-amino acids are those encoded by the genetic code as well as those amino acids that are later modified (e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine). Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally- occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D- aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D- isoleucine (D-lle), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-GIn), D-serine (D-Ser), D-threonine (D- Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0034] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. For example, an L-amino acid may be represented herein by its commonly known three letter symbol (e.g., Arg for L-arginine) or by an upper-case one-letter amino acid symbol (e.g., R for L-arginine). A D-amino acid may be represented herein by its commonly known three letter symbol (e.g., D-Arg for D-arginine) or by a lower-case one-letter amino acid symbol (e.g., r for D-arginine).

[0035] According to some embodiments, the Ap targeting moiety is an antibody. By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the target molecule (e.g., a cell surface molecule of a target cell), including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., humanized scFv); and fusion proteins comprising an antigen-binding portion of an antibody and a non- antibody protein. In certain embodiments, the antibody is selected from an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv’)2, or a single variable domain located on a heavy chain (VHH). According to some embodiments, the antibody is a VHH (sometimes referred to herein and elsewhere as a “nanobody”). The antibody may be detectably labeled, e.g., with an in vivo imaging agent, a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.

[0036] In certain embodiments, the A targeting moiety is an aptamer. By “aptamer” is meant a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for the target molecule. Aptamers exhibit certain desirable properties for targeted delivery of the bifunctional molecules of the present disclosure, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Aptamers that bind to targets in vivo are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C). Aptamers that find use in the context of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1 ):39-45; Sun et al. (2014) Mol. Then Nucleic Ac / ofe 3:e182; and Zhang et al. (2011 ) Curr. Med. Chem. 18(27) :4185-4194.

[0037] According to some embodiments, the Ap targeting moiety is a nanoparticle. As used herein, a “nanoparticle” is a particle having at least one dimension in the range of from 1 nm to 1000 nm, from 20 nm to 750 nm, from 50 nm to 500 nm, including 100 nm to 300 nm, e.g., 120- 200 nm. The nanoparticle may have any suitable shape, including but not limited to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped, prism-shaped, or any other suitable geometric or non-geometric shape. In certain aspects, the nanoparticle includes on its surface one or more of the other targeting moieties described herein, e.g., antibodies, ligands, aptamers, small molecules, etc. Nanoparticles that find use in the context of the present disclosure include those described in Wang et al. (2010) Pharmacol. Res. 62(2):90-99; Rao et al. (2015) ACS Nano 9(6) :5725-5740; and Byrne et al. (2008) Adv. Drug Deliv. Rev. 60(15):1615-1626.

[0038] In some embodiments, the Ap targeting moiety is a small molecule. By “small molecule” is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In some instances, the small molecule is not made of repeating molecular units such as are present in a polymer. Small molecules that find use as targeting moieties in vivo are known. As just one example, folic acid (FA) derivatives have been shown to effectively target certain types of cancer cells by binding to the folate receptor, which is overexpressed, e.g., in many epithelial tumors. See, e.g., Vergote et al. (2015) Then Adv. Med. Oncol. 7(4):206-218. In another example, the small molecule sigma-2 has proven to be effective in targeting cancer cells. See, e.g., Hashim et al. (2014) Molecular Oncology 8(5):956-967. Sigma-2 is the small molecule ligand for sigma-2 receptors, which are overexpressed in many proliferating tumor cells including pancreatic cancer cells.

[0039] In certain embodiments, the Ap targeting moiety specifically binds Ap. In some instances, the Ap targeting moiety specifically binds Ap42. As used herein, a first molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances, e.g., in a sample. In certain embodiments, the Ap targeting moiety “specifically binds” the target molecule if it binds to or associates with the Ap target molecule with an affinity or Ka (that is, an association rate constant of a particular binding interaction with units of 1 / M) of, for example, greater than or equal to about 104M1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 102M to 1013M, or less). In certain aspects, specific binding means the targeting moiety binds to the target molecule with a KD of less than or equal to about 105M, less than or equal to about 106M, less than or equal to about 107M, less than or equal to about 108M, or less than or equal to about 109M, 1010M, 1011M, or 1012M or less. The binding affinity of the Ap targeting moiety for the Ap target molecule can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0040] In some instances, the Ap targeting moiety binds to the KLVFFAE region of Ap42. When the Ap targeting moiety binds to the KLVFFAE region of Ap42, in certain embodiments, the Ap targeting moiety is a peptide or polypeptide comprising the sequence KLVFFAE, wherein the KLVFFAE amino acids are D-amino acids. According to some embodiment, such a targeting moiety has a length of from 7-30 amino acids, e.g., from 7-25, from 7-20, from 7-15, or from 7-10 amino acids. Supporting such embodiments, a crystal structure of a KLVFFAE:klvffae rippled sheet is shown in FIG. 2.

[0041] As summarized above, a bifunctional molecule of the present disclosure comprises a deamidation agent or a deamidation agent-binding moiety. As used herein, a “deamidation agent” is any substance capable of removing an amide functional group from the side chain of an amino acid (e.g., from the side chain of glutamine or asparagine), or converting the amide functional group to a different functional group. In some instances, the deamidation agent hydrolyzes the amide side chain of one or more glutamine and / or asparagine residues of Ap to form their corresponding carboxylic acid derivatives. According to some embodiments, the deamidation agent is a deamidase. Non-limiting examples of deamidases include glutaminases and asparaginases. Details regarding deamidation agents (including but not limited to deamidases such as glutaminases and asparaginases) are provided in, e.g., Gervais (2015) Journal of Chemical Technology & Biotechnology 91 (3):569-575; De Sciscio et al. (2023) J. Phys. Chem. B 127(44):9550-9559; Riggs et al. (2019) Anal. Chem. 91 (20) :13032-13038; Giles et al. (2018) Molecular Therapy 26(12):2848-2862; and Peters & Trout (2006) Biochemistry 45(16):5384-92; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0042] A “deamidation agent-binding moiety” is any moiety capable of binding a deamidation agent. In certain embodiments, the deamidation agent-binding moiety is a moiety capable of recruiting, upon administration to a subject, an endogenous or co-administered deamidation agent (e.g., an endogenous glutaminase, an endogenous asparaginase, or the like) to the bifunctional molecule of the present disclosure, e.g., a bifunctional molecule bound or targeted to an endogenous Ap polypeptide (e.g., endogenous A|342) in the subject. Suitable deamidation agent-binding moieties include, but are not limited to, a peptide, a polypeptide, an antibody, a ligand, an aptamer, a nanoparticle, and a small molecule. Details regarding such binding moieties are described above in the context of Ap targeting moieties.

[0043] In certain embodiments, a bifunctional molecule of the present disclosure is configured to target and deamidate endogenous Ap (e.g., endogenous Ap42) at position N27 (e.g., resulting in AP42-N27D), Q15 (e.g., resulting in AP42-Q15E), or both N27 and Q15 upon administration to a subject, e.g., in the brain of the subject having or at risk of developing Alzheimer’s disease.

[0044] As summarized above, the bifunctional molecules comprise the Ap targeting moiety stably associated with the deamidation agent or deamidation agent-binding moiety. By “stably associated” is meant a physical association between two entities in which the mean half-life of association is one day or more in phosphate buffered saline (PBS) at 4°C. In some embodiments, the physical association between the two entities has a mean half-life of one day or more, one week or more, one month or more, including six months or more, e.g., 1 year or more, in PBS at 4°C. According to some embodiments, the stable association arises from a covalent bond between the two entities, a non-covalent bond between the two entities (e.g., an ionic or metallic bond), or other forms of chemical attraction, such as hydrogen bonding, Van der Waals forces, and the like.

[0045] In certain embodiments, the Ap targeting moiety is stably associated with the deamidation agent or deamidation agent-binding moiety via fusion of a protein domain comprising the Ap targeting moiety and a protein domain comprising the deamidation agent or deamidation agentbinding moiety. In other words, the Ap targeting moiety may be part of a fusion protein comprising the Ap targeting moiety fused directly or indirectly to the deamidation agent or deamidation agentbinding moiety. According to some embodiments, the protein domain comprising the Ap targeting moiety is fused indirectly via a linker to the protein domain comprising the deamidation agent or deamidation agent-binding moiety. Non-limiting examples of a linker that may be employed include a glycine-serine linker.

[0046] According to some embodiments, the Ap targeting moiety is stably associated with the deamidation agent or deamidation agent-binding moiety via conjugation. The term “conjugation” or “conjugated” generally refers to a chemical linkage, either covalent or non-covalent, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. In certain embodiments, the Ap targeting moiety is conjugated to the deamidation agent or deamidation agent-binding moiety via a linker. If present, the linker molecule(s) may be of sufficient length to permit the A targeting moiety and deamidation agent or deamidation agentbinding moiety to allow some flexible movement between the Ap targeting moiety and deamidation agent or deamidation agent-binding moiety. Linker molecules may be, e.g., about 6- 50 atoms long. Linker molecules may also be, e.g., aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.

[0047] Where the linkers are peptides, the linkers can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids in length.

[0048] Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest, and may serve as a neutral tether between components. The ordinarily skilled artisan will recognize that design of conjugates can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure.

[0049] According to some embodiments, the Ap targeting moiety is conjugated to the deamidation agent or deamidation agent-binding moiety via a non-cleavable linker. Non- cleavable linkers of interest include, but are not limited to, thioether linkers. An example of a thioether linker that may be employed includes a succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) linker.

[0050] In certain embodiments, the Ap targeting moiety is conjugated to the deamidation agent or deamidation agent-binding moiety via a cleavable linker. According to some embodiments, the linker is a chemically-labile linker, such as an acid-cleavable linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the mildly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of a target cell (e.g., a neuron). Chemically- labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, etc. In certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into a target cell, e.g., a neuron. Enzyme- labile linkers include, but are not limited to, linkers that include peptidic bonds, e.g., dipeptide- based linkers such as valine-citrulline (VC) linkers, such as a maleimidocaproyl-valine-citruline- p-ami nobenzyl (MC-vc-PAB) linker, a valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, and the like. Chemically-labile linkers, enzyme-labile, and non-cleavable linkers are known and described in detail, e.g., in Ducry & Stump (2010) Bioconjugate Chem. 21 :5-13; Nolting, B. (2013) Methods Mol Biol. 1045:71 -100; Tsuchikama and An (2018) Protein & Cell 9(1 ):33-46; and elsewhere.

[0051] Numerous strategies are available for linking the Ap targeting moiety and deamidation agent or deamidation agent-binding moiety directly, or indirectly via a linker. For example, the Ap targeting moiety may be derivatized by covalently attaching a linker to the Ap targeting moiety, where the linker has a functional group capable of reacting with a “chemical handle” on the deamidation agent or deamidation agent-binding moiety. Also by way of example, the deamidation agent or deamidation agent-binding moiety may be derivatized by covalently attaching a linker to the deamidation agent or deamidation agent-binding moiety, where the linker has a functional group capable of reacting with a “chemical handle” on the Ap targeting moiety. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the Ap targeting moiety or deamidation agent or deamidation agent-binding moiety. According to one embodiment, the chemical handle is provided by incorporation of an unnatural amino acid having the chemical handle into the Ap targeting moiety or deamidation agent or deamidation agent-binding moiety. Unnatural amino acids which find use for preparing the conjugates of the present disclosure include those having a functional group selected from an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, and boronic acid functional group. Unnatural amino acids which may be incorporated into an Ap targeting moiety or deamidation agent or deamidation agent-binding moiety of a conjugate of the present disclosure, which unnatural amino acid may be selected to provide a functional group of interest, are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9):1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65; and elsewhere. An unnatural amino acid may be incorporated into an Ap targeting moiety or deamidation agent or deamidation agent-binding moiety via chemical synthesis or recombinant approaches, e.g., using a suitable orthogonal amino acyl tRNA synthetase-tRNA pair for incorporation of the unnatural amino acid during translation of an Ap targeting moiety or deamidation agent or deamidation agent-binding moiety in a host cell.

[0052] The functional group of an unnatural amino acid present in the Ap targeting moiety or deamidation agent or deamidation agent-binding moiety may be an azide, alkyne, alkene, aminooxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). As just one example, an azide- bearing unnatural amino acid (e.g., 5-azido-L-norvaline, or the like) may be incorporated into the Ap targeting moiety or deamidation agent or deamidation agent-binding moiety and the linker portion of a linker-agent moiety may include an alkyne functional group, such that the A(3 targeting moiety or deamidation agent or deamidation agent-binding moiety and linker-agent moiety are covalently conjugated via azide-alkyne cycloaddition. Conjugation may be carried out using, e.g., a copper-catalyzed azide-alkyne cycloaddition reaction.

[0053] In certain embodiments, the chemical handle on the Ap targeting moiety or deamidation agent or deamidation agent-binding moiety does not involve an unnatural amino acid. An Ap targeting moiety or deamidation agent or deamidation agent-binding moiety containing no unnatural amino acids may be conjugated by utilizing, e.g., nucleophilic functional groups of the Ap targeting moiety or deamidation agent or deamidation agent-binding moiety (such as the N- terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a moiety bearing a reactive leaving group or other electrophilic group. An example would be to prepare an Ap targeting moiety-linker moiety bearing an N-hydroxysuccinimidyl (NHS) ester and allow it to react with the deamidation agent or deamidation agent-binding moiety under aqueous conditions at elevated pH (—10) or in polar organic solvents such as DMSO with an added non-nucleophilic base, such as N,N- diisopropylethylamine.

[0054] It will be appreciated that the particular approach for attaching a linker, Ap targeting moiety and / or deamidation agent or deamidation agent-binding moiety to each other may vary depending upon the particular linker, Ap targeting moiety and / or deamidation agent or deamidation agent-binding moiety and functional groups selected and employed for conjugating the various components to each other.

[0055] COMPOSITIONS

[0056] Aspects of the present disclosure further include compositions. According to some embodiments, a composition of the present disclosure comprises a bifunctional molecule of the present disclosure. The bifunctional molecule may be any of the bifunctional molecules described in the bifunctional molecule section hereinabove or in the Experimental section below, which descriptions are incorporated but not reiterated herein for purposes of brevity.

[0057] In certain aspects, a composition of the present disclosure includes the bifunctional molecule present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI2, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions. Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure includes a bifunctional molecule of the present disclosure, and a pharmaceutically acceptable carrier.

[0058] The bifunctional molecule can be incorporated into a variety of formulations for therapeutic administration. More particularly, the bifunctional molecules can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

[0059] Formulations of the bifunctional molecules for administration to a subject (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0060] In pharmaceutical dosage forms, the bifunctional molecules can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0061] For oral preparations, the bifunctional molecules can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0062] The bifunctional molecules can be formulated for oral, parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, intrathecal, subarachnoid, subcutaneous, intranasal, etc.) administration. In some instances, the bifunctional molecules is formulated for administration to the brain of a subject (e.g., a subject having or at risk of developing Alzheimer’s Disease), e.g., via intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, subarachnoid, or intranasal administration.

[0063] According to some embodiments, a composition of the present disclosure comprises an agent that facilitates crossing of the blood-brain barrier by the bifunctional molecule, e.g., upon systemic (e.g., intravenous) administration to the subject, thereby facilitating contacting of A|3 in the brain of the subject with the bifunctional molecule for targeted deamidation thereof in the brain. In some instances, the bifunctional molecules are formulated for injection by dissolving, suspending or emulsifying the bifunctional molecules in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0064] Pharmaceutical compositions that include the bifunctional molecules may be prepared by mixing the bifunctional molecules having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0065] The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

[0066] An aqueous formulation of the bifunctional molecules may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0067] A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0068] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0069] A lyoprotectant may also be added in order to protect the bifunctional molecules against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

[0070] In some embodiments, the pharmaceutical composition includes the bifunctional molecule, and one or more of the above-identified components (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% weight / volume (w / v).

[0071] METHODS OF USE

[0072] Aspects of the present disclosure further include methods of using the bifunctional molecules of the present disclosure. For example, provided are methods of deamidating Ap (e.g., Ap42) in a subject in need thereof, the method comprising administering to the subject a composition of the present disclosure in an amount effective to deamidate Ap in the subject. Also by way of example, provided are methods of reducing or preventing Ap (e.g., Ap42) neurotoxicity in a subject in need thereof, the method comprising administering to the subject a composition of the present disclosure in an amount effective to reduce or prevent Ap neurotoxicity in the subject. According to any of the methods, in certain embodiments, the subject has Alzheimer's Disease (AD) or is at risk of developing AD.

[0073] The bifunctional molecules of the present disclosure may be administered via any suitable route of administration. In some instances, the bifunctional molecule is administered parenterally, e.g., by intravenous, intra-arterial, intraosseous, intramuscular, intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, intrathecal, subarachnoid, subcutaneous, intranasal, etc.) administration. In some instances, the bifunctional molecule is formulated for administration to the brain of the subject (e.g., a subject having or at risk of developing Alzheimer’s Disease), e.g., via intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, subarachnoid, or intranasal administration.

[0074] According to some embodiments, the composition administered to the subject comprises an agent that facilitates crossing of the blood-brain barrier by the bifunctional molecule, e.g., upon systemic (e.g., intravenous) administration to the subject, thereby facilitating contacting of Ap in the brain of the subject with the bifunctional molecule for targeted deamidation thereof in the brain.

[0075] The bifunctional molecules of the present disclosure may be administered (e.g., in a pharmaceutical composition) in a therapeutically effective amount. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of Alzheimer’s Disease, as compared to a control. An effective amount can be administered in one or more administrations.

[0076] According to some embodiments, provided are methods of treating Alzheimer’s Disease in a subject in need thereof. Such methods comprise administering to the subject a composition of the present disclosure in an amount effective to treat the Alzheimer’s Disease. By treatment is meant at least an amelioration of one or more symptoms associated with Alzheimer’s Disease, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with Alzheimer’s Disease. As such, treatment also includes situations where the Alzheimer’s Disease, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from Alzheimer’s Disease, or at least the symptoms that characterize the Alzheimer’s Disease.

[0077] A bifunctional molecule of the present disclosure may be administered to the subject alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use in treating Alzheimer’s Disease.

[0078] When a bifunctional molecule of the present disclosure is administered with a second agent, the bifunctional molecule and the second agent may be administered to the subject according to any suitable administration regimen. According to certain embodiments, the bifunctional molecule and the second agent are administered according to a dosing regimen approved for individual use. In some embodiments, the administration of the bifunctional molecule permits the second agent to be administered according to a dosing regimen that involves one or more lower and / or less frequent doses, and / or a reduced number of cycles as compared with that utilized when the second agent is administered without administration of the bifunctional molecule. In certain aspects, the administration of the second agent permits the bifunctional molecule to be administered according to a dosing regimen that involves one or more lower and / or less frequent doses, and / or a reduced number of cycles as compared with that utilized when the bifunctional molecule is administered without administration of the second agent.

[0079] In some embodiments, one or more doses of the bifunctional molecule and the second agent are administered concurrently to the subject. By “concurrently” is meant the bifunctional molecule and the second agent are either present in the same pharmaceutical composition, or the bifunctional molecule and the second agent are administered as separate pharmaceutical compositions within 1 hour or less, 30 minutes or less, or 15 minutes or less.

[0080] In some embodiments, one or more doses of the bifunctional molecule and the second agent are administered sequentially to the subject.

[0081] In some embodiments, the bifunctional molecule and the second agent are administered to the subject in different compositions and / or at different times. For example, the bifunctional molecule may be administered prior to administration of the second agent, e.g., in a particular cycle. Alternatively, the second agent may be administered prior to administration of the bifunctional molecule, e.g., in a particular cycle. The second agent to be administered may be administered a period of time that starts at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more after the administration of the first agent to be administered. In one example, the second agent is administered to the subject for a desirable period of time prior to administration of the bifunctional molecule.

[0082] In some embodiments, administration of one agent is specifically timed relative to administration of the other agent. For example, in some embodiments, the bifunctional molecule is administered so that a particular effect is observed (or expected to be observed, for example based on population studies showing a correlation between a given dosing regimen and the particular effect of interest).

[0083] In certain aspects, desired relative dosing regimens for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in vivo and / or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., so that a correlation is established), or alternatively in a particular subject of interest.

[0084] In some embodiments, the bifunctional molecule and the second agent are administered according to an intermittent dosing regimen including at least two cycles. Where two or more agents are administered in combination, and each by such an intermittent, cycling, regimen, individual doses of different agents may be interdigitated with one another. In certain aspects, one or more doses of a second agent is administered a period of time after a dose of the first agent. In some embodiments, each dose of the second agent is administered a period of time after a dose of the first agent. In certain aspects, each dose of the first agent is followed after a period of time by a dose of the second agent. In some embodiments, two or more doses of the first agent are administered between at least one pair of doses of the second agent; in certain aspects, two or more doses of the second agent are administered between at least one pair of doses of the first agent. In some embodiments, different doses of the same agent are separated by a common interval of time; in some embodiments, the interval of time between different doses of the same agent varies. In certain aspects, different doses of the bifunctional molecule and the second agent are separated from one another by a common interval of time; in some embodiments, different doses of the different agents are separated from one another by different intervals of time.

[0085] One exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the bifunctional molecule is administered to the subject; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the second agent is administered to the subject; and (d) a second resting period. A second exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the second agent is administered to the subject; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the bifunctional molecule is administered to the subject; and (d) a second resting period.

[0086] In some embodiments, the first resting period and second resting period may correspond to an identical number of hours or days. Alternatively, in some embodiments, the first resting period and second resting period are different, with either the first resting period being longer than the second one or, vice versa. In some embodiments, each of the resting periods corresponds to 120 hours, 96 hours, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour, or less. In some embodiments, if the second resting period is longer than the first resting period, it can be defined as a number of days or weeks rather than hours (for instance 1 day, 3 days, 5 days, 1 week, 2, weeks, 4 weeks or more).

[0087] If the first resting period’s length is determined by existence or development of a particular biological or therapeutic event, then the second resting period’s length may be determined on the basis of different factors, separately or in combination. Exemplary such factors may include type and / or stage of Alzheimer’s Disease against which the therapy is administered; properties (e.g. , pharmacokinetic properties) of the bifunctional molecule, and / or one or more features of the patient’s response to therapy with the bifunctional molecule. In some embodiments, length of one or both resting periods may be adjusted in light of pharmacokinetic properties (e.g., as assessed via plasma concentration levels) of one or the other of the administered agents. For example, a relevant resting period might be deemed to be completed when plasma concentration of the relevant agent is below a pre-determined level, optionally upon evaluation or other consideration of one or more features of the subject’s response.

[0088] In certain aspects, the number of cycles for which a particular agent is administered may be determined empirically. Also, in some embodiments, the precise regimen followed (e.g., number of doses, spacing of doses (e.g., relative to each other or to another event such as administration of another therapy), amount of doses, etc.) may be different for one or more cycles as compared with one or more other cycles.

[0089] The bifunctional molecule and the second agent may be administered together or independently via any suitable route of administration. The bifunctional molecule and the second agent may be administered via a route of administration independently selected from oral, intravenous, intra-arterial, intraosseous, intramuscular, intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, intrathecal, subarachnoid, subcutaneous, intranasal, etc.) administration either concurrently (in the same pharmaceutical composition or separate pharmaceutical compositions) or sequentially.

[0090] KITS

[0091] Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in practicing the methods of the present disclosure, e.g., methods of treating Alzheimer’s Disease in a subject in need thereof.

[0092] Accordingly, in certain embodiments, a kit of the present disclosure comprises any of the bifunctional molecules of the present disclosure (e.g., present in a pharmaceutical composition), and instructions for administering the bifunctional molecule to a subject in need thereof. As will be appreciated, the kits of the present disclosure may include any of the bifunctional molecules having any of the features (e.g., targeting moieties, deamidation agents, deamidation agent binding moieties, etc.) described above in the section relating to the bifunctional molecules of the present disclosure, which are not reiterated herein for purposes of brevity.

[0093] The kits of the present disclosure may include a quantity of the bifunctional molecule, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of a bifunctional molecule of the present disclosure. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the bifunctional molecule calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the bifunctional molecule employed, the effect to be achieved, and the pharmacodynamics associated with the bifunctional molecule, in the subject. In yet other embodiments, the kits may include a single multi dosage amount of the bifunctional molecule.

[0094] The instructions (e.g., instructions for use (I FU)) included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0095] NON-HUMAN ANIMALS

[0096] Aspects of the present disclosure further include non-human animals. In certain embodiments, provided are non-human animals that express a mutant Ap polypeptide (e.g., Ap42) comprising an N27D amino acid substitution, a Q15E amino acid substitution, or both.

[0097] In some instances, the non-human animal is a rodent. For example, the non-human animal may be a mouse, e.g. a mouse of a C57BL strain (e.g. C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 1 OScSn, C57BL / 10Cr, C57BL / Ola, etc.); a mouse of the 129 strain (e.g. 129P1 , 129P2, 129P3, 129X1 , 129S1 (e.g., 129S1 / SV, 129S1 / Svlm),129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1 , 129T2); a mouse of the BALB strain; e.g., BALB / c; and the like. See, e.g., Festing et al. (1999) Mammalian Genome 10:836, see also, Auerbach et al (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines).

[0098] According to some embodiments, the non-human animal comprises an amyloid precursor protein (APP) knock-in allele encoding a mutant APP which is processed to generate the mutant A 42 polypeptide. In some instances, the non-human animal may comprise a knock-out of the endogenous APP gene and comprise a transgene that encodes a mutant APP which is processed to generate the mutant Ap42 polypeptide. In further embodiments, the non-human animal may comprise a knock-out of the endogenous APP gene and comprises a transgene that encodes the mutant Ap42 polypeptide. For a detailed description of methods known in the art useful for generating knock-in and knock-out alleles, see Nagy et al., (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821 ), U.S. Pat. No. 7,576,259, U.S. Pat. No. 7,659,442, U.S. Pat. No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0099] In some instances, the endogenous APP gene of the non-human animal may be edited to encode a mutant APP which is processed to generate the mutant Ap42 polypeptide. Any convenient and appropriate genetic modification system may be employed to introduce one or more of the genetic modifications described herein. Methods of site-directed introduction of a desired genetic modification will vary and may include introducing one or more site directed cleavage events, e.g., through the use of one or more site-directed nucleases (e.g., a CRISPR / Cas9 nuclease, a TALEN nuclease, a ZFN, and the like). Site-directed cleavage may include double and / or single strand breaks where applicable. In some instances, site-directed cleavage is followed by a specific repair event at the site cleaved by the site-directed nuclease, e.g., to introduce a desired edit, such as e.g., a substitution, insertion, deletion, or the like. Such methods of specific repair may include, e.g., homologous recombination, including homology directed repair (HDR), e.g., in the presence of a nucleic acid that includes homology regions to guide the repair. In some instances, site-directed cleavage may be employed to introduce a gene disruption and / or knock-out, e.g., without employing a specific repair event, e.g., through cellular processes following site-directed cleavage such as e.g., non-homologous end joining (NHEJ). In some instances, site-directed introduction of a desired genetic modification may employ a base editing system that does not introduce a double strand cleavage event, such as but not limited to e.g., CRISPR protein-guided based editing systems, such as e.g., dCas9-deaminase fusion protein systems including cytosine base editor (CBE) and adenine base editor (ABE) systems. In some instances, useful base editing systems introduce a single base change, e.g., without cleavage of the phosphodiester nucleic acid backbone.

[0100] Various genetic modification compositions may be employed and such compositions will vary, e.g., based on the genetic modification system employed, the type of genetic modification desired, the sequence of a targeted locus or loci, etc. Useful genetic modification compositions may include e.g., CRISPR / Cas9 editing compositions, e.g., including a Cas9 protein, or a nucleic acid encoding a Cas9 protein, and gRNAs or a sgRNA or a nucleic acid encoding the gRNAs or sgRNA; TALEN editing compositions, including e.g., a TALEN nuclease or TALEN nuclease pair, or a nucleic acid encoding a TALEN nuclease or TALEN nuclease pair; ZFN editing compositions, including e.g., a ZFN nuclease or ZFN nuclease pair, or a nucleic acid encoding a ZFN nuclease or ZFN nuclease pair; base-editing editing compositions e.g., including a CRISPR-protein- guided-base-editing protein, or a nucleic acid encoding a CRISPR-protein-guided-base-editing protein, and gRNAs or a sgRNA or a nucleic acid encoding the gRNAs or sgRNA; and the like.

[0101] According to some embodiments, useful genetic modification (sometimes referred to herein as “editing compositions”) will include a CRISPR-Cas protein, such as e.g., a Cas9 protein, or a polynucleotide encoding a CRISPR-Cas protein and guide RNA (gRNA) or a polynucleotide encoding gRNA. As used herein, the term “gRNA” generally encompasses either two-component guide systems (e.g., two gRNAs) as well as single guide RNA (sgRNA) systems, unless inappropriate and / or denoted otherwise. In some instances, the gRNA or multiple gRNAs may be configured and employed to target a desired locus as described herein or one or more elements thereof such as one of more exons of a gene present at the locus. For example, in some instances, a gRNA or multiple gRNAs may be configured and employed to target a locus or one or more elements thereof, such as e.g., one or more exons of the locus. In certain embodiments, the genetic modification may include the use of a Cas9 nuclease, including natural and engineered Cas9 nucleases, as well as nucleic acid sequences encoding the same. Useful Cas9 nucleases include but are not limited to e.g., Streptococcus pyogenes Cas9 and variants thereof, Staphylococcus aureus Cas9 and variants thereof, Actinomyces naeslundii Cas9 and variants thereof, Cas9 nucleases also include those discussed in PCT Publications Nos. WO 2013 / 176772 and W02015 / 103153 and those reviewed in e.g., Makarova et al. (201 1 ) Nature Reviews Microbiology 9:467-477, Makarova et al. (2011 ) Biology Direct 6:38, Haft et al. (2005) PLOS Computational Biology 1 :e60 and Chylinski et al. (2013) RNA Biology 10:726-737, the disclosures of which are incorporated herein by reference in their entirety. In some instances, a non-Cas9 CRISPR nuclease (or engineered variant thereof) may be employed, including but not limited to e.g., Cpf1 or Cpf 1 variant.

[0102] The CRISPR system offers significant versatility in gene editing in part because of the small size and high frequency of necessary sequence targeting elements within host genomes. CRISPR guided Cas9 nuclease requires the presence of a protospacer adjacent motif (PAM), the sequence of which depends on the bacteria species from which the Cas9 was derived (e.g. for Streptococcus pyogenes the PAM sequence is "NGG") but such sequences are common throughout various target nucleic acids. The PAM sequence directly downstream of the target sequence is not part of the guide RNA but is obligatory for cutting the DNA strand. Synthetic Cas9 nucleases have been generated with novel PAM recognition, further increasing the versatility of targeting, and may be used in the methods described herein. Cas9 nickases (e.g., Cas9 (D10A) and the like) that cleave only one strand of target nucleic acid as well as endonuclease deficient (i.e., “dead”) dCas9 variants with additional enzymatic activities added by an attached fusion protein have also been developed.

[0103] In certain embodiments, a method of genetic modification may include the use of a zinc- finger nuclease (ZFN). ZFNs consist of the sequence-independent Fokl nuclease domain fused to zinc finger proteins (ZFPs). ZFPs can be altered to change their sequence specificity. Cleavage of targeted dsDNA involves binding of two ZFNs (designated left and right) to adjacent half-sites on opposite strands with correct orientation and spacing, thus forming a Fokl dimer. Dimerization increases ZFN specificity significantly. Three or four finger ZFPs target about 9 or 12 bases per ZFN, or about 18 or 24 bases for the ZFN pair. The specificity, efficiency and versatility of targeting and replacement of homologous recombination is greatly improved through the combined use of various homology-directed repair strategies and ZFNs (see e.g., Urnov et al. (2005) Nature. 435(7042):646-5; Beumer et al (2006) Genetics. 172(4) :2391 -2403; Meng et al (2008) Nat Biotechnol. 26(6):695-701 ; Perez et al. (2008) Nat Biotechnol. 26(7):808-816; Hockemeyer et al. (2009) Nat Biotechnol. 27(9):851 -7; the disclosures of which are incorporated herein by reference in their entirety). In general, one ZFN site can be found every 125-500 bp of a random genomic sequence, depending on the assembly method. Methods for identifying appropriate ZFN targeting sites include computer-mediated methods e.g., as described in e.g., Cradick et al. (2011 ) BMC Bioinformatics. 12:152, the disclosure of which is incorporated herein by reference in its entirety.

[0104] According to some embodiments, a method of genetic modification may include the use of a transcription activator-like effector nuclease (TALEN). Similar in principle to the ZFN nucleases, TALENs utilize the sequence-independent Fokl nuclease domain fused to Transcription activator-like effectors (TALEs) proteins that, unlike ZNF, individually recognize single nucleotides. TALEs generally contain a characteristic central domain of DNA-binding tandem repeats, a nuclear localization signal, and a C-terminal transcriptional activation domain. A typical repeat is 33-35 amino acids in length and contains two hypervariable amino acid residues at positions 12 and 13, known as the "repeat variable di-residue" (RVD). An RVD is able to recognize one specific DNA base pair and sequential repeats match consecutive DNA sequences. Target DNA specificity is based on the simple code of the RVDs, which thus enables prediction of target DNA sequences. Native TALEs or engineered / modified TALEs may be used in TALENs, depending on the desired targeting. TALENs can be designed for almost any sequence stretch. Merely the presence of a thymine at each 5' end of the DNA recognition site is required. The specificity, efficiency and versatility of targeting and replacement of homologous recombination is greatly improved through the combined use of various homology-directed repair strategies and TALENs (see e.g., Zu et al. (2013) Nature Methods. 10:329-331 ; Cui et al. (2015) Scientific Reports 5:10482; Liu et al. (2012) J. Genet. Genomics. 39:209-215, Bedell et al. (2012) Nature. 491 :1 14-118, Wang et al. (2013) Nat. Biotechnol. 31 :530-532; Ding et al. (2013) Cell Stem Cell. 12:238-251 ; Wefers et al. (2013) Proc. Natl. Acad. Sci. U.S.A, 1 10:3782-3787; the disclosures of which are incorporated herein by reference in their entirety).

[0105] In certain embodiments, a method of genetic modification may include the use of a base editor system, including but not limited to e.g., base editor systems employing a fusion protein comprising a programable DNA binding protein, a nucleobase editor and gRNA, and the like. Base editing will generally not rely on HDR and / or NHEJ and will generally not result in or require the cleavage of phosphodiester bonds on both backbones of dsDNA. Thus, based editing may, in some instances, employ RNA-guided (i.e., “programable”) DNA binding proteins, such as Cas nucleases, that do not cause double-strand breaks, such as e.g., nuclease-deficient or nucleasedefective Cas proteins, such as e.g., a dCas9 or a Cas9 nickase. Useful examples of base editors and base editing systems, including base editor encoding nucleic acids, include but are not limited to BE1 , BE2, BE3 (Komor et al., 2016); Target-AID (Nishida et al., 2016); SaBE3, BE3 PAM variants, BE3 editing window variants (Kim et al., 2017); HF-BE3 (Rees et al., 2017); BE4 and BE4-Gam; AID, CDA1 and APOBEC3G BE3 variants (Komor et al., 2017); BE4max, ArcBe4max, ABEmax (Koblan et al., 2018); Adenine base editors (ABE7.10) (Gaudelli et al., 2017); ABE8 (Richter et al., 2020); ABE8e (Gaudelli et al., 2020); A&C-BEmax (Zhang et al., 2020); SPACE (Grunewald et al., 2020); and the like; the preceding references being incorporated by reference herein in their entirety. Also provided by the present disclosure are methods comprising comparing one or more parameters of one or more non-human animals of the present disclosure to those of a control non-human animal that expresses wild-type A 42 polypeptide. Non-limiting examples of such parameters include A 42 deposition, A 42 toxicity, or both.

[0106] For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

[0107] 1 . A bifunctional molecule comprising: an Ap targeting moiety stably associated with (1 ) a deamidation agent or (2) a deamidation agent-binding moiety.

[0108] 2. The bifunctional molecule of clause 1 , wherein the Ap targeting moiety is an Ap42 targeting moiety.

[0109] 3. The bifunctional molecule of clause 2, wherein the Ap targeting moiety binds to the KLVFFAE region of Ap42.

[0110] 4. The bifunctional molecule of clause 3, wherein the Ap targeting moiety is a peptide comprising the sequence KLVFFAE, wherein the KLVFFAE amino acids are D-amino acids.

[0111] 5. The bifunctional molecule of any one of clauses 1-4, wherein the deamidation agent is a deamidase.

[0112] 6. The bifunctional molecule of clause 5, wherein the deamidase is a glutaminase or an asparaginase.

[0113] 7. The bifunctional molecule of any one of clauses 1-6, wherein the Ap targeting moiety is conjugated to the deamidation agent or deamidation agent-binding moiety.

[0114] 8. A composition comprising the bifunctional molecule of any one of clauses 1 -7.

[0115] 9. The composition of clause 8, wherein the composition is formulated for administration to a subject in need thereof.

[0116] 10. The composition of clause 9, wherein the composition is formulated for parenteral administration to a subject.

[0117] 11 . The composition of clause 10, wherein the composition is formulated for intravenous administration.

[0118] 12. The composition of clause 11 , wherein the composition comprises an agent that facilitates crossing of the blood-brain barrier by the bifunctional molecule.

[0119] 13. The composition of clause 10, wherein the composition is formulated for intranasal administration. 14. The composition of clause 10, wherein the composition is formulated for intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, and / or subarachnoid administration to the subject.

[0120] 15. A method of deamidating Ap in a subject in need thereof, the method comprising administering to the subject the composition of any one of clauses 9-14 in an amount effective to deamidate Ap in the subject.

[0121] 16. A method of reducing or preventing Ap neurotoxicity in a subject in need thereof, the method comprising administering to the subject the composition of any one of clauses 9-14 in an amount effective to reduce or prevent Ap neurotoxicity in the subject.

[0122] 17. The method of clause 15 or 16, wherein the subject has Alzheimer's Disease (AD) or is at risk of developing AD.

[0123] 18. A non-human animal that expresses a mutant Ap42 polypeptide comprising an N27D amino acid substitution, a Q15E amino acid substitution, or both.

[0124] 19. The non-human animal of clause 18, wherein the non-human animal is a rodent.

[0125] 20. The non-human animal of clause 19, wherein the rodent is a mouse.

[0126] 21 . The non-human animal of any one of clauses 18-20, wherein the non-human animal comprises an amyloid precursor protein (APP) knock-in allele encoding a mutant APP which is processed to generate the mutant Ap42 polypeptide.

[0127] 22. The non-human animal of any one of clauses 18-20, wherein the endogenous APP gene of the non-human animal is edited to encode a mutant APP which is processed to generate the mutant Ap42 polypeptide.

[0128] 23. The non-human animal of any one of clauses 18-20, wherein the non-human animal comprises a knock-out of the endogenous APP gene and comprises a transgene that encodes a mutant APP which is processed to generate the mutant Ap42 polypeptide.

[0129] 24. The non-human animal of any one of clauses 18-20, wherein the non-human animal comprises a knock-out of the endogenous APP gene and comprises a transgene that encodes the mutant A 42 polypeptide.

[0130] 25. The non-human animal of clause 24, wherein the expression of the transgene is under the control of APP gene regulatory elements.

[0131] 26. The non-human animal of clause 25, wherein the expression of the transgene is under the control of the non-human animal APP gene regulatory elements.

[0132] 27. A method comprising comparing one or more parameters of the non-human animal of any one of clauses 18-26 to those of a control non-human animal that expresses wild-type A 42 polypeptide. 28. The method of clause 27, wherein the one or more parameters comprise A 42 deposition, Ap42 toxicity, or both.

[0133] The following examples are offered by way of illustration and not by way of limitation.

[0134] EXPERIMENTAL

[0135] Example 1 - Deamidation Reduces AB42 Neurotoxicity

[0136] The present study was inspired by the existence of three distinct familial mutations within the Ap framework that increase the peptide’s sidechain amidation content and cause Alzheimer’s Disease (AD). Those mutations are K16N, E22Q and D23N. Based on this, it was hypothesized that decreasing the sidechain amidation content within wildtype Ap may lower its neurotoxicity.

[0137] Cell culture experiments were performed in which neurotoxicity of Ap42 and AP42-N27D against SH-SY5Y cells was assessed at 20 pM and 50 pM. The results are shown in FIG. 1. As shown, deamidating Ap42 at site N27 indeed produces a peptide with reduced neurotoxicity (i.e., AP42-N27D).

[0138] Example 2 - Investigation of an AP42 Targeting Moiety

[0139] Described herein is the development of moieties capable of targeting Ap42. In this particular example, mirror-image peptide recognition moieties were investigated. Obtained was a crystal structure in which the KLVFFAE peptide is bound to its mirror-image counterpart. The structure is shown in FIG. 2.

[0140] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:1 . A bifunctional molecule comprising: an Ap targeting moiety stably associated with (1 ) a deamidation agent or (2) a deamidation agent-binding moiety.

2. The bifunctional molecule of claim 1 , wherein the A targeting moiety is an Ap42 targeting moiety.

3. The bifunctional molecule of claim 2, wherein the Ap targeting moiety binds to the KLVFFAE region of Ap42.

4. The bifunctional molecule of claim 3, wherein the Ap targeting moiety is a peptide comprising the sequence KLVFFAE, wherein the KLVFFAE amino acids are D-amino acids.

5. The bifunctional molecule of claim 1 , wherein the deamidation agent is a deamidase.

6. The bifunctional molecule of claim 5, wherein the deamidase is a glutaminase or an asparaginase.

7. The bifunctional molecule of claim 1 , wherein the Ap targeting moiety is conjugated to the deamidation agent or deamidation agent-binding moiety.

8. A composition comprising the bifunctional molecule of claim 1 .

9. The composition of claim 8, wherein the composition is formulated for administration to a subject in need thereof.

10. The composition of claim 9, wherein the composition is formulated for intracerebroventricular (ICV), intracerebral, intraventricular, intraparenchymal, and / or subarachnoid administration to the subject.

11. A method of deamidating Ap in a subject in need thereof, the method comprising administering to the subject the composition of claim 9 in an amount effective to deamidate Ap in the subject.

12. A method of reducing or preventing A0 neurotoxicity in a subject in need thereof, the method comprising administering to the subject the composition of claim 9 in an amount effective to reduce or prevent Ap neurotoxicity in the subject.

13. The method of claim 11 , wherein the subject has Alzheimer's Disease (AD) or is at risk of developing AD.

14. A non-human animal that expresses a mutant AP42 polypeptide comprising an N27D amino acid substitution, a Q15E amino acid substitution, or both.

15. The non-human animal of claim 14, wherein the non-human animal is a rodent.

16. The non-human animal of claim 14, wherein: the non-human animal comprises an amyloid precursor protein (APP) knock-in allele encoding a mutant APP which is processed to generate the mutant Ap42 polypeptide; the endogenous APP gene of the non-human animal is edited to encode a mutant APP which is processed to generate the mutant Ap42 polypeptide; and / or the non-human animal comprises a knock-out of the endogenous APP gene and comprises a transgene that encodes a mutant APP which is processed to generate the mutant A042 polypeptide.

17. The non-human animal of claim 14, wherein the non-human animal comprises a knockout of the endogenous APP gene and comprises a transgene that encodes the mutant A 42 polypeptide.

18. The non-human animal of claim 17, wherein the expression of the transgene is under the control of APP gene regulatory elements.

19. The non-human animal of claim 18, wherein the expression of the transgene is under the control of the non-human animal APP gene regulatory elements.

20. A method comprising comparing one or more parameters of the non-human animal of claim 14 to those of a control non-human animal that expresses wild-type Ap42 polypeptide.

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