WO2025199495A1 - Compositions and methods for treatment and prevention of alzheimer's disease - Google Patents

Abstract

The present invention provides methods and compositions for reducing internalization and/or trafficking of tan in neuronal cells comprising contacting the cells with an effective amount of VLDL receptor antagonist. The invention further provides a method of treating or preventing Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of a VLDL receptor antagonist.

Description

COMPOSITIONS AND METHODS FOR TREATMENT AND PREVENTION OF

ALZHEIMER’S DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional U.S. Appl. No.: 63/568,862, filed March 22, 2024, the contents of which are incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under Grant Numbers HL135743 and AG073236 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer- readable sequence listing submitted concurrently herewith and identified as follows: One 4,944 Byte XML file named “sequence_listing.xml,” created on March 21, 2025.

FIELD OF THE INVENTION

The field of the invention relates to medicine, in particular therapeutics for the treatment and prevention of Alzheimer’s disease.

BACKGROUND OF THE INVENTION

Alzheimer’s Disease (AD) is a leading cause of dementia and a major public health crisis impacting millions of people worldwide, and its etiology remains poorly understood. AD is a neurodegenerative disorder characterized by the accumulation of extracellular plaques and intracellular neurofibrillary tangles, which are comprised primarily of aggregated amyloid P and tau proteins, respectively. Tau pathology spreads across the brain as AD progresses, and this process is mediated by cell-to-cell transfer of seed competent tau. A potential mechanism responsible for this is the release of seed-competent tau into the extracellular space and subsequent internalization of pathogenic tau via receptor- mediated endocytosis. The internalized pathogenic tau ultimately reaches the cytoplasm to seed aggregation of endogenous tau. Multiple genetic mutations have been identified as risk factors for AD, including a) inheritance of the c4 allele of apolipoprotein E (ApoE4), which is the strongest genetic risk factor for AD, and b) several single nucleotide polymorphisms in the SORL1 gene, which encodes the sortilin-related receptor 1 and shares structural homology with LDL receptor family members. Several AD-associated proteins bind and are regulated by members of the LDL-receptor family, including LRP1, VLDLr, and ApoER2. Furthermore, LRP1 was recently identified as major endocytic receptor for tau and regulates tau internalization, degradation, and seeding in a manner that is modified differentially by ApoE isoforms.

The VLDL receptor is a member of the low density lipoprotein receptor family. It functions as a peripheral lipoprotein receptor involved in the delivery of triglyceride- rich lipoproteins to peripheral tissue (Takahashi S, et al., Proc Natl Acad Sci USA 1992; 89: 9252-9256; Sakai J, et al., J Biol Chem 1994; 269: 2173-2182) and also plays an important role in reelin signaling (Trommsdorff M, et al., Cell 1999; 97: 689-701; Herz J, Chen Y., Nat Rev Neurosci 2006; 7: 850-859), angiogenesis and tumor growth (Hembrough TA, et al., Blood 2004; 103: 3374-3380), and fibrin-dependent inflammation (Yakovlev S, et al., Blood 2012; 119: 637-644). VLDLR consists of one polypeptide chain that forms the extracellular portion, the transmembrane domain, and the cytoplasmic domain (Takahashi S, et al., J Atheroscler Thromb 2004; 11: 200-208; Lillis AP, et al., Physiol Rev 2008; 88: 887-918). The extracellular' portion, which includes 8 complement-type repeats (CR-domains), and EGF-like, P-propeller, and the O-linked sugar domains, has been expressed in the insect expression system (Ruiz J, et al., J Lipid Res 2005; 46: 1721-1731). The ligand-binding region of VLDLR including all 8 CR-domains has been expressed in the bacterial expression system and used in functional studies, as well as an antigen for preparation of anti- VLDLR monoclonal antibodies (Ruiz J, etal., J Lipid Res 2005; 46: 1721-1731). Three such antibodies, 1H10, 1H5, and 5F3, have been prepared and partially characterized (Ruiz J, et al., J Lipid Res 2005; 46: 1721-1731; Yakovlev S, et al., Thromb Haemost 2016; 116: 1122-1130).

The very low density receptor (VLDLr) was discovered as a receptor for apoE containing lipoproteins (1), and is highly conserved between different species (e.g., mouse VLDLr is 96% identical to the amino acid sequence of human VLDLr). The cDNA for the VLDLr can be alternatively spliced such that 4 variants in mammals are detected; one variant lacks LDL ligand binding repeat 3; another lacks the O-linked sugar domain; a third variant lacks both LDL ligand binding repeat 3 along with the O-linkcd sugar domain (for review see (2)). In 1999, the Herz laboratory prepared double knockout mice that included the VLDLr and a related receptor, ApoE Receptor 2 (also known as LRP8) (3). These mice displayed a “reeler” phenotype which led to the discovery that these two receptors are critical for reelin signaling. This signaling pathway plays a critical role in the development of laminated structures of the brain and in synaptic plasticity of the adult brain (3).

Reelin signaling has also been discovered to be relevant for Alzheimer’s disease. Upon binding of reelin multimers to the VLDLr, the phosphorylation of Disabled 1 (Dabl) occurs which has a number of important consequences (for review see (4)) that ultimately leads to phosphorylation of glycogen synthase kinase 3P (GSK3P) at its inhibitory serine- 9 site. This reduces phosphorylation of tau. Hyperphosphorylation of tau results in tau aggregation and tangle formation in neurons, which is thought to drive progression of Alzheimer’s disease.

What is needed are new compositions and methods that are useful to treat and prevent AD. The foregoing description of the background is provided to aid in understanding the invention, and is not admitted to be or to describe prior art to the invention.

SUMMARY OF THE INVENTION

It is to be understood that both the foregoing general description of the invention and the following detailed description are exemplary, and thus do not restrict the scope of the invention.

In one aspect, the invention provides a method of reducing tau internalization and/or trafficking in neuronal cells comprising contacting the cells with an effective amount of a VLDL receptor antagonist.

In another aspect, the invention provides a method of treating or preventing Alzheimer’s disease in a subject in need thereof, comprising administering to the subject an effective amount of a VLDL receptor antagonist.

In some embodiments, the VLDL receptor antagonist blocks the interaction of tau and VLDL receptor. In some embodiments, the VLDL receptor antagonist inhibits the expression of VLDL receptor.

In some embodiments, the VLDL receptor antagonist is an VLDL receptor antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is selected from the group consisting of 1H5, 1H10, 5F3 and combinations thereof. In some embodiments, the antibody is a humanized antibody and comprises CDR sequences of an antibody selected from the group consisting of 1H5, 1H10, and 5F3.

In some embodiments, the VLDL receptor antagonist is a nucleic acid that inhibits the expression of VLDL receptor. In some embodiments, the nucleic acid is an RNA, a DNA, or a combination thereof. In some embodiments, the nucleic acid is a ribozyme, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA). In some embodiments, the nucleic acid is delivered as a viral vector.

In some embodiments, the VLDLR antagonist is administered to a subject by topical, intravenous, subcutaneous, intramuscular', intracutaneous, transcutaneous, intrathecal, intranasal, intra-arterial, rectal, intragastric, parenteral, or oral administration.

In some embodiments, the method further comprises administering one or more additional active agents. In some embodiments, the one or more additional active agents comprises i) an effective amount of an LRP1 antagonist; ii) an effective amount of a SorLA antagonist; or iii) an effective amount of an LRP1 antagonist and a SorLA antagonist.

In another aspect, the invention provides a method of screening for potential antagonists of VLDLR that reduce internalization and/or trafficking of tau in cells, comprising i) providing a cell expressing VLDLR or a functional equivalent of VLDLR; ii) providing tau protein or a fragment or derivative thereof to the cell, wherein the tau protein or fragment or derivative thereof is extracellular; iii) treating the cell with a potential VLDLR antagonist; and iv) assaying the cellular uptake of tau or the fragment or derivative thereof. In some embodiments, the method further comprises comparing the cellular uptake of tau in the cell with cellular uptake of tau in cells that have not been treated with the potential antagonist.

In some embodiments, the cells are neuronal cells. In some embodiments, the cells are non-neuronal cells. In some embodiments, the cells are from mammals, yeast, Drosophila or E. coli. In some embodiments, the cells express VLDLR endogenously. In some embodiments, the cells have been transfected or engineered to express VLDLR.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1. a) Tau binds VLDLr ligand binding repeats 1-8. Surface plasmon resonance was used to assess the binding of increasing concentrations of 2N4R tau to recombinantly produced VLDLr protein fragment composed of the ligand binding repeats 1-8 (s VLDLr 1- 8). Shown are means ± SD, n = 3 b,c) Binding of 2N4R tau to the soluble ectodomain of VLDLr in the presence of (b) EDTA or (c) lOOnM RAP.

FIG. 2. VLDLr mediates tau internalization. The uptake of 20 nM ¹²⁵I-labeled 2N4R tau was quantified in LRP1 -deficient CHO cells (13-5-1) that stably express VLDLr, and compared to non-expressing 13-5- 1 cells. I p.M receptor associated protein (RAP) was used to inhibit LDL receptor family member function. Shown are means +/- SD, ; one-way ANOVA followed by Tukey’ multiple comparison test, n = 3.

FIG. 3. Monoclonal antibodies block the binding of tau to VLDLr. (a) Schematic representing the binding sites of antibodies 5F3, 1H10, and 1H5 to the ligand binding repeats 1-8 of VLDLr. (b) Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr FL) was immobilized to the surface of a CM5 sensorchip, and then the binding of 20nM tan +/1 100nM anti-VLDLr antibodies (1H10, 1H5, or 5F3) was assessed via SPR. Each experiment was repeated 3 times (except 5F3), shown are representative images.

FIG. 4. Affinities of VLDLr antibodies for sVLDLr. Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr) was immobilized to the surface of a CM5 sensorchip, and then SPR was used to determine the binding affinities of the VLDLr antibodies to sVLDLr. Shown are means +/- SD, n-3.

EIG. 5. Anti-VLDLr antibodies inhibit tau uptake. LRP1 -deficient CHO 13-5-1 cells were incubated with 20nM ¹²⁵Llabeled 2N4R tau in the presence or absence of anti- VLDLr antibodies (1H10, 1H5, or 5E3, 100 nM) or 1 pM RAP. Shown are means +/- SEM, one-way ANOVA followed by Tukey’ multiple comparison test, n = 3.

EIG. 6. Tau binds to VLDLr. (a) Surface plasmon resonance (SPR) analysis of binding of increasing concentrations of 2N4R tau to soluble VLDLr (sVLDLr) (Kd = 190.2) or VLDLr ligand binding repeats 1-8 (Kd = 56.83) coupled to a CM5 sensorchip. Analysis was performed using a nonlinear regression fit using Graphpad Prism’s equation for one site - specific binding. Shown are means ± SEM, n - 3 (b) Binding of 2N4R tau to VLDLr 1-8 in the presence or absence of EDTA. (c) Experiment in (b) was repeated 3 times. Shown are means ± SEM, n = 3.

FIG. 7. ApoE binds VLDLr. Recombinantly produced VLDLr protein containing the ligand binding repeats (VLDLr 1-8) was immobilized to the surface of a CM5 sensorchip, and increasing concentrations of ApoE isoforms were flowed over the flow cells in order to assess binding. Analysis was performed using a nonlinear regression fit using Graphpad Prism’s equation for one site - specific binding. This experiment was performed three times. Shown are means ± SEM.

FIG. 8. ApoE blocks the binding of tau to VLDLr. Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr) was immobilized to the surface of a CM5 sensorchip, and then the binding of 20 nM tau +/- 100 nM (a) apoE2, (b) apoE3, or (c) apoE4 was assessed via SPR. Each experiment was repeated 3 times, shown are representative images. Experiments were repeated at least 3 times, (d) shown are means ± SEM.

FIG. 9. Monomer vs Fibrils experiment. FIG. 10. Binding of pseudo HMW (D20Q3) and pseudo LMW (D8Q2) tau, compared to unmodified 2N4R tau, to LRP1 assessed using the Biacorc 8K surface plasmon resonance (SPR) system. Full-length LRP1 purified from human placenta was immobilized via amine coupling on the surface of a CM5 Biacore sensorchip. Increasing concentrations (3.7, 11.1, 33.3 nM) of recombinant 2N4R, D20Q3, or D8Q2 tau were flowed over the sensorchip in a single cycle kinetic titration experiment at pH7.4. (A) Representative image of single experiment. (B) Nonlinear regression analysis of Rmax vs concentration.

FIG. 11. Effect of pH on tau binding to LRP1 and SORL1. Tau cannot bind LRP1 at endosomal pH (5.5) (A) and (B), but continues to bind to SORL1 with similar affinity as seen at pH 7.4 (C) and (D).

FIG. 12. Reelin blocks the binding of tau to VLDLr. Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr) was immobilized to the surface of a CMS sensorchip, and then the binding of 20 nM tau +/- 100 nM reelin was assessed via SPR. Shown is a representative image.

FIG. 13. ApoE3 vs ApoE3 CH binding LRP1 or VLDLr.

FIG. 14. LRP1 regulates tau uptake, degradation, and seeding. Uptake of tau in LRP1 -deficient CHO cells confirms the existence of additional receptors for tau uptake. A, steady-state levels of 1251-labeled tau (20 nM) internalized in WT or LRP1 -deficient 13- 5-1 CHO cells when incubated in the absence or the presence of 1 uM RAP for 2 h at 37 °C. B and C, time course for internalization of 1251-labeled tau (20 nM) in CHO WT (B) and CHO 13-5-1 (C) cells in the presence or the absence of RAP (1 mM) or heparin (20 mg/ml). D, WT, 13-5-1 and HSPG-deficient (CHO HSPG) CHO cells were incubated with 20 nM 1251-labeled tau in the absence or the presence of RAP (1 mM) or heparin (20 mg/ml) at 37 °C for 2 h, and internalized tau was measured. E, SPR analysis of 10 nM tau binding to LRP1 in the absence or the presence of 20 mg/ml heparin. A-D, means ± SEM; two-way ANOVA followed by Sidak's multiple comparisons test, (A) ***p < 0.0001 compared with WT control, n = 3; (B and C) *p < 0.0001 comparison of tau versus tau + RAP, n = 3; ( ) significance reported compared with *CHO WT, #CHO 13-5-1, or CHO HSPG (one symbol p < 0.03; two symbols p < 0.007; and three symbols p < 0.0001). CHO, Chinese hamster ovary; HSPG, heparan sulfate proteoglycan; LRP1, low-density lipoprotein receptor-related protein 1 ; RAP, receptor-associated protein; SPR, surface plasmon resonance.

FIG. 15. SORL1 provides a mechanism of uptake that supports tau proteopathic seeding in the cytoplasm. Tau binds to SORL1 and SORL1 transfection reconstitutes pathogenic internalization and seeding in HEK293T reporter cells, (a) SPR equilibrium analysis of the binding of increasing concentrations of recombinant 2N4R tau to full length SORL1 (blue circles) and the SORL1 VPS 10 Domain (orange triangles), (b) H4 Cells were incubated with 40 nM tau labeled with Alexaflour594 for 2 h, then fixed and immunostained with anti-SORLl antibody to label endogenous SORL1. (c) CHO cells were transfected with SORL1 plasmid or empty vector (Mock), then incubated with 20 nM 1251-labelled tau ± 1 pM RAP for 2 hours, and the amount of tau internalized by the cells was quantified, (d) HEK293T FRET reporter cells were transfected with SORL1 plasmid, then incubated with HMW SEC fractions from AD patient brain (AD) or healthy control (Ctrl) and tau seeding quantified, (e) siRNA was used to knockdown SORL1 in H4 cells that stably express the FRET reporter system, then cells were incubated with 300ng/well AD brain derived HMW tau seeding material and tau seeding was quantified. (Means ± SEM; 2-way ANOVA). (f) Immunoblots confirming transfection and knockdown.

FIG. 16. Phosphorylated forms of tau bind weakly to LRP1. Binding of tau to LRP1 was assessed by surface plasmon resonance (SPR) experiments. LRP1 was coupled to a CM5 sensor chip and then increasing concentrations of various forms of tau over the chip.

A, inhibition of tau binding to LRP1 by excess RAP as assessed by coinjection experiment.

B, single-cycle kinetic experiment quantifying binding of monomeric tau (3.8, 11.5, 34.4, 103.3, and 310 nM) to LRP1 in the presence of Ca2+ (blue line) or EDTA (black line). C, binding of tau isoforms 2N4R, 2N3R, and tau MBD to LRP1 assessed by SPR equilibrium analysis. D, about 1 pg of recombinant tau produced in Escherichia coli or SF9 cells was ran on a 4 to 12% gel and stained with colloidal Coomassie. Image was captured using Licor. Quantification of bands reveals a signal of 2270 for E. coli tau and 2280 for SF9 tau. E, the binding of tau produced by Sf9 cells along with two mutant forms of tau to full- length human LRP1 was measured by SPR; 6A (T181, S199, S202, S396, S400, and S404 are all converted to alanine) and 6E, in which all these residues are converted to glutamic acid. F, binding of mutant forms of tau to LRP1: 3XKQ in which lysine residues 311, 317, and 321 were converted to glutamine residues and 9XKQ tau in which lysine residues 311 , 217, 321, 340, 343, 347, 353, 369, and 375 arc all converted to glutamine residues. G, binding of monomeric tau to LRP1 clusters II, III, or IV by SPR equilibrium analysis. For all experiments, n = 3 (biological replicates), (A and B) show representative data, (C, E, and F) show means ± SEM. LRP1, low-density lipoprotein receptor-related protein 1; MBD, microtubule-binding domain; RAP, receptor-associated protein; SPR, surface plasmon resonance.

FIG. 17. Less impact of phosphorylation on tau binding to SORLl’s VPS10P domain. Recombinant monomeric tau binds to SORL1 and the VPS 10 domain of SORL1. (a) Equilibrium analysis of the binding of increasing concentrations of recombinant 2N4R tau to full length SORL1 (blue line) and the SORL1 VPS 10 domain (orange line) coupled to a Biacore CM5 sensor chip, (b) Binding of tau isoforms 2N4R, 0N3R, 2N3R and tau MBD to SORL1 VPS 10 domain assessed by SPR equilibrium analysis, (c) The binding of tau produced by Sf9 cells along with two mutant forms of tau to SORL1 VPS 10 domain was measured by SPR; 6A (T181, S199, S202, S396, S400, and S404 are all converted to alanine) and 6E, in which all these residues are converted to glutamic acid, (d) Binding of mutant forms of tau to SORL1 VPS10 domain: 3XKQ in which lysine residues 311, 317, and 321 were converted to glutamine residues and 9XKQ tau in which lysine residues 311, 217, 321, 340, 343, 347, 353, 369, and 375 are all converted to glutamine residues. The data was normalized to Rmax to correct for slight differences in coupling to the SPR surfaces. Each experiment was repeated at least 3 time. Shown are means ± SEM. MBD, microtubule-binding domain; SPR, surface plasmon resonance.

FIG. 18. Impact of pH on tau binding LRP1 and SORL1. SPR analysis of tau binding LRP1 and full-length (FL) SORL1 at pH 7.4 and pH 5.5. LRP1 or SORL1 was immobilized on the surface of a CMS sensorchip and increasing concentrations (11.1, 33.3, 100, 300, 900 nM) of 2N4R tau were flowed over the surface in the presence of either HEPES buffer pH 7.4 or MES buffer pH 5.5.

FIG. 19. LRP1 binds tau isolated from AD patient brains. Using a tau capture assay, we investigated the binding of LRP1 to HMW and LMW tau isolated by SEC from Alzheimer’s disease (AD) patient brains or age-matched control (CT) individuals. We used recombinantly produced 2N4R tau as a positive control. (A&B) Single injections of 300nM LRP1 demonstrate that LRP1 binds to LMW tau from AD and CT brains, but no binding was detected to HMW tau from AD brains. No tau was captured from HMW CT samples, consistent with the low amount of HMW tau present in these samples. (C) Plotted is the amount of LRP1 bound divided by the amount of tau captured.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of therapeutic agents that can block tau binding to VLDLR and suppress or inhibit tau internalization in cells.

Tau is an intracellular microtubule-associated protein that is hyperphosphorylated and forms “tangles” in neurons of Alzheimer’s disease patients. Experiments have shown that aggregated tau can spread from cell to cell and from one region of the brain to other regions. Tau is secreted by neurons and is taken up by receptor-mediated endocytosis. Thus, a significant interest in how tau is taken up by neurons exists. The interest in tau receptors arise from the fact that neuronal transfer of pathological forms of tau has been proposed as a mechanism of Alzheimer’s disease (AD) progression. The present inventors have discovered that the very low density lipoprotein receptor (VLDL receptor) can bind tau and mediate its uptake. The VLDL receptor is one of two reelin receptors that participate in reelin signaling. This signaling is required for development of laminated structures of the brain that occur during development. It is shown herein that two monoclonal antibodies (1H10 and 1H5) that are directed against the VLDL receptor block tau binding. Without being bound by theory, it is believed that these antibodies (alone and/or together) may cluster the VLDL receptor and trigger reelin signaling. Thus, these antibodies may not only block tau transmission, but also reduce tau phosphorylation.

Accordingly, the interaction of tau with VLDL receptor has important implications for the progression of Alzheimer’s disease, and the further identification of agents capable of blocking the interaction between tau and VLDL receptor can also be beneficial for the treatment or prevention of Alzheimer’s disease.

Reference will now be made in detail to embodiments of the invention which, together with the drawings and the following examples, serve to explain the principles of the invention. These embodiments describe the invention in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and/or “consisting of.” As used herein, the term "about" means at most plus or minus 10% of the numerical value of the number with which it is being used.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Current Protocols in Molecular Biology (Ausubel et. al., eds. John Wiley & Sons, N.Y. and supplements thereto), Current Protocols in Immunology (Coligan et al., eds., John Wiley St Sons, N.Y. and supplements thereto), Current Protocols in Pharmacology (Enna ct al., cds. John Wiley & Sons, N.Y. and supplements thereto) and Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilicins, 2Vt edition (2005)), for example.

In one embodiment, the invention provides a method of reducing tau internalization and/or trafficking in neuronal cells comprising contacting the cells with an effective amount of a Very Low Density Lipoprotein Receptor (VLDLR) antagonist.

In another embodiment, the invention provides a method of treating or preventing Alzheimer’s disease in a subject in need thereof, comprising administering to the subject an effective amount of a Very Low Density Lipoprotein Receptor (VLDLR) antagonist.

In some embodiments, the present invention provides a method of reducing the cellular uptake or trafficking of tau and thereby preventing and/or treating Alzheimer’s disease, the method comprising administering to the subject an effective amount of an agent that inhibits the activity of VLDLR.

In another embodiment, the invention provides a method of screening for potential agents that reduce internalization and/or trafficking of tau in cells, comprising i) providing a cell expressing VLDLR or a functional equivalent of VLDLR; ii) providing tau protein or a fragment or derivative thereof to the cell, wherein the tau protein or a fragment or derivative thereof is extracellular; iii) treating the cell with an effective amount of a test agent that may inhibit binding of tau to VLDLR or reduce expression of VLDLR in cells; and iv) assaying the cellular uptake of tau or the fragment or derivative thereof.

Alzheimer's disease is a neurodegenerative disease and the most common cause of dementia. This disease appears as a gradual but progressive decline in memory, thinking ability, and behavior that is accelerated compared to normal aging. There are two major types of this disease. Familial Alzheimer's disease is usually caused by a dominant mutation in one of three genes (APP, PSEN1 or PSEN2). This type of disease is a rare but devastating disease that occurs in middle age. The second and much more prevalent form of this disease is sporadic or late-onset Alzheimer's disease. The onset of Alzheimer's disease usually occurs after age 62.

Symptoms of Alzheimer's disease are mainly characterized by memory impairment, language dysfunction, and cognitive impairment including visual and spatial abilities, which can extend to occupational and social problems (e.g., activities of daily living); and depression. Behavioral symptoms, including anxiety, aggression and psychosis, may manifest as a progression of disease severity.

Currently, a clear diagnosis of Alzheimer's disease requires clinical findings of cognitive impairment consistent with AD and autopsy identification of brain lesions consistent with AD. The term "AD dementia" is used to describe dementia due to the pathophysiology of Alzheimer's disease. The term "probable Alzheimer's disease" refers to when a subject exhibits the clinical features of Alzheimer's disease and other possible biological causes of dementia (e.g., Parkinson's disease or stroke). Used during life if excluded.

Currently, there are a variety of art-accepted methods for diagnosing probable Alzheimer's disease. Usually, these methods are used in combination. These methods include determining an individual's ability to perform daily activities and identifying behavioral and personality changes. Dementia of the AD type is usually also characterized by amnestic symptoms (memory impairment) or impairment of language, visual spatial or executive function. Cognitive/dysfunction includes global cognition (e.g., improved minimental state test (3MS-E)), and visual and verbal memory (e.g., simple visual spatial memory test (revised version) (BVMT-), respectively. R) and Hopkins language learning test (revised version) (HVLT-R)), language (e.g., utterance fluency test (GVFT)) and executive function and attention (e.g., digit span test (DST)). It can be ascertained by art- accepted methods, including but not limited to equipment useful for evaluation. Dementia due to AD is also defined by the history of insidious development and deterioration of cognitive ability.

As used herein, the terms "effective amount" or "therapeutically effective amount" are interchangeable and refer to an amount that results in an improvement or remediation of at least one symptom of the disease or condition. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition. In some embodiments, the term “effective amount” corresponds to an amount administered that reduces the internalization or trafficking of tau in cells, mediated by VLDLR. The “effective amount” can correspond to an amount administered to subjects or to cells directly. As used herein, the term "inhibit" refers to the ability of the compound to block, partially block, interfere, decrease, reduce or deactivate a receptor such as VLDLR. Thus, one of skill in the art understands that the term inhibit encompasses a complete and/or partial loss of activity of the receptor. Receptor activity may be inhibited by blockage of ligand binding sites on the receptor, by interference with the mechanism of expression of the receptor protein, or by other means. For example, a complete and/or partial loss of activity of the receptor may be indicated by a reduction in the extent of tau internalization or trafficking into neurons or other mammalian cells or a reduction in NFTs or senile plaques in the brain of a subject.

As used herein, "treat" and all its forms and tenses (including, for example, treat, treating, treated, and treatment) refer to both therapeutic treatment and prophylactic or preventative treatment. A subject in need of treatment includes those already with a pathological condition of the invention as well as those in which a pathological condition of the invention is to be prevented. In some embodiments, Alzheimer’s disease is treated by delaying the development or progression of the disease.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of activity compared to normal.

The subject to be administered the therapeutic agent is not limiting. In some embodiments, the subject is a mammal including for example, a dog, cat, monkey, goat, pig, chimpanzee, cow, horse, sheep, rabbit, guinea pig, rat, hamster, mouse, and human. In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with Alzheimer’s disease or is at risk of developing the disease. In some embodiments, the subject has or is at risk of developing late onset disease. In some embodiments, the subject has or is at risk of developing early onset disease, or Familial Alzheimer's (which can develop well before the senile period, e.g., between 35 and 60 years of age). See, e.g., Selkoe, TINS 16:403 (1993); Hardy et a , WO 92/13069 ; Selkoe, J. Neuropathol. Exp. Neurol. 53:438 (1994); Duff et al., Nature 373:476 (1995); Games et al., Nature 373:523 (1995). In some embodiments, the subject is at risk of developing the disease, c.g., due to a family history, genetic predisposition, lifestyle, or due to the presence of one or more early markers or symptoms of the disease.

The VLDLR antagonist is not particularly limiting. In some embodiments, the VLDLR antagonist can be a protein, a peptide, a lipid, a carbohydrate, an organic molecule, or an inorganic molecule. Exemplary inhibitors of VLDLR function include, without limitation, soluble VLDLR receptor polypeptides.

In some embodiments, the antagonist inhibits binding of tau to Very Low Density Lipoprotein Receptor (VLDLR). In some embodiments, the antagonist inhibits expression of VLDLR in cells.

In some embodiments, the therapeutically effective amount of an agent that inhibits binding of tau to Very Low Density Lipoprotein Receptor (VLDLR) is an antibody.

The term "antibody" as used herein refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof. The term "antibody" refers to a homogeneous molecular entity, or a mixture such as a polyclonal serum product made up of a plurality of different molecular entities, and broadly encompasses naturally- occurring forms of antibodies (for example, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multispecific antibodies. The term "antibody" also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatized variants thereof that retains the ability to specifically bind an epitope. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. A monoclonal antibody is capable of selectively binding to a target antigen or epitope. Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, single chain antibodies (scEvs), Lab fragments, E(ab').sub.2 fragments, disulfide-linked Evs (sdEv) fragments, for example, as produced by a Lab expression library, anti-idiotypic (anti-Id) antibodies, intrabodies, nanobodies, synthetic antibodies, and epitope-binding fragments of any of the above.

Monoclonal antibodies (mAbs) as used herein also include sequences corresponding to human antibodies, animal antibodies, and combinations thereof. The term “chimeric antibody,” as used herein, includes antibodies that have variable regions derived from an animal antibody, such as a rat or mouse antibody, fused to another molecule, for example, the constant domains derived from a human antibody. One type of chimeric antibodies, “humanized antibodies,” have had the variable regions altered (through mutagenesis or CDR grafting) to match (as much as possible) the known sequence of human variable regions. CDR grafting involves grafting the CDRs from an antibody with desired specificity onto the FRs of a human antibody, thereby replacing much of the nonhuman sequence with human sequence. Humanized antibodies, therefore, more closely match (in amino acid sequence) the sequence of known human antibodies. By humanizing mouse monoclonal antibodies, the severity of the human anti-mouse antibody, or HAMA, response is diminished.

In some embodiments, the antibody can bind at least one complement-type repeat (CR) domain of VLDLR selected from the group consisting of CR-2, CR-3 and CR-4, or any combination thereof. In some embodiments, the antibody can bind to CR domains 3- 6. In some embodiments, the antibody can bind to CR domains 1-2 and 5-6. In some embodiments, the antibody can bind to CR domains 2 and 5-6.

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a mouse monoclonal antibody. In some embodiments, the antibody is 1H10. In some embodiments, the antibody is 1H5. In some embodiments, the antibody is 5F3. In some embodiments, the antibody comprises one or more complementarity determining regions (CDRs) identical to the CDRs of 1H10, 1H5 or 5F3. Antibodies 1H10, 1H5 and 5F3 are available commercially from Molecular Innovations (Novi, MI). See also Ruiz et al. (2005) J. Lipid Res. 46:1721-1731; and Oganesian et al. (2008) Mol. Biol. Cell 19:563-571.

In some embodiments, it may be desirable to “humanize” the antibody in order to attenuate any adverse immune reaction. Humanized antibodies can be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e., chimeric antibodies). See, e.g., Robinson et al., WO/1987/002671; Akira et al., EP Application 184,187; Taniguchi, EP Application 171,496; Morrison etal., EP Application 173,494; Neuberger etal., C) 86/01533; Cabilly et al., EP Application 125,023, all of which are incorporated herein by reference. In some embodiments, the agent that inhibits binding of fibrin to Very Low Density Lipoprotein Receptor (VLDLR) is a humanized antibody. Tn some embodiments, the antibody is a humanized antibody of antibody 1H10, 1H5 or 5F3, that harbors the CDR sequences of antibody 1H10, 1H5, or 5F3. In some embodiments, a combination of antibodies are administered. In some embodiments, the antibody is a fully human antibody.

In some embodiments, the antagonist is an inhibitory nucleic acid that inhibits the expression of VLDLR. The nucleic acid can be an RNA, a DNA, or a combination thereof. For example, the inhibitory nucleic acid that inhibits the expression of VLDLR can be a small interfering RNA (“siRNA”), a short hairpin RNA (“shRNA”), an antisense RNA, or a ribozyme. The siRNA, shRNA, or other inhibitory RNA can be designed with the aid of a computer program specifically prepared therefor. As appropriate, the inhibitory nucleic acid can be delivered in a viral vector, for example, a neurotropic viral vector. In some embodiments, the inhibitor of VLDLR is a siRNA or shRNA that specifically inhibits the expression of VLDLR. In some embodiments, the siRNA or shRNA can be delivered in a lentiviral vector, a herpesvirus vector or an adenoviral vector. In some embodiments, the siRNA or shRNA can be delivered in pharmaceutical compositions comprising particles (e.g., nanoparticles) comprising one or more lipids, such as cationic lipids, such as ionizable amino lipids, or lipid-like molecules. See, e.g., U.S. Patent No. 10,646,549, which is incorporated by reference herein.

In some embodiments, the VLDLR antagonist comprises a nucleic acid molecule that comprises a nucleotide sequence that binds to at least a portion of a nucleotide sequence of VLDLR. The nucleic acid molecule can be of any length, so long as at least part of the molecule hybridizes sufficiently to VLDLR nucleic acid such as mRNA. The nucleic acid molecule can bind to any region of VLDLR mRNA. In some embodiments, the nucleic acid molecule binds to a particular domain of VLDLR mRNA.

In some embodiments, the nucleic acid sequence encoding VLDLR can be found in GenBank Accession No. BC144245, which is incorporated herein by reference in its entirety, and corresponding to SEQ ID NO:1.

In some embodiments, a region of the nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary to at least a portion of SEQ ID NO:1. In some embodiments, the portion of SEQ ID NO:1 comprises a nucleic acid sequence corresponding to a portion of Homo sapiens VLDLR. In some embodiments, the composition can comprise a DNA molecule, such as an antisense DNA molecule. In some embodiments, the composition can comprise an RNA molecule, such as an anti-sense RNA molecule, a small interfering RNA (siRNA) molecule, or small hairpin RNA (shRNA) molecule, which may or may not be comprised on a vector, including a viral vector (such as an adeno-associated viral vector, an adenoviral vector, a retroviral vector, or a lentiviral vector) or a non-viral vector.

A target sequence on a target mRNA can be selected from a given cDNA sequence corresponding to the VLDLR, in some embodiments, beginning 50 to 100 nt downstream (z.e., in the 3' direction) from the start codon. The target sequence can, however, be located in the 5' or 3' untranslated regions, or in the region nearby the start codon.

In one embodiment, the VLDLR inhibitory agent comprises a nucleic acid molecule that comprises a nucleotide sequence that binds to at least a portion of a nucleotide sequence of VLDLR mRNA. In some embodiments, the nucleic acid molecule is a DNA. In some embodiments, the nucleic acid molecule is an RNA.

In some embodiments, the composition comprises an anti-sense DNA. Anti-sense DNA binds with mRNA and prevents translation of the mRNA. The anti-sense DNA can be complementary to a portion of VLDLR mRNA. In some embodiments, the anti-sense DNA is complementary to the entire reading frame of VLDLR. In some embodiments, the anti-sense DNA is complementary to the entire reading frame of SEQ ID NO:1. In some embodiments, the antisense DNA is complementary to a portion of SEQ ID NO: 1. In some embodiments, the antisense DNA is at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1500 nucleotides, at least about 2000 nucleotides, at least about 2500 nucleotides, at least about 3000 nucleotides, at least about 3500 nucleotides, or at least about 4000 nucleotides. In some embodiments, the composition comprises an anti-sense RNA. Anti-sense RNA binds with mRNA and prevents translation of the mRNA. The anti-sense RNA can be complementary to a portion of VLDLR mRNA. In some embodiments, the anti-sense RNA is complementary to the entire reading frame of VLDLR. In some embodiments, the anti-sense RNA is complementary to the entire reading frame of SEQ ID NO:1. In some embodiments, the antisense RNA is complementary to a portion of SEQ ID NO: 1. In some embodiments, the antisense RNA is at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1500 nucleotides, at least about 2000 nucleotides, at least about 2500 nucleotides, at least about 3000 nucleotides, at least about 3500 nucleotides, or at least about 4000 nucleotides.

It is also contemplated in the present invention that double-stranded RNA is used as an interference molecule, e.g., RNA interference (RNAi). In some embodiments, RNA interference is used to "knock down" or inhibit a particular gene of interest by simply injecting, bathing or feeding to the organism of interest the double- stranded RNA molecule. This technique selectively "knock downs" gene function without requiring transfection or recombinant techniques.

Another type of RNAi is often referred to as small interfering RNA (siRNA), which may also be utilized to inhibit VLDLR. A siRNA may comprise a double stranded structure or a single stranded structure, the sequence of which is "substantially identical" to at least a portion of the target gene (see WO 04/046320, which is incorporated herein by reference in its entirety). "Identity," as known in the ail, is the relationship between two or more polynucleotide (or polypeptide) sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match of the order of nucleotides between such sequences. Identity can be readily calculated. See, for example: Computational Molecular Biology, Lesk, A. M., ed. Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ca., Academic Press, New York, 1993, and the methods disclosed in WO 99/32619, WO 01/68836, WO 00/44914, and WO 01/36646, specifically incorporated herein by reference. While a number of methods exist for measuring identity between two nucleotide sequences, the term is well known in the art. Methods for determining identity are typically designed to produce the greatest degree of matching of nucleotide sequence and are also typically embodied in computer programs. Such programs are readily available to those in the relevant art. For example, the GCG program package (Devereux et al.), BLASTP, BLASTN, and FASTA and CLUSTAL are applicable.

Preferably, the siRNA contains a nucleotide sequence that is completely identical to at least a portion of the target gene. Of course, when comparing an RNA sequence to a DNA sequence, an "identical" RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine.

One of skill in the art will appreciate that two polynucleotides of different lengths may be compared over the entire length of the longer fragment. Alternatively, small regions may be compared. Normally sequences of the same length are compared for a final estimation of their utility in the practice of the present invention. It is preferred that there be 100% sequence identity between the double- stranded RNA (“dsRNA”) for use as siRNA and at least 15 contiguous nucleotides of the target gene (e.g., X'LDLR). although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater may also be used in the present invention. A siRNA that is essentially identical to a least a portion of the target gene may also be a dsRNA wherein one of the two complementary strands (or, in the case of a self-complementary RNA, one of the two self-complementary portions) is either identical to the sequence of that portion or the target gene or contains one or more insertions, deletions or single point mutations relative to the nucleotide sequence of that portion of the target gene. siRNA technology thus has the property of being able to tolerate sequence variations that might be expected to result from genetic mutation, strain polymorphism, or evolutionary divergence. There are several methods for preparing siRNA, such as chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes. Irrespective of which method one uses, the first step in designing an siRNA molecule is to choose the siRNA target site, which can be any site in the target gene. In certain embodiments, one of skill in the art may manually select the target selecting region of the gene, which may be an ORF (open reading frame) as the target selecting region and may preferably be 50- 100 nucleotides downstream of the "ATG" start codon. However, there are several readily available programs available to assist with the design of siRNA molecules, for example siRNA Target Designer by Promega, siRNA Target Finder by GenScript Corp., siRNA Retriever Program by Imgenex Corp., EMBOSS siRNA algorithm, siRNA program by Qiagen, Ambion siRNA predictor, Ambion siRNA predictor, Whitehead siRNA prediction, and Sfold. Thus, it is envisioned that any of the above programs may be utilized to produce siRNA molecules that can be used in the present invention.

In some embodiments, the composition is an siRNA targeting VLDLR. The VLDLR siRNA contains a nucleotide sequence that is essentially identical to at least a portion of the target gene. In some embodiments, the siRNA contains a nucleotide sequence that is completely identical to at least a portion of the VLDLR gene. Of course, when comparing an RNA sequence to a DNA sequence, an "identical" RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine.

In some embodiments, a VLDLR siRNA comprises a double stranded structure, the sequence of which is "substantially identical" to at least a portion of the target gene. "Identity," as known in the art, is the relationship between two or more polynucleotide (or polypeptide) sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match of the order of nucleotides between such sequences. Identity can be readily calculated by standard practices in the art.

One of skill in the art will appreciate that two polynucleotides of different lengths may be compared over the entire length of the longer fragment. Alternatively, small regions may be compared. Normally sequences of the same length are compared for a final estimation of their utility in the practice of the present invention. In some embodiments, there is 100% sequence identity between the dsRNA for use as siRNA and at least 15 contiguous nucleotides of the target gene, although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater may also be used in the present invention. A siRNA that is essentially identical to a least a portion of the target gene may also be a dsRNA wherein one of the two complementary strands (or, in the case of a self-complementary RNA, one of the two self-complementary portions) is either identical to the sequence of that portion or the target gene or contains one or more insertions, deletions or single point mutations relative to the nucleotide sequence of that portion of the target gene. siRNA technology thus has the property of being able to tolerate sequence variations that might be expected to result from genetic mutation, strain polymorphism, or evolutionary divergence.

In some embodiments, the invention provides a VLDLR siRNA that is capable of triggering RNA interference, a process by which a particular RNA sequence is destroyed (also referred to as gene silencing). In specific embodiments, VLDLR siRNA are dsRNA molecules that are 100 bases or fewer in length (or have 100 base pairs or fewer in its complementarity region). In some embodiments, a dsRNA may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides or more in length. In certain embodiments, VLDLR siRNA may be approximately 21 to 25 nucleotides in length. In some cases, it has a two nucleotide 3' overhang and a 5' phosphate. The particular VLDLR RNA sequence is targeted as a result of the complementarity between the dsRNA and the particular VLDLR RNA sequence. It will be understood that dsRNA or siRNA of the disclosure can effect at least a 20, 30, 40, 50, 60, 70, 80, 90 percent or more reduction of expression of a targeted VLDLR RNA in a cell. dsRNA of the invention (the term "dsRNA" will be understood to include "siRNA" and/or "candidate siRNA") is distinct and distinguishable from antisense and ribozyme molecules by virtue of the ability to trigger RNAi. Structurally, dsRNA molecules for RNAi differ from antisense and ribozyme molecules in that dsRNA has at least one region of complementarity within the RNA molecule. In some embodiments, the complementary (also referred to as "complementarity") region comprises at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,

40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,

190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 contiguous bases. In some embodiments, long dsRNA are employed in which "long" refers to dsRNA that are 1000 bases or longer (or 1000 base pairs or longer in complementarity region). The term "dsRNA" includes "long dsRNA", "intermediate dsRNA" or "small dsRNA" (lengths of 2 to 100 bases or base pairs in complementarity region) unless otherwise indicated. In some embodiments of the disclosure, dsRNA can exclude the use of siRNA, long dsRNA, and/or "intermediate" dsRNA (lengths of 100 to 1000 bases or base pairs in complementarity region).

It is specifically contemplated that a dsRNA may be a molecule comprising two separate RNA strands in which one strand has at least one region complementary to a region on the other strand. Alternatively, a dsRNA includes a molecule that is single stranded yet has at least one complementarity region as described above (such as when a single strand with a hairpin loop is used as a dsRNA for RNAi). For convenience, lengths of dsRNA may be referred to in terms of bases, which simply refers to the length of a single strand or in terms of base pairs, which refers to the length of the complementarity region. It is specifically contemplated that embodiments discussed herein with respect to a dsRNA comprised of two strands are contemplated for use with respect to a dsRNA comprising a single strand, and vice versa. In a two-stranded dsRNA molecule, the strand that has a sequence that is complementary to the targeted mRNA is referred to as the "antisense strand" and the strand with a sequence identical to the targeted mRNA is referred to as the "sense strand." Similarly, with a dsRNA comprising only a single strand, it is contemplated that the "antisense region" has the sequence complementary to the targeted mRNA, while the "sense region" has the sequence identical to the targeted mRNA. Furthermore, it will be understood that sense and antisense region, like sense and antisense strands, are complementary (i.e., can specifically hybridize) to each other. Strands or regions that are complementary may or may not be 100% complementary ("completely or fully complementary"). It is contemplated that sequences that are "complementary" include sequences that are at least 50% complementary, and may be at least 50%, 60%, 70%, 80%, or 90% complementary. In some embodiments, siRNA generated from sequence based on one organism may be used in a different organism to achieve RNAi of the cognate target gene. In other words, siRNA generated from a dsRNA that corresponds to a human gene may be used in a mouse cell if there is the requisite complementarity, as described above. Ultimately, the requisite threshold level of complementarity to achieve RNAi is dictated by functional capability. It is specifically contemplated that there may be mismatches in the complementary strands or regions. Mismatches may number at most or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 residues or more, depending on the length of the complementarity region.

In some embodiments, the single RNA strand or each of two complementary double strands of a dsRNA molecule may be of at least or at most the following lengths: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, or more (including the full-length of a particular's gene's mRNA without the poly-A tail) bases or base pairs. If the dsRNA is composed of two separate strands, the two strands may be the same length or different lengths. If the dsRNA is a single strand, in addition to the complementarity region, the strand may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more bases on either or both ends (5' and/or 3') or as forming a hairpin loop between the complementarity regions. In some embodiments, the strand or strands of dsRNA are 100 bases (or base pairs) or less. In specific embodiments, the strand or strands of the dsRNA arc less than 70 bases in length. With respect to those embodiments, the dsRNA strand or strands may be from 5-70, 10-65, 20-60, 30-55, 40-50 bases or base pairs in length. A dsRNA that has a complementarity region equal to or less than 30 base pairs (such as a single stranded hairpin RNA in which the stem or complementary portion is less than or equal to 30 base pairs) or one in which the strands are 30 bases or fewer in length is specifically contemplated, as such molecules evade a mammalian's cell antiviral response. Thus, a hairpin dsRNA (one strand) may be 70 or fewer bases in length with a complementary region of 30 base pairs or fewer. In some cases, a dsRNA may be processed in the cell into siRNA.

In some embodiments, the siRNA of the invention can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.

One or both strands of the siRNA of the disclosure can comprise a 3' overhang. As used herein, a "3' overhang" refers to at least one unpaired nucleotide extending from the 3'- end of a duplexed RNA strand.

Thus, in some embodiments, the VLDLR siRNA of the invention comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length.

In some embodiments in which both strands of the VLDLR siRNA molecule comprise a 3' overhang, the length of the overhangs can be the same or different for each strand. In some embodiments, the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the VLDLR siRNA of the invention can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").

In order to enhance the stability of the present VLDLR siRNA, the 3' overhangs can be also stabilized against degradation. In some embodiments, the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, c.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the absence of a 2' hydroxyl in the 2'-deoxythymidine significantly enhances the nuclease resistance of the 3' overhang in tissue culture medium.

In some embodiments, the VLDLR siRNA of the invention comprises the sequence AA(N19)TT or NA(N21), where N is any nucleotide. These VLDLR siRNA comprise approximately 30-70% GC, and in some embodiments comprise approximately 50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3 to 23), respectively. In the latter case, the 3' end of the sense siRNA is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs. The antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.

Because position 1 of the 23-nt sense strand in these embodiments is not recognized in a sequence- specific manner by the antisense strand, the 3'-most nucleotide residue of the antisense strand can be chosen deliberately. However, the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23-nt sense strand in either embodiment) is generally complementary to the targeted sequence.

In another embodiment, the VLDLR siRNA of the invention comprises the sequence NAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a pyrimidine e.g., C or U/T). The respective 21 -nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide. Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.

In some embodiments, the VLDLR siRNA of the disclosure can be targeted to any stretch of approximately 19-25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T et al., "The siRNA User Guide," revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. "The siRNA User Guide" is available on the worldwide web at a website maintained by Dr. Thomas Tuschl, Department of Cellular Biochemistry, AG 105, Max-Planck-Institutc for Biophysical Chemistry, 37077 Gottingen, Germany, and can be found by accessing the website of the Max Planck Institute and searching with the keyword "siRNA." Thus, in some embodiments, the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.

Transcription factors are regulatory proteins that bind to a specific DNA sequence (e.g., promoters and enhancers) and regulate transcription of an encoding DNA region. Thus, transcription factors can be used to modulate the expression of VLDLR. Typically, a transcription factor comprises a binding domain that binds to DNA (a DNA-binding domain) and a regulatory domain that controls transcription. Where a regulatory domain activates transcription, that regulatory domain is designated an activation domain. Where that regulatory domain inhibits transcription, that regulatory domain is designated a repression domain.

In some embodiments, a transcription factor may be targeted by a composition of the invention. The transcription factor may be one that is associated with a pathway in which VLDLR is involved. The transcription factor may be targeted with an antagonist of the invention, including siRNA to downregulate the transcription factor. Such antagonists can be identified by standard methods in the art, and in particular embodiments the antagonist is employed for treatment and or prevention of an individual in need thereof. In an additional embodiment, the antagonist is employed in conjunction with an additional compound, such as a composition that modulates ApoE. For example, the VLDLR antagonist may be used in combination with an inhibitor of ApoE. When employed in combination, the antagonist of a transcription factor of a VLDLR-related pathway may be administered prior to, during, and/or subsequent to the additional compound.

In some embodiments, an antisense molecule that binds to a translational or transcriptional staid site, or splice junctions, can be used as an inhibitor. Antisense, ribozyme, and double- stranded RNA molecules target a particular sequence to achieve a reduction or elimination of a particular polypeptide, such as VLDLR. Thus, it is contemplated that antisense, ribozyme, and double-stranded RNA, and RNA interference molecules are constructed and can be used to modulate VLDLR expression. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with complementary sequences. By complementary, it is meant that polynucleotides arc those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others, in hybridizing sequences does not interfere with pairing.

Targeting double- stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNAs, are employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense constructs are designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs may include regions complementary to intron/exon splice junctions. Thus, in some embodiments, antisense constructs with complementarity to regions within 50-200 bases of an intron-exon splice junction are used. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

It is advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

Ribozymes are RNA-protein complexes that cleave nucleic acids in a site- specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction. Ribozyme catalysis has primarily been observed as part of sequence specific cleavage/ligation reactions involving nucleic acids. For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence- specific ribozyme- mediated inhibition of gene expression is particularly suited to the therapeutic applications. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme. In light of the information included herein and the knowledge of one of ordinary skill in the art, the preparation and use of additional ribozymes that are specifically targeted to a given gene will now be straightforward.

Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art. The identification of operative and preferred sequences for use VLDLR targeted ribozymes is simply a matter of preparing and testing a given sequence, and is a routinely practiced screening method known to those of skill in the art.

Combination therapy

The VLDLR antagonists can be administered alone or in combination with effective amounts of one or more active pharmaceutical agents. In some embodiments, the one or more active pharmaceutical agents are other drugs that are useful for treating Alzheimer’s disease in the subject, such as cholinesterase inhibitors, N-methyl-D-aspartic acid (NMDA) receptor antagonists or antibodies directed against beta-amyloid.

In some embodiments, the one or more additional active agents comprises i) an effective amount of an LRP1 antagonist; ii) an effective amount of a SorLA antagonist; or iii) an effective amount of an LRP1 antagonist and a SorLA antagonist. Use of LRP1 and SorLA antagonists for the treatment of Alzheimer’s disease arc disclosed in U.S. Application Pub. No.: 2022/0332813 Al, which is incorporated by reference herein.

In some embodiments, the one or more additional active agents comprises receptor associated protein (RAP).

In some embodiments, the one or more additional active pharmaceutical agents comprises aminocaproic acid, acamprosate, amlodipine, argatroban, baclofen, cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam, leflunomide, mepacrine, methimazole, phenformin, prilocaine, rifabutin, sulfisoxazole, tadalafil, terbinafine, cinnarizine, ciclopirox, eplerenone, carbenoxolone, sulodexide, carbamazine, amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide, risedronate, enprofylline, oxtriphylline, paramethadione, cefmenoxime, aprindine, etomidate, mitiglinide, benidipine, levosimendan or zonisamide.

In some embodiments, the subject is administered one or more atypical antipsychotics, beta-amyloid antibodies, cholinesterase inhibitors, or NMDA antagonists. In some embodiments, the subject is administered lecanemab, brexpiprazole, rivastigmine, donepezil, galantamine, memantine, or a combination of memantine and donepezil.

Routes of administration

The VLDLR antagonist or therapeutic agent can be administered in a variety of ways and is not particularly limiting. In some embodiments, the administration of the therapeutic agent and/or the therapies of the present invention may include systemic, local and/or regional administrations. In some embodiments, the agent is administered directly (topically), intravenously, subcutaneously, transcutaneously, intrathecally, intramuscularly, intracutaneously, intragastrically, intranasally, rectally, intra-arterially, parenterally, orally, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

In some embodiments, the agent is administered topically (dermally, transdermally), via catheters, implantable pumps, dermal patches, transdermal patches, etc. Other routes of administration arc also contemplated such as, for example, arterial perfusion, intracavitary, intraperitoneal, intrapleural, intraventricular and/or intrathecal. The skilled artisan is aware of determining the appropriate administration route using standard methods and procedures.

In some embodiments, the therapeutic compound is administered intrathecally. In some embodiments, the compound is administered intrathecally via an implantable pump. In one embodiment, the implantable pump comprises a SynchroMed™ II pump that stores and delivers medication into the intrathecal space (Medtronic).

In some embodiments, the antagonist can be administered parenterally or alimentarily. Parenteral administrations include, but are not limited to intravenously, intradermally, transdermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally. See, e.g., U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety). Alimentary administrations include, but are not limited to orally, buccally, rectally, or sublingually.

In some embodiments, the VLDLR antagonist or therapeutic agent can be modified to facilitate transport across the blood-brain barrier. See, e.g., Zhao el al., Antib Ther. 2022 Oct; 5(4): 311-331.

Dosing

Treatment methods involve administering to a subject an effective amount of therapeutic agents as described herein.

As is well known in the art, a specific dose level of active compounds such as an antagonist of VLDLR for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.

In some embodiments, the compound(s) or composition(s) can be administered to the subject once, such as by a single injection or deposition at or near the site of interest. In some embodiments, the compound(s) or composition(s) can be administered to a subject over a period of days, weeks, months or even years. In some embodiments, the compound(s) or composition(s) is administered at least once a day to a subject. Where a dosage regimen comprises multiple administrations, it is understood that the effective amount of the compound(s) or composition(s) administered to the subject can comprise the total amount of the compound(s) or composition(s) administered over the entire dosage regimen.

In some embodiments, an effective amount of the antagonist of VLDLR that is administered includes a dose of about 0.0001 nM to about 2000 pM. In some embodiments, amount administered is from about 0.01 nM to about 2000 pM; about 0.01 pM to about 0.05 pM; about 0.05 pM to about 1.0 pM; about 1.0 pM to about 1.5 pM; about 1.5 pM to about 2.0 pM; about 2.0 pM to about 3.0 pM; about 3.0 pM to about 4.0 pM; about 4.0 pM to about 5.0 pM; about 5.0 pM to about 10 pM; about 10 pM to about 50 pM; about 50 pM to about 100 pM; about 100 pM to about 200 pM; about 200 pM to about 300 pM; about 300 pM to about 500 pM; about 500 pM to about 1000 pM; about 1000 pM to about 1500 pM; and about 1500 pM to about 2000 pM. Of course, all of these amounts are exemplary, and any amount in-between these points is also expected to be of use in the invention.

In some embodiments, the total daily dose of the antagonist of VLDLR of the present invention administered to a subject in single or in divided doses can be in amounts, for example, from 0.01 to 25 mg/kg body weight or more usually from 0.1 to 15 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a human or other mammal in need of such treatment from about 1 mg to about 1000 mg of the active substance(s) of this invention per day in multiple doses or in a single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg.

The treatments may include various "unit doses." Unit dose is defined as containing a predetermined quantity of the therapeutic composition (an antagonist of VLDLR) calculated to produce the desired responses in association with its administration, e.g., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. Also of importance is the subject to be treated, in particular, the state of the subject and the protection desired. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.

Pharmaceutical formulations In some embodiments, the VLDLR antagonists are formulated as pharmaceutical compositions comprising a therapeutically effective amount of one or more of the active agents along with a pharmaceutically acceptable carrier.

For example, in one embodiment, the invention is directed to a method of treating or preventing Alzheimer’ s disease in a subject by administering to the subject a an effective amount of a composition comprising an antagonist of VLDLR and a pharmaceutically acceptable carrier.

The compositions can comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection.

The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one Alzheimer’s disease drug or related compounds or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. Exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, intrathecal, subcutaneous, intramuscular, intracutancous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.

Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Examples of pharmaceutically acceptable antioxidants include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, a-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

The antagonists can be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

Further in accordance with the present invention, the compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the ail.

In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of pharmaceutical lipid vehicle compositions that include compounds or compositions of the invention such as Alzheimer’s therapeutics, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the ail, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester- linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the Alzheimer’s therapeutics may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

In some embodiments of the present invention, the compounds and compositions of the invention, such as Alzheimer’s therapeutics are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. See, e.g., U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each specifically incorporated herein by reference in its entirety. The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar', or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water, isotonic solutions, or saline. Such compositions may also comprise adjuvants, such as wetting agents; emulsifying and suspending agents; sweetening, flavoring and perfuming agents.

For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

Additional formulations that are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycol, which are solid at ordinary temperature but liquid at the rectal temperature and will, therefore, melt in the rectum and release the drug.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. In some embodiments, the injectable composition can be administered as a nanoparticle formulation.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of a drug from subcutaneous or intramuscular injection. The most common way to accomplish this is to inject a suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug becomes dependent on the rate of dissolution of the drug, which is, in turn, dependent on the physical state of the drug, for example, the crystal size and the crystalline form. Another approach to delaying absorption of a drug is to administer the drug as a solution or suspension in oil. Injectable depot forms can also be made by forming microcapsule matrices of drugs and biodegradable polymers, such as polylactide-polyglycoside. Depending on the ratio of drug to polymer and the composition of the polymer, the rate of drug release can be controlled. Examples of other biodegradable polymers include polyorthoesters and poly anhydrides. The depot injectables can also be made by entrapping the drug in liposomes or microemulsions, which arc compatible with body tissues.

Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, dimethyl sulfoxide (DMSO), polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

For the preparation of fluids for parenteral administration, suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria- retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use. The active compound is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent. In other embodiments, the compounds and compositions of the invention, such as Alzheimer’ s therapeutics may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.

Dosage forms for topical or transdermal administration of a compound of this invention further include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. Transdermal patches have the added advantage of providing controlled delivery of active compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Screening assays

In another embodiment, the invention provides a method of screening for potential agents that reduce internalization and/or trafficking of tau in cells, comprising i) providing a cell expressing VLDLR or a functional equivalent of VLDLR; ii) providing tau protein or a fragment or derivative thereof to the cell, wherein the tau protein or a fragment or derivative thereof is extracellular; iii) treating the cell with an effective amount of a test agent that may inhibit binding of tau to VLDLR or reduce expression of VLDLR in cells; and iv) assaying the cellular uptake of tau or the fragment or derivative thereof.

In some embodiments, tau and VLDLR or a functional equivalent thereof can be used in screening assays for compounds which bind one or more of the proteins and which inhibit their interaction.

In some embodiments, the screening methods can be conducted in cells, cell-free preparations, cellular homogenates, animals, or on one or more substrates, for example on surface plasmon resonance sensor chips. In some embodiments any of a tau antibody, tau or a fragment or derivative thereof (including fractions from brain, including from Alzheimer’s patients), VLDLR, and the potential antagonist/agent can be coupled to a solid surface to assay competitive binding. In some embodiments, one or more domains of VLDLR are assayed for competition binding of tau or a fragment or derivative thereof using a test compound.

In some embodiments, a tau antibody is bound to a surface, such as a surface plasmon resonance chip, and a sample comprising tau or a fragment or derivative thereof is added to the surface. In some embodiments, the source of tau comprises a sample from brain, e.g., of an Alzheimer’s patient, such as a homogenate, or size exclusion purified fraction, to bind tau to the antibody. Tau binding can be confirmed by a second tau antibody, in some embodiments. VLDLR, or tau binding fragments or derivatives thereof are added to the immobilized tau, and can be added in combination with a test agent to be assayed for competitive binding to displace the bound VLDLR from the surface or to prevent binding.

In some embodiments, the invention provides a screening assay to test for compounds that inhibit the interaction of tau with VLDLR comprising i) providing a tau antibody bound to a substrate; ii) adding a sample to the substrate comprising tau or a fragment or derivative thereof; iii) adding VLDLR and a test compound to the substrate; iv) detecting binding of VLDLR to the substrate or detecting the absence or reduction of binding of VLDLR to the substrate in the presence of the test compound.

In another embodiment, the invention provides a screening assay to test for compounds that inhibit the interaction of tau with VLDLR comprising i) providing a VLDLR antibody bound to a substrate; ii) adding a sample to the substrate comprising VLDLR or a fragment or derivative thereof; iii) adding a source of tau or a fragment or derivative thereof and a test compound to the substrate; iv) detecting binding of tau or a fragment or derivative thereof to the substrate or detecting the absence or reduction of binding of tau or a fragment or derivative thereof to the substrate in the presence of the test compound.

In some embodiments, the substrate is a surface plasmon resonance sensor chip. In some embodiments, tau or VLDLR binding can be confirmed by a second tau or VLDLR antibody. In some embodiments, the invention provides a method of screening for potential antagonists of VLDLR that reduce internalization and/or trafficking of tau in cells. In some embodiments, the method comprises providing a cell expressing VLDLR or a functional equivalent of VLDLR; providing tau or a fragment or derivative thereof protein to the cell, wherein the tau protein or fragment or derivative thereof is extracellular; treating the cell a potential VLDLR antagonist; and assaying the cellular uptake of tau or the fragment or derivative thereof. In some embodiments, the method comprises comparing the cellular uptake of tau or the fragment or derivative thereof in the cell with cells that have not been treated with the antagonist.

In some embodiments, the screening procedures involve producing appropriate cells, which can be neuronal cells which express VLDLR or functional equivalents thereof. Such cells can include neuronal or non-neuronal cells from mammals, yeast, Drosophila or E. coli. In some embodiments, the cells express the polypeptide endogenously. In other embodiments, the cells have been transfected or engineered to express the polypeptide. In some embodiments, cells expressing the protein (or extracts or purified preparations from cells) are contacted with a test compound to observe stimulation or inhibition of a functional response. In some embodiments, for assaying compounds that inhibit expression of VLDLR, the levels of VLDLR mRNA or protein can be assayed after contacting the cells with the test compound. In some embodiments, the expression level of an endogenous VLDLR target gene is assayed. In some embodiments, the cells can comprise a reporter gene located downstream of one or more VLDLR promoter elements and inhibition of the reporter gene is assayed.

In some embodiments, assays test binding of a candidate compound to VLDLR or tau or assays involving competition with a labeled competitor. In some embodiments, inhibitors of activation can be tested in the presence of an agonist and the effect on activation by the agonist in the presence of the candidate compound is observed.

Examples of antagonists can include antibodies, peptides, carbohydrates, lipids, or small molecules which bind to one or more of the proteins so that binding between tau and VLDLR is inhibited. These agents can be selected and screened 1) at random, 2) by a rational selection or 3) by design using for example, protein or ligand modeling techniques (preferably, computer modeling). All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

EXAMPLES

Example 1. VLDL receptor interacts with tau and mediates its cellular uptake.

The low-density lipoprotein receptor-related protein (LRP1) has been established as an endocytic receptor for tau that facilitates tau uptake and promotes the seeding of endogenous tau aggregation (Cooper et al 2021). Recently, we have shown that another receptor related to LRP1, sortilin-related receptor (SORLA), can also mediate these functions. We tested the hypothesis that other members of this receptor family might serve a similar function, and focused studies on the very low-density lipoprotein receptor (VLDLr), a receptor associated with lipid metabolism and reelin signaling. Here, we investigate VLDLr’ s role in tau binding and it's internalization.

Methods

Surface plasmon resonance (SPR) was used to investigate binding interactions between tau and VLDLr. To determine if VLDLr mediates tau uptake, we assessed the uptake of recombinant ¹²⁵I-labeled 2N4Rtau labeled in LRP1 -deficient CHO cells (CHO 13-5-1) that stably expressed VLDLr, and compared the uptake to non-expressing control cells. Cells were incubated with ¹²⁵I-labeled tau for two hours, and the amount of tau internalized was quantified.

Results

SPR data showed that tau binds soluble forms of the VLDLr containing the LDL- ligand binding repeats with high affinity (KD = 22 ± 3 nM). The binding was inhibited by receptor associated protein (RAP), which inhibits binding of ligands to LDL receptor family members. Cells expressing VLDLr internalized significantly more ¹²⁵I-labeled tan than control cells in a process that was blocked by RAP.

Conclusions

These experiments identify VLDLr as a third receptor capable of binding tau and mediating its internalization. This activity of the VLDLr’ s raises the possibility that the VLDLr may contribute to the spreading of pathogenic forms of tau in the AD brain.

Example 2. The VLDL receptor binds and internalizes tau in a process that is inhibited by monoclonal antibodies 1H10 and 1H5.

This example describes the discovery of another receptor that mediates tau uptake, the VLDLr. The interest in tau receptors arise from the fact that neuronal transfer of pathological forms of tau has been proposed as a mechanism of Alzheimer’s disease (AD) progression. The accumulation of misfolded tau aggregates initiates in the entorhinal cortex and spreads across connected neural pathways (7-13).

In addition, the VLDLr is one of two receptors that participate in reelin signaling, which decreases the extent of tau phosphorylation. Phosphorylation generates the pathogenic forms of tau. Since receptor dimerization using antibodies have also been shown to activate the reelin signaling pathway (5), we anticipate that 1H10 and 1H5 will not only block tau uptake, but also activate the reelin signaling pathway decreasing tau phosphorylation.

Results

Tau binds tightly to the VLDLr. To test the hypothesis that the VLDLr is capable of binding tau, we immobilized a soluble form of the VLDLr that contains the ligand binding domain (sVLDLrl-8) (6) on a SPR chip. In a single cycle experiment, increasing amounts of tau were injected over the surface, and the Req was measured at each tau concentration. The results confirm that tau binds to the VLDLr with high affinity (Figure 1).

Cells that express the VLDLr mediate tau internalization. Having demonstrated a direct interaction between tau and the VLDLr, we next tested if the VLDLr can mediate tau internalization. For this experiment, we selected Chinese hamster ovary (CHO) cells lacking LRP1 (a major endocytic receptor for tau) that were stably transfected with the VLDLr. These cells, along with parental 13-5-1 cells were incubated with 20 nM of ¹²⁵I- labclcd tau for 2h, and the amount of tau internalized quantified. The results arc shown in Figure 2 and demonstrate significant increases in tau internalization in cells expressing the VLDLr. The VLDLr-mediated internalization was blocked with the receptor-binding protein (RAP), a molecule that antagonizes binding of ligands to this receptor and other LDL-receptor family members.

Monoclonal antibodies 1H10 and 1H5 block tau binding to the VLDLr and VLDLr- mediated tau uptake. We have developed monoclonal antibodies to the VLDLr (6), and conducted experiments to determine if these antibodies could block tau binding to the VLDLr. In SPR experiments, we found that 100 nM 1H10 was highly effective in blocking 20 nM tau from binding to immobilized VLDLr (Fig 3a). In contrast, antibody 1H5 was less effective at blocking binding, while 5F3 was not able to block binding. While these antibodies bind to different regions of the ligand binding domain of the VLDLr, the lack of inhibition of 5F3 might result from its low affinity for the VLDLr (Figure 4).

We next checked if these antibodies were capable of blocking VLDLr-mediated tau uptake using the 13-5-1 CHO cells stably transfected with VLDLr. The results of this experiment confirm that 1H10 and 1H5 are both effective at blocking VLDLr-mediated tau uptake (Figure 5). 5F3 resulted in a 50% reduction in tau uptake.

Example 3. Administration of VLDL antibodies in a pathogenic tau animal model.

6 week old Tg30tau mice (which express human 1N4R tau protein with two pathogenic mutations (P301S and G272V) under the control of the neuron- specific Thy 1.2 promotor and/or HtauP301L transgenic mice (which express human 2N4R tau protein with the P301L mutation under the Thy 1.2 promotor) will receive stereotaxic injections of tau isolated from human Alzheimer patients, with or without anti- VLDLr antibodies 1H10, 1H5, 5F3 (30mg/kg) into the CAI layer of the hippocampus. 2 days after injection, mice will be sacrificed and whole brains processed for immunohistochemical (IHC) analysis using human specific anti-tau antibodies. The amount of tau internalized will be quantified based on the integrated tau signal. Simultaneous injection of patient-derived tau with RAP or nonspecific antibody will be used as positive and negative controls. 6 mice per condition will be assayed. This experimental protocol is adapted from Albert et al., Brain 142(6): 1736-1750 (2019).

Literature cited

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2. Dlugosz, P., and Nimpf, J. (2018) The Reelin Receptors Apolipoprotein E receptor 2 (ApoER2) and VLDL Receptor. Int. J. Mol. Sci. 19, 3090

3. Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R. E., Richardson, J. A., and Herz, J. (1999) Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2. Cell. 97, 689-701

4. Herz, J., and Chen, Y. (2006) Reelin, lipoprotein receptors and synaptic plasticity. Nat. Rev. Neurosci. 7, 850-859

5. Strasser, V., Fasching, D., Hauser, C., Mayer, H., Bock, H. H., Hiesberger, T., Herz, J., Weeber, E. J., Sweatt, J. D., Pramatarova, A., Howell, B., Schneider, W. J., and Nimpf, J. (2004) Receptor Clustering Is Involved in Reelin Signaling. Mol. Cell. Biol. 24, 1378-1386

6. Ruiz, J., Kouiavskaia, D., Migliorini, M., Robinson, S., Saenko, E. L., Gorlatova, N., Li, D., Lawrence, D., Hyman, B. T., Weisgraber, K. H., and Strickland, D. K. (2005) The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor. J. Lipid Res. 46, 1721-1731

7. Hyman, B. T., Hoesen, G. W. Van, Damasio, A. R., and Clifford, L. (1984) Alzheimer ’ s Disease : Cell-Specific Pathology Isolates the Hippocampal Formation. Science. 225, 1168-1170

8. Serrano-Pozo, A., Qian, J., Monsell, S. E., Frosch, M. P., Betensky, R. A., and Hyman, B. T. (2013) Examination of the clinicopathologic continuum of Alzheimer disease in the autopsy cohort of the national alzheimer coordinating center. J. Neuropathol. Exp. Neurol. 72, 1182-1192

9. Braak, H., and Braak, E. (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 82, 239-259

10. Polydoro, M., de Calignon, A., Suarez-Calvet, M., Sanchez, L., Kay, K. R., Nicholls, S. B., Roe, A. D., Pitstick, R., Carlson, G. A., Gdmez-Isla, T., Spires-Jones, T. L., and Hyman, B. T. (2013) Reversal of neurofibrillary tangles and tau-associated phenotype in the rTgTauEC model of early Alzheimer’s disease. J. Neurosci. 33, 13300- 13311

11. De Calignon, A., Polydoro, M., Suarez-Calvet, M., William, C., Adamowicz, D. H., Kopeikina, K. J., Pitstick, R., Sahara, N., Ashe, K. H., Carlson, G. A., Spires-Jones, T. L., and Hyman, B. T. (2012) Propagation of Tau Pathology in a Model of Early Alzheimer’s Disease. Neuron. 73, 685-697

12. Liu, L., Drouet, V., Wu, J. W., Witter, M. P., Small, S. A., Clelland, C., and Duff, K. (2012) Trans-synaptic spread of tau pathology in vivo. PLoS One. 7, e31302 13. Harris, J. A., Koyama, A., Maeda, S., Ho, K., Devidze, N., Dubai, D. B., Yu, G. Q., Masliah, E., and Mucke, L. (2012) Human P301L-Mutant Tau Expression in Mouse Entorhinal-Hippocampal Network Causes Tau Aggregation and Presynaptic Pathology but No Cognitive Deficits. PLoS One. 7, e45881

-SO-

Claims

WHAT IS CLAIMED IS:

1. A method of reducing tau internalization and/or trafficking in neuronal cells comprising contacting the cells with an effective amount of a VLDL receptor antagonist.

2. The method of claim 1, wherein the VLDL receptor antagonist blocks the interaction of tau and VLDL receptor.

3. The method of claim 1, wherein the VLDL receptor antagonist inhibits the expression of VLDL receptor.

4. The method of any of claims 1-2, wherein the VLDL receptor antagonist is an VLDL receptor antibody.

5. The method of claim 4, wherein the antibody is a monoclonal antibody.

6. The method of claim 5, wherein the antibody is selected from the group consisting of 1H5, 1H10, 5F3 and combinations thereof.

7. The method of claim 5, wherein the antibody is a humanized antibody and comprises CDR sequences of an antibody selected from the group consisting of 1H5, 1H10, and 5F3.

8. The method of claim 3, wherein the VLDL receptor antagonist is a nucleic acid that inhibits the expression of VLDL receptor.

9. The method of claim 8, wherein the nucleic acid is an RNA, a DNA, or a combination thereof.

10. The method of claim 8, wherein the nucleic acid is an RNA.

11. The method of claim 8, wherein the nucleic acid is a ribozyme, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

12. The method of any of claims 8-11, wherein the nucleic acid is delivered as a viral vector.

13. The method of any of claims 1-12, wherein the VLDLR antagonist is administered to a subject by topical, intravenous, subcutaneous, intramuscular, intracutaneous, transcutaneous, intrathecal, intranasal, intra-arterial, rectal, intragastric, parenteral, or oral administration.

14. The method of any of claims 1-13, wherein the method further comprises administering one or more additional active agents.

15. The method of claim 14, wherein the one or more additional active agents comprises i) an effective amount of an LRP1 antagonist; ii) an effective amount of a SorLA antagonist; or iii) an effective amount of an LRP1 antagonist and a SorLA antagonist.

16. A method of treating or preventing Alzheimer’s disease in a subject in need thereof, comprising administering to the subject an effective amount of a VLDL receptor antagonist.

17. The method of claim 16, wherein the VLDL receptor antagonist blocks the interaction of tau and VLDL receptor.

18. The method of claim 16, wherein the VLDL receptor antagonist inhibits the expression of VLDL receptor.

19. The method of any of claims 16-17, wherein the VLDL receptor antagonist is an VLDL receptor antibody.

20. The method of claim 19, wherein the antibody is a monoclonal antibody.

21. The method of claim 20, wherein the antibody is selected from the group consisting of 1H5, 1H10, 5F3 and combinations thereof.

22. The method of claim 20, wherein the antibody is a humanized antibody and comprises CDR sequences of an antibody selected from the group consisting of 1H5, 1H10, and 5F3.

23. The method of claim 18, wherein the VLDL receptor antagonist is a nucleic acid that inhibits the expression of VLDL receptor.

24. The method of claim 23, wherein the nucleic acid is an RNA, a DNA, or a combination thereof.

25. The method of claim 23, wherein the nucleic acid is an RNA.

26. The method of claim 23, wherein the nucleic acid is a ribozyme, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

27. The method of any of claims 23-26, wherein the nucleic acid is delivered as a viral vector.

28. The method of any of claims 16-27, wherein the VLDLR antagonist is administered to a subject by topical, intravenous, subcutaneous, intramuscular, intracutaneous, transcutaneous, intrathecal, intranasal, intra-arterial, rectal, intragastric, parenteral, or oral administration.

29. The method of any of claims 16-28, wherein the method further comprises administering one or more additional active agents.

30. The method of claim 29, wherein the one or more additional active agents comprises i) an effective amount of an LRP1 antagonist; ii) an effective amount of a SorLA antagonist; or iii) an effective amount of an LRP1 antagonist and a SorLA antagonist.

31. A method of screening for potential antagonists of VLDLR that reduce internalization and/or trafficking of tau in cells, comprising i) providing a cell expressing VLDLR or a functional equivalent of VLDLR; ii) providing tau protein or a fragment or derivative thereof to the cell, wherein the tau protein or fragment or derivative thereof is extracellular; iii) treating the cell with a potential VLDLR antagonist; and iv) assaying the cellular uptake of tau or the fragment or derivative thereof.

32. The method of claim 31, further comprising comparing the cellular uptake of tau in the cell with cellular uptake of tau in cells that have not been treated with the potential antagonist.

33. The method of any of claims 31-32, wherein the cells are neuronal cells.

34. The method of any of claims 31-32, wherein the cells are non-neuronal cells.

35. The method of any of claims 31-32, wherein the cells are from mammals, yeast, Drosophila or E. coli.

36. The method of any of claims 31-35, wherein the cells express VLDLR endogenously.

37. The method of any of claims 31-35, wherein the cells have been transfected or engineered to express VLDLR.