WO2023049762A1

WO2023049762A1 - Compositions and methods to modulate transfer across the blood-brain barrier

Info

Publication number: WO2023049762A1
Application number: PCT/US2022/076800
Authority: WO
Inventors: Gaya AMARASINGHE; Safder GANAIE; Daisy LEUNG; Herbert VIRGIN; Amy HARTMAN
Original assignee: University of Pittsburgh; Washington University in St Louis WUSTL
Current assignee: University of Pittsburgh; Washington University in St Louis WUSTL
Priority date: 2021-09-21
Filing date: 2022-09-21
Publication date: 2023-03-30
Anticipated expiration: 2024-03-21

Abstract

The present disclosure relates to compositions and methods for treating or preventing a viral infection. The present disclosure also provide compositions and method for improving transfer of a therapeutic or imaging agent transfer across the blood-brain-barrier. In addition the present disclosure provides compositions and methods for treating various central nervous system disease and disorders including, for example, tauopathies.

Description

COMPOSITIONS AND METHODS TO MODULATE TRANSFER ACROSS THE BLOOD-BRAIN BARRIER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of U.S. Provisional Application number 63/261 ,447, filed September 21 , 2022, the disclosure of which is herein incorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

[0002] This invention was made with government support under AI106688, Al 144033, Al 150792 and NS101100 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE TECHNOLOGY

[0003] This present disclosure generally relates to compositions and methods useful for treating viral infections, modulating the transfer across the bloodbrain barrier, and preventing tau aggregation.

REFERENCE TO SEQUENCE LISTING

[0004] This application contains a Sequence Listing that has been submitted in ASCII format via Patent Center and is hereby incorporated by reference in its entirety. The ASCII copy, created on September 21 , 2022, is named 740076. xml, and is 12,288 bytes in size.

BACKGROUND

[0005] Blood vessels are critical to deliver oxygen and nutrients to all of the tissues and organs throughout the body. The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood-brain barrier, which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. This precise control of CNS homeostasis allows for proper neuronal function and also protects the neural tissue from toxins and pathogens, and alterations of these barrier properties are an important component of pathology and progression of different neurological diseases. The physiological barrier is coordinated by a series of physical, transport, and metabolic properties possessed by the endothelial cells (ECs) that form the walls of the blood vessels, and these properties are regulated by interactions with different vascular, immune, and neural cells.

Understanding the barrier properties is essential for understanding how the brain functions during health and disease.

[0006] Therefore, a need in the art exists for compositions and methods of modulating transfer across the blood-brain barrier, e.g., preventing viral infection of the CNS, increasing the transfer of therapeutic agents into the CNS and treatment of various CNS disorders.

SUMMARY

[0007] One aspect of the present disclosure provides methods of reducing or treating a viral infection in a subject, the methods generally comprise administering to the subject an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and decoy receptor comprising an LRP1 polypeptide or fragment thereof.

[0008] In some embodiments, the viral infection is a bunyaviral infection. In each of the above embodiments, the viral infection can be a Rift Valley Fever virus (RVFV) infection, a oropouche virus (OROV) infection, or a La Crosse virus (LACV) infection. In each of the above embodiments, the subject is having symptoms of a viral infection or is suspected of having a viral infection. In each of the above embodiments, the decoy receptor comprises one or more LRP1 CLiv domains. In each of the above embodiments, the decoy receptor comprises one or more LRP1 CLn domains. In each of the above embodiments, the decoy receptor comprises a LRP1 polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 1 . In each of the above embodiments, the decoy receptor comprises an extracellular domain of a LRP1 polypeptide. In each of the above embodiments, the decoy receptor comprises a LRP1 polypeptide as a fusion protein. In some embodiments, the fusion protein is a LRP1 polypeptide-Fc fusion protein. In some embodiments, the fusion protein is a LRP1 polypeptide-SpyTag/SpyCatcher fusion.

[0009] In some embodiments, the methods further comprises administering to the subject an additional anti-viral agent. In each of the above embodiments, infectivity of the virus for a host cell is reduced. In each of the above embodiments, the infectivity of the virus is reduced by reducing internalization of a virus into the cell. In each of the above embodiments, infectivity of the virus is reduced by reducing replication or internalization of a viral genome into the cell. In each of the above embodiments, infectivity of the virus is reduced by disrupting or preventing an interaction between a viral surface protein and a host receptor protein. In each of the above embodiments, the viral surface protein is a Gn viral glycoprotein protein and the host receptor protein is LRP1.

[0010] In another aspect, the present disclosure provides a decoy receptor composition comprising a recombinant LRP1 polypeptide. In some embodiments, the decoy receptor comprises one or more LRP1 CLiv domains. In each of the above embodiments, the decoy receptor comprises one or more LRP1 CLn domains. In each of the above embodiments, the decoy receptor comprises a LRP1 polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 1 . In each of the above embodiments, the decoy receptor comprises an extracellular domain of a LRP1 polypeptide. In each of the above embodiments, the decoy receptor comprises a LRP1 polypeptide as a fusion protein. In some embodiments, the fusion protein is a LRP1 polypeptide-Fc fusion protein. IN some embodiments, the fusion protein is a LRP1 polypeptide-SpyTag/SpyCatcher fusion. In each of the above embodiments, the decoy receptor is for use in treating a viral infection.

[0011 ] In still another aspect, the present disclosure provides a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a decoy receptor as described herein. [0012] In yet another aspect, the present disclosure provides methods of reducing or treating a viral infection in a subject, the method generally comprises administering to the subject an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and decoy viral inhibitor comprising a viral Gn polypeptide or fragment thereof. In some embodiments, the viral infection is a bunyaviral infection. In each of the above embodiments, the viral infection may be a Rift Valley Fever virus (RVFV) infection, a oropouche virus (OROV) infection, or La Crosse virus (LACV) infection. In each of the above embodiments, the subject is having symptoms of a viral infection or is suspected of having a viral infection. In each of the above embodiments, the decoy viral inhibitor comprises a Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 2. In each of the above embodiments, the decoy viral inhibitor comprises the extracellular domain of a Gn polypeptide. In each of the above embodiments, the decoy viral inhibitor comprises amino acids 1 to 316 of SEQ ID NO: 2. In each of the above embodiments, wherein the decoy viral inhibitor is a RVFV Gn polypepide. In each of the above embodiments, the decoy viral inhibitor comprises a Gn polypeptide as a fusion protein. In some embodiments, the fusion protein is a Gn polypeptide-Fc fusion protein. In some embodiments, the fusion protein is a Gn polypeptide-SpyTag/SpyCatcher fusion.

[0013] In some embodiments, the method further comprises administering to the subject an additional anti-viral agent. In each of the above embodiments, infectivity of the virus for a host cell is reduced. In each of the above embodiments, infectivity of the virus is reduced by reducing internalization of a virus into the cell. In each of the above embodiments, infectivity of the virus is reduced by reducing replication or internalization of a viral genome into the cell. In each of the above embodiments, infectivity of the virus is reduced by disrupting or preventing an interaction between a viral surface protein and a host receptor protein. In each of the above embodiments, the viral surface protein is a Gn viral glycoprotein protein and the host receptor protein is LRP1. [0014] In still yet another aspect, the present disclosure provides a decoy viral inhibitor composition comprising a recombinant viral Gn polypeptide. In some embodiments, the decoy viral inhibitor comprises a Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 2. In each of the above embodiments, the decoy viral inhibitor can comprise the extracellular domain of a Gn polypeptide. In each of the above embodiments, the decoy viral inhibitor can comprise amino acids 1 to 316 of SEQ ID NO: 2. In each of the above embodiments, the decoy viral inhibitor can be a RVFV Gn polypeptide. In each of the above embodiments, the decoy viral inhibitor can comprise a Gn polypeptide as a fusion protein. In some embodiments, the fusion protein is a Gn polypeptide-Fc fusion protein. In some embodiments, the decoy viral inhibitor is for use in treating a viral infection or tauopathy.

[0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a decoy viral inhibitor as described herein.

[0016] In still another aspect, the present disclosure provides methods of reducing or treating a tauopathy or reducing a tau-related pathology in a subject, the methods generally comprise administering to the subject an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and decoy viral inhibitor comprising a viral Gn polypeptide or fragment thereof. In some embodiments, the subject is amyloid negative. In some embodiments, the subject has no dementia. In some embodiments, the subject has dementia. In some embodiments, the subject is amyloid positive. In some embodiments, the tauopathy is progressive supranuclear palsy (PSP), dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease, Parkinson-dementia complex of Guam, tangle- predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden- Spatz disease, lipofuscinosis, Pick’s disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Frontotemporal lobar degeneration (FTLD), Alzheimer’s disease (AD), or frontotemporal dementia (FTD). In a certain embodiment, the tauopathy is AD.

[0017] In each of the above embodiments, the decoy viral inhibitor can comprise a Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 2. In each of the above embodiments, the decoy viral inhibitor can comprise the extracellular domain of a Gn polypeptide. In each of the above embodiments, the decoy viral inhibitor can comprise amino acids 1 to 316 of SEQ ID NO: 2. In each of the above embodiments, the decoy viral inhibitor can be a RVFV Gn polypeptide. In some embodiments the decoy viral inhibitor comprises a Gn polypeptide as a fusion protein. In some embodimets the fusion protein is a Gn polypeptide-Fc fusion protein. In each of the above embodiments, cell-to-cell spread of pathogenic tau is reduced relative to the spread of tau in the absence of the decoy viral inhibitor. In each of the above embodiments, cell-to-cell spread of pathogenic tau is reduced by reducing internalization of a pathogenic tau into the cell. In each of the above embodiments, cell- to-cell spread of the pathogenic tau is reduced by disrupting or preventing an interaction between the pathogenic tau and a host receptor protein. In each of the above embodiments, the host receptor protein is LRP1 .

[0018] In still yet another aspect, the present disclosure provides methods of increasing the amount of an imaging agent or therapeutic agent in the central nervous system of a subject, the methods generally comprises administering to the subject a composition comprising the imaging agent or therapeutic agent conjugated to a viral Gn polypeptide, thereby improving transfer of a therapeutic or imaging agent transfer across the blood-brain-barrier. In some embodiments, the Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full- length amino acid sequence of SEQ ID NO: 2. In some embodiments the viral Gn polypeptide comprises the extracellular domain of a Gn polypeptide. In some embodiments, the Gn polypeptide comprises amino acids 1 to 316 of SEQ ID NO: 2. In some embodiments, the viral Gn polypeptide is a RVFV Gn polypeptide. In some embodiments, the imaging agent or therapeutic agent is directly or indirectly conjugated to the viral Gn polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

[0019] The application or patent file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] FIG. 1A-1 E show a pooled genome-scale CRISPR screen identifies Lrp1 and Lrp1 -associated proteins RAP and Grp94 as critical proteins for RVFV infection. FIG. 1A shows a schematic of the CRISPR/Cas9 screen in BV2 cells. FIG. 1 B shows Light microscope images (4X) of BV2 cells before infection and at 4 different time points post-infection. FIG. 1C shows at 18 dpi, surviving cells were reseeded into new flasks for reinfection on 19 dpi and imaged at 4X by light microscopy. FIG. 1 D shows Volcano plot analysis of the BV2 screen results of surviving cells from the initial infection at an MOI 0.1 . FIG. 1E shows a summary of key interactions that modulate Lrp1 surface presentation, including RAP and Grp94. FIG. 1 F shows Lrp1 is identified as an essential host entry factor for Rift Valley fever virus and a potential target for therapy against this pathogen.

[0021 ] FIG. 2A-2H show LRP1 is essential for RVFV infection of BV2 cells. FIG. 2A shows a western blot of BV2 Lrp1 knockout clones (Lrp1 ^K0 C3, Lrp1 ^K0 R1 , Lrp1 ^K0 R2, Lrp1 ^K0 R4, Lrp1 ^K0 R5, and Lrp1 ^K0 R6), and partial knockout (Lrp1 P^K0 R3) generated using either single gRNA or dual gRNA CRISPR/Cas9 approaches, as described in methods. FIG. 2B shows BV2 wildtype (WT) and Lrp1 ^K0 clones were infected with RVFV ZH501 at a MOI of 0.1 . After 18 hours, the cells were harvested for RNA extraction and subjected to qRT-PCR analysis. Data shown are viral RNA (vRNA) titers normalized to wildtype BV2 cells. FIG. 2C shows microscopic images showing the WT and LRP1 ^K0 R4 cells infected with RVFV MP12^GFP in fluorescence images (top panels) and bright-field images (bottom panels). Images were taken at 20X magnification. FIG. 2D shows flow cytometry of WT, Lrp1 P^K0 R3, and Lrp1 ^K0 R4 cells infected with RVFV MP12^GFP. FIG. 2E shows corresponding analysis of flow cytometry histograms in D. FIG. 2F shows western blot of mouse embryonic fibroblasts (MEFs) from Lrp1 ^+/+ and Lrp1^F/F mice infected with Ad^Cre. FIG. 2G shows representative flow cytometry of MEFs Lrp1 ^+/+ and Lrp^F/F cells 5 dpi with Ad^Cre and then infected with RVFV- MP12^GFP at MOI of 1 for 15 hours. FIG. 2H shows corresponding analysis of flow cytometry histogram data in G.

[0022] FIG. 3A-3F show RAP and GRP94 can reduce RVFV infection indirectly by modulating Lrp1 levels. FIG. 3A shows Western blot of BV2 knockout clones for RAP probed with an anti-Lrp1 antibody. FIG. 3B shows BV2 knockout clones in A were infected with RVFV-MP12^GFP 439 at an MO1 1 for 15 hours. The cells were examined for virus infection (GFP) using flow cytometry. FIG. 3C shows quantitative analysis of flow data in (B). Data are expressed as % infection relative to BV2 WT cells. FIG. 3D shows western blot of BV2 knockout clones for Grp94 probed with an anti-Lrp1 antibody. FIG. 3E shows BV2 knockout clones in D were infected with RVFV-MP12^GFP at an MO1 1 for 15 hours. The cells examined for virus infection (GFP) using flow cytometry. FIG. 3F shows quantification of the flow data in E. Data are expressed as % infection relative to BV2 WT cells. Experiments were done at least three times. ****, p<0.0001.

[0023] FIG. 4A-4I show Lrp1 binds RVFV glycoprotein Gn. FIG. 4A shows LRP1 comprises of four clusters, CL¹, CL" , CL¹¹¹ , and CL^IV, and the cytoplasmic and transmembrane domains (left). Mini-domains CL¹, CL", CL¹", and CL^IV were generated as-Fc fusions (top right) and were used in these studies. Lentiviruses carrying either pLVX- empty vector or pLVX-expressing minidomains mini-LRP1 CL¹, CL", CL¹", and CL^IV were also generated (bottom right). FIG. 4B shows BV2 WT and Lrp1 ^K0 cells were transduced with lentiviruses carrying either pLVX- empty vector (EV) or pLVX- expressing mini-LRP1 CL¹, CL", CL¹", and CL^IV prior to infection with RVFV MP12^GFP. The bar graph shows the quantification of % infectivity. Biolayer interferometry sensorgrams of RVFV Gn binding to immobilized: FIG. 4C shows the Fc control. FIG. 4D shows Fc-hLrp1 CLn. FIG. 4E shows Fc-hLrp1 CLm. FIG. 4F shows Fc-hLrp1 CLiv. Impact on MP12 GFP infection in the presence of exogenous (FIG. 4G) Fc control; (FIG. 4H) shows Fc-hLrp1 CLn; (FIG. 4I) shows Fc-hLrp1 CLm_; Fc-hLrp1 CLiv.. BV2 WT cells were pre-incubated for 1 hour with 2.5 pg/mL of hl_rp1 CLII-specific (15408 and 15409) or CLIV-specific antibodies (15430 and 15438) and then infected with RVFV MP12GFP. Cells were analyzed for virus infection after 16 hours. Bar graph represents % cells infected after the antibody treatment, compared to the infection of untreated cells. Dose-response curve showing the inhibition of RVFV MP12^GFP infection of BV2 cells (y-axis) with EC50 936 ± 78 ng/mL after treatment with serial dilutions of IgG 15408 (x-axis)

[0024] FIG. 5A-5F show Lrp1 is critical for virus binding and internalization and anti-l_rp1 Abs inhibit RVFV infection. FIG. 5A shows to evaluate binding versus internalization, BV2 WT and BV2 Lrp1 KO R4 cells were incubated with RVFV MP12^GFP at 4 C for binding assay. FIG. 5B shows 37 C for internalization assay. FIG. 5C shows quantification of Alexa Fluor labeled viral particles binding with BV2 WT and BV2 Lrp1 ^K0 R4 cells. FIG. 5D shows 37 C were evaluated and normalized to respective levels of BV2 WT cells. FIG. 5E shows BV2 WT cells were pre-incubated for 1 h with 2.5 mg/mL of hl_rp1 CLn-specific (15409), CLiv-specific antibodies (15438), and bi-specific (15408 and 15430) and then infected with RVFV MP12GFP . Cells were analyzed for virus infection after 16 h. Bar graph represents % cells infected after the antibody treatment compared to the infection of untreated cells. FIG. 5F shows dose-response curve showing the inhibition of RVFV MP12^GFP infection of BV2 cells with ECso 936 ± 78 ng/mL after treatment with serial dilutions of IgG 15408.

[0025] FIG. 6A-6K mRAPos competes with RVFV glycoprotein Gn for binding to Lrp 1 and inhibits RVFV infection. FIG. 6A shows domain organization of mouse RAP (mRAP) protein. FIG. 6B shows BLI sensograms of mRAPD3 binding to immobilized. FIG. 6C shows LRP1 CLn and immobilized LRP1 CLiv. FIG. 6D shows mRAPos competition assay to assess relative binding of Gn to LRP1 CLiv in the presence of 1 , 3, 6, or 10 mg/mL concentrations of mRAPos. FIG. 6E shows flow cytometry data for BV2 cells infected with RVFV MP12^GFP in the presence of increasing concentrations of mRAPos. FIG. 6F shows analysis of relative infectivity as a function of mRAPos concentration. ECso is 0.59 ± 0.2 mg/mL. FIG. 6G shows BLI sensograms showing the binding of FTIRAPDS (blue, black) and mutant mRAPos (cyan, red) with LRP1 CLn and FIG. 6H shows LRP1 CLIV. FIG. 6I shows RVFV MP12^GFP infection of BV2 cells in presence of mRAPos and mutant mRAPos. FIG. 6J shows cell lines from different species were infected with RVFV-MP12^GFP at an MO1 1 in the absence ( ) or presence (+) of 5 mg/mL of mRAPD3(10³ ECso). Infection was assessed 15 hpi by flow cytometry. FIG. 6K shows mouse (BV2) and human (HepG2 and SH-SY5Y) cell lines were infected with RVFV ZH501 at an MO1 1 in the absence ( ) or presence (+) of mRAPos. Infection was assessed at 18 hpi by RT-qPCR on cell supernatants and intracellular flow cytometry for viral Gn protein.

[0026] FIG. 7A-7E show mRAP binding to Lrp1 protects mice from intracranial infection of RVFV ZH501 . FIG. 7A shows survival of mice infected intracranially with 10 PFU of RVFV ZH501 in absence or presence of 215 mg of recombinant mRAPD3 protein, 210 mg of mutant mRAPD3, and 250 mg of control protein (Ebola VP30). FIG. 7B shows in a second experiment, 3 mice/group were euthanized at 3 dpi, and liver, spleen, brain, and serum were harvested at necropsy and assessed for (FIG. 7B) viral RNA levels by RT-qPCR or FIG. 7C shows infectious virus by plaque assay. Heatmaps show average log-transformed titer for each tissue (indicated by the number in each cell of the heatmap) and are also represented visually by the color shading in the legend. X through the cell indicates samples that were not available for analysis. FIG. 7D shows pathology in liver and FIG. 7E shows brain tissue was assessed by immunofluorescence for viral antigen using an anti-NP antibody (top panels) or H&E staining (lower panels) in presence or absence of the indicated proteins. Images were taken at 203 magnification. The liver and brain tissues shown in (FIG. 7D) and (FIG. 7E) are from respective animals; IF and H&E are from the same tissues.

[0027] FIG. 8A-8F show OROV and RVFV show reduced infection in multiple cell lines that are KO for Lrp 1 . FIG. 8A shows infection of Lrp 1 KO and RAP KO cell lines described above, with RVFV and OROV at MOI 0.1. Infection of WT + and Lrp1 KO - versions of FIG. 8B shows HEK293T, FIG. 8C shows A549, and FIG. 8D shows N2a cells with OROV, RVFV, and ZIKV at MOI 0.1. OROV and RVFV samples were harvested at 24 hpi, and infectious virus was measured by viral plaque assay. ZIKV samples were harvested at 48 hpi and viral RNA (vRNA) was evaluated by qRT- PCR. Fluorescent microscopy (20) of FIG. 8E shows OROV and FIG. 8F shows RVFV infection of A549 WT and Lrp1 KO cells at MOI 0.1 at 24 hpi. Scale bars, 250 pm. Statistical significance was determined using an unpaired t test on log-transformed data. Experiments were repeated three times. **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001 .

[0028] FIG. 9A-9C show the Lrp1 -binding chaperone RAP can inhibit OROV infection of Vero E6 cells and undifferentiated SH-SY5Y cells. FIG. 9A shows RAP is a 39-kDa ER-resident protein consisting of 3 domains (D1 -3) that chaperones LDLR family proteins, including LRP1. Recombinant mRAPD3 WT and mutant mRAPD3 Mut (K256A and K270E) were expressed and purified from BL21 (DE3) cells using an N-terminal His-tag. mRAPD3 or mutant mRAPD3 was added to FIG. 9B shows Vero E6 nonhuman primate cells or FIG. 9C shows SH-SY5Y human neuroblastoma cells 1 h before infection with MOI 0.1 of RVFV, OROV, or ZIKV. Samples were harvested at 24 hpi (for RVFV and OROV) or 48 hpi (for ZIKV), and infectious virus was measured by plaque assay or qRT-PCR. Statistical significance was determined using two-way ANOVA on log-transformed data. Experiments were repeated three times. **P < 0.01 ; ****P < 0.0001.

[0029] FIG. 10A-10D show Lrp1 KO reduces VSV-OROV infection in BV2 cells and VSV-OROV binds to Lrp1 CLiv. BV2 WT and BV2 Lrp1 KO R4 cells were infected with MO1 1 of VSV or MOI of 5 of VSV-OROV. Samples were collected at 6 and 8 hpi to be processed by FIG. 10A shows flow cytometry or FIG. 10B shows imaging by fluorescent microscopy (20*). Scale bars, 50 pm. FIG. 10C shows LRP1 consists of a 515-kDa extracellular alpha chain (blue/tan) and an 85-kDa intracellular beta chain (not shown) connected by a transmembrane domain (gray). The alpha chain is further divided into four complement-type repeat clusters (CLi-iv; blue), and epidermal growth factor (EGF)-like and YWTD domains (tan). Recombinant Fc-fused LRP1 CLn and CLiv were expressed and purified from Expi293 cells for the experiments presented here. FIG. 10D shows AHC sensors coated with either Fc or Lrp1 -CLiv-Fc and incubated with VSV-OROV particles. Sensograms show the over-time association and dissociation of virus particles to coated sensors. Significance was determined using an unpaired t test. Experiments were repeated two times. **P < 0.01 ; ****P < 0.0001 . [0030] FIG. 11A-11C show soluble Fc-bound Lrp1 CLII and CLiv inhibit cellular infection by OROV. FIG. 11 A shows soluble Fc-bound CLn, CLiv, or Fc control proteins were added to Vero E6 cells 1 h before infection with OROV, FIG. 11 B shows RVFV, or FIG. 11 C shows ZIKV at MOI 0.1. Samples were harvested at 24 hpi (OROV and RVFV) or 48 hpi (ZIKV), and virus was measured by plaque assay and qRT-PCR. Data are expressed as a percentage of untreated control titers. Statistical significance was determined using two-way ANOVA. Experiments were repeated two times. ****P < 0.0001.

[0031 ] FIG. 12A-12B show RVFV Gn inhibits cellular infection by OROV. FIG. 12A shows RVFV Gn was added to BV2 mouse microglia cells 1 h before infection with MOI 0.1 of OROV or RVFV ZH501 . Samples were collected at 24 hpi and processed by viral plaque assay. FIG. 12B shows RVFV Gn was added to Vero E6 nonhuman primate cells 1 h before infection with MOI 0.1 of OROV, RVFV, or ZIKV. Samples were harvested at 24 hpi (for RVFV and OROV) or 48 hpi (for ZIKV) and infectious virus was measured by plaque assay and qRT-PCR. Data are expressed as a percentage of untreated control titers. Statistical significance was determined using oneway ANOVA. Experiments were repeated two times. ***P < 0.001 .

[0032] FIG. 13A-13C show mRAPos protects mice from lethal OROV IC infection and significantly reduces infectious virus in the brain at 3 dpi. FIG. 13A shows mice were infected with 100 PFU of OROV IC alone or in combination with either mRAPos, mutant mRAPos, or the control protein VP30. They were monitored for 15 d to determine percentage of survival in each group. FIG. 13B shows a subset of mice from each group was euthanized at 3 dpi to collect brain tissue, which was processed by viral plaque assay. FIG. 13C shows immunofluorescent microscopy of brain tissues (cerebral cortex) from mice euthanized at 3 dpi (20x). Scale bars, 250 pm. Statistical significance was determined using a Mantel-Cox test for survival and two-way ANOVA for log- transformed data. Experiments were repeated four times. **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001. No tx, No treatment; mut, mutant.

[0033] FIG. 14A shows a schematic of experiment LRP1 CLIV-Fc was immobilized on AHC BLI pins and sequentially dipped into wells containing 22- 200 nM 2N4R tau (produced in E coli). FIG. 14B shows sensorgrams (black) with corresponding 1 : 1 kinetic fits revealing KD = 9.5 ± 0.2 nM. Similar results were obtained using 2N4R tau produced in HEK293 cells (not shown).

[0034] FIG. 15A shows Fc-LRP1 CLIV was immobilized on anti-human-Fc (AHC) BLI pins and sequentially dipped into RVFV Gn (100 nM) followed by 2N4R tau (100 nM). Competition binding experiments are shown in blue while non-blocked controls are shown in red. FIG. 15B shows a schematic of experiment and results observed. FIG. 15C shows immobilized Fc-LRP1 CLIV dipped into 2N4R tau (100 nM) followed by RVFV Gn (100 nM). Similar results were obtained using 10 nM RVFV Gn (data not shown). FIG. 15D shows schematic of experiment and results. Binding buffer: PBS with 0.005% Tween 20, 0.1 % BSA.

[0035] FIG. 16 shows engineered decoy viral inhibitors (DEVi). SpyTag/SpyCatcher-based LRP1 binding RVFV Gn multimer development as Tau inhibitors. OD- oligomerization domain. Protein-RVFV Gn or RAP protein, to prevent Tau spread by Lrp1 .

DETAILED DESCRIPTION

[0036] The present disclosure is based, at least in part, on the discovery that low-density lipoprotein receptor-related protein 1 (Lrp1 ) is a mediator of Rift Valley Fever Virus infection (RVFV). As shown herein, Lrp1 activity is required for RVFV infectivity. Interestingly, when the activity of Lrp1 is reduced or absent (e.g., reduced expression or binding), RVFV infection and viral production is significantly reduced. Moreover, fusion proteins comprising Lrp1 domains or RAP domains are shown to be useful in reducing viral infection by competing with Lrp1 binding. In addition, RVFV glycoprotein Gn or fragments thereof are shown to mediate viral entry via Lrp1 . Thus the Gn peptide or fragments thereof are useful as a carrier of therapeutic agents in the CNS, enhancing the ability to cross the blood-brain barrier. Modifications to Gn allow for tuning of the complex to target specific cells or regions of interest. Lastly, Gn is shown to be inhibitor of Tau, by competing with Tau for biologically significant binding sites.

[0037] Understanding the mechanics of macromolecular entry into cells is crucial to the study of many different types of diseases. From infectious agents to progressive neurologic disorders, characterizing and targeting the mechanisms by which the causative agents of a disease enter the target cells has proven to be a strong therapeutic strategy. Unfortunately, the entry mechanisms of many agents remain woefully understudied and warrant further investigation.

[0038] The present disclosure provides the entry mechanisms of arthropod-borne agents with the discovery of LDL receptor related protein 1 (LRP1 ) as a receptor of the highly pathogenic bunyaviruses. For example, the importance of LRP1 in RVFV infections was first uncovered through the use of genome-wide CRISPR screening, which implicated both LRP1 and its essential host chaperones, receptor- associated protein (RAP) and glucose-regulated protein 94 (GRP94), as key components of RVFV infection. LRP1^F/F BV2 cells (microglia) and LRP1 -deficient mouse embryonic fibroblasts (MEFs) infected with RVFV had drastically reduced viral replication. In both systems, diminished viral entry was observed, suggesting that LRP1 as a viral entry factor. Utilizing the high-affinity interaction of LRP1 and extracellular RAP to block RVFV infection, LRP1 was demonstrated to be a key receptor in several cell lines, achieving nearly complete inhibition of infection. Treating the susceptible mice with extracellular RAP, to block LRP1 , resulted in >60% survival after RVFV infection, as opposed to 0% survival after infection in control animals. Collectively, these data demonstrate a significant biological importance of LRP1 in RVFV infection.

[0039] The present disclosure provides significant strides in deciphering the biophysical mechanisms of the LRP1 : RVFV interaction. Previous entry factors, including DC-SIGN and heparan sulfate, were demonstrated to act in a glycosylationdependent manner. The present disclosure demonstrated that the removal of glycosylation on both RVFV Gn/Gc glycoproteins and LRP1 had little effect on viral entry. These data support a glycosylation-independent, receptor-mediated mechanism of RVFV entry.

[0040] Recent structural advances in our understanding of RVFV Gn/Gc glycoproteins have elucidated a lipid-binding pocket that appears to be conserved across many arthropod-borne viruses. Given the ability of LRP1 to bind various lipid species, lipids may play a role in the observed interaction of LRP1 and RVFV Gn/Gc.

[0041 ] The factors determining vector competence and reservoir hosts’ susceptibility for arthropod-borne viruses remain poorly understood. LRP1 has significant sequence conservation across species, suggesting common functionality. The LRP1 protein of certain livestock, such as Bos taurus, shows significant homology to that of humans, implying a potential biological function in bovine infection with RVFV.

[0042] More broadly, the role of LRP1 in other diseases, specifically Alzheimer’s disease, has been demonstrated that LRP1 plays a significant role in tau uptake. In the presence of functional LRP1 , increased seeding and aggregate formation was observed. Although tau is not the only component of severe Alzheimer’s disease, prevention of its aggregation is believed to be an important component of successful treatment. The implication of LRP1 in these two unrelated diseases, RVFV and Alzheimer’s, implies a broader functionality of LRP1 as a cellular entry receptor.

[0043] Collectively, these observations solidify the role of LRP1 in the pathogenesis of multiple types of illnesses. Discoveries of this nature are infrequent, making the therapeutic targeting of LRP1 a promising strategy in the battle of various diseases.

[0044] Altogether, the present disclosure provides multiple lines of evidence showing the presently disclosed compositions and methods to be useful in the in the prevention of viral infection, transport of therapeutic agents and the inhibition of Tau in the central nervous system. Other aspects and iterations of the invention are described more thoroughly below.

[0045] Disclosed herein are compositions, methods, and treatment plans for treating an individual who is at risk of having a viral infection, has symptoms of a viral infection, or treating or preventing a tauopathy. A composition of the present disclosure comprising a Lrp I decoy receptor or bunyavirus Gn polypeptide disclosed herein may be used to treat, prevent, or reduce the infectivity of a viral infection. A treatment plan may comprise administering a composition of the disclosure to an individual having or at risk of having a viral infection or tauopathy, thereby preventing or treating the viral infection or tauopathy. In some embodiments, a viral infection may be prevented by reducing the amount of virus capable of binding to a host cell or tissue. For example, a composition of the present disclosure may comprise a decoy receptor or decoy viral inhibitor and a viral infection may be prevented by disrupting interactions between a viral surface proteins and host cell proteins that activate or enhance insertion of the viral genetic material into the host cell. For example, interactions between a viral bunyavirus Gn protein, and a host cell Lrp1 receptor.

I. Definitions

[0046] The term "a" or "an" entity refers to one or more of that entity; for example, a "polypeptide subunit" is understood to represent one or more polypeptide subunits. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0047] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0048] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[0049] Where applicable, units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. Nucleic acid sequences are written from 5’ to 3’, left to right.

[0050] The headings provided herein are not limitations of the various aspects and embodiments of the disclosure, which can be had by reference to the specification as a whole.

[0051 ] Terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0052] As used herein, the term “non-naturally occurring” substance, composition, entity, and/or any combination of substances, compositions, or entities, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the substance, composition, entity, and/or any combination of substances, compositions, or entities that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”

[0053] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by peptide bonds (also known as amide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0054] A “protein” as used herein can refer to a single polypeptide, i.e. , a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, hydrophobic interactions, etc., to produce, e.g., a multimeric protein.

[0055] As used herein, the term “non-naturally occurring” polypeptide, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the polypeptide that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”

[0056] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" when referring to polypeptide subunit or multimeric protein as disclosed herein can include any polypeptide or protein that retain at least some of the activities of the complete polypeptide or protein, but which is structurally different. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments. Variants include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur spontaneously or be intentionally constructed. Intentionally constructed variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, insertions, and/or deletions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the native polypeptide, such as increased resistance to proteolytic degradation. Examples include fusion proteins. Variant polypeptides can also be referred to herein as "polypeptide analogs." As used herein a "derivative" also refers to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides that contain one or more standard or synthetic amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0057] A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0058] As used herein, the term “binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. As described further herein, a binding molecule can comprise one of more “binding domains.” As used herein, a "binding domain" is a two- or three-dimensional polypeptide structure that cans specifically bind a given antigenic determinant, or epitope. A non-limiting example of a binding molecule is an antibody or fragment thereof that comprises a binding domain that specifically binds an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody comprising a first binding domain binding to a first epitope, and a second binding domain binding to a second epitope.

[0059] Disclosed herein are certain binding molecules, or antigen-binding fragments, variants and/or derivatives thereof. Unless specifically referring to full-sized antibodies such as naturally-occurring antibodies, the term "binding molecule" encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally-occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.

[0060] By "specifically binds," it is meant that a binding molecule, e.g., an antibody or antigen-binding fragment thereof binds to an epitope via its antigen binding domain, and that the binding entails some recognition between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding domain binds more readily than it would bind to a random, unrelated epitope.

[0061 ] The terms “treat,” "treating," or "treatment" as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition, or disorder or those in which the disease, condition or disorder is to be prevented.

[0062] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and does not contain components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.

[0063] An "effective amount" as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0064] Bunyavirales is an order of single-strand, spherical, enveloped RNA viruses (formerly the Bunyaviridae family). The virus families in the Bunyavirales order that cause viral hemorrhagic fevers include Phenuiviridae, Arenaviridae, Nairoviridae, and Hantaviridae. Distribution of these viruses is determined by the distribution of the vector and host species. Non-limiting examples of Bunyavirus include Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Hantavirus, Oropouche virus, and La Crosse virus (LACV).

[0065] Most bunyaviruses contain a tripartite genome, consisting of a large (L), medium (M) and small (S) RNA segment. The L segment encodes the L protein, which has the RNA-dependent RNA polymerase (RdRp) and endonuclease functions, the M segment the glycoprotein precursor (GPC), and the S segment the nucleocapsid protein (N), which encapsidates the genomic RNA. In addition, some bunyaviruses encode nonstructural (NS) proteins, such as NSm (whose gene resides on the M segment) and NSs (on the S segment).

[0066] Degenerate variant: In the context of the present disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0067] In one example, a desired response is to inhibit or reduce or prevent a bunyaviral infection. The infection does not need to be completely eliminated or reduced or prevented for the method to be effective. For example, administration of an effective amount of a composition of the disclosure decreases the infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the virus) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infection), as compared to a suitable control.

[0068] As used herein, the term “subject” refers to a mammal, preferably a human. The mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.

[0069] The term “tau” refers to a plurality of isoforms encoded by the gene MAPT (or homolog thereof), as well as species thereof that are C-terminally truncated in vivo, N-term inally truncated in vivo, post-translationally modified in vivo, or any combination thereof. As used herein, the terms “tau” and “tau protein” and “tau species” may be used interchangeably. In many animals, including but not limited to humans, non-human primates, rodents, fish, cattle, frogs, goats, and chicken, tau is encoded by the gene MAPT. In animals where the gene is not identified as MAPT, a homolog may be identified by methods well known in the art.

[0070] In humans, there are six isoforms of tau that are generated by alternative splicing of exons 2, 3, and 10 of MAPT. These isoforms range in length from 352 to 441 amino acids. Exons 2 and 3 encode 29-amino acid inserts each in the N- terminus (called N), and full-length human tau isoforms may have both inserts (2N), one insert (1 N), or no inserts (ON). All full-length human tau isoforms also have three repeats of the microtubule binding domain (called R). Inclusion of exon 10 at the C- terminus leads to inclusion of a fourth microtubule binding domain encoded by exon 10. Hence, full-length human tau isoforms may be comprised of four repeats of the microtubule binding domain (exon 10 included: R1 , R2, R3, and R4) or three repeats of the microtubule binding domain (exon 10 excluded: R1 , R3, and R4). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau may be phosphorylated, ubiquinated, glycosylated, and glycated. Human tau also may or may not be proteolytically processed in vivo at the C-terminus, at the N-terminus, or at the C-terminus and the N-terminus. Accordingly, the term “human tau” encompasses the 2N3R, 2N4R, 1 N3R, 1 N4R, 0N3R, and 0N4R isoforms, as well as species thereof that are C-term inally truncated in vivo, N-terminally truncated in vivo, post-translationally modified in vivo, or any combination thereof. Alternative splicing of the gene encoding tau similarly occurs in other animals.

[0071 ] A disease associated with tau deposition in the brain is referred to herein as a “tauopathy”. The term “tau deposition” is inclusive of all forms pathological tau deposits including but not limited to neurofibrillary tangles, neuropil threads, and tau aggregates in dystrophic neurites. Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy (PSP), dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease, Parkinson-dementia complex of Guam, tangle-predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden- Spatz disease, lipofuscinosis, Pick’s disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Frontotemporal lobar degeneration (FTLD), Alzheimer’s disease (AD), and frontotemporal dementia (FTD).

[0072] Tauopathies are classified by the predominance of tau isoforms found in the pathological tau deposits. Those tauopathies with tau deposits predominantly composed of tau with three MTBRs are referred to as “3R-tauopathies”. Pick’s disease is a non-limiting example of a 3R-tauopathy. For clarification, pathological tau deposits of some 3R-tauopathies may be a mix of 3R and 4R tau isoforms with 3R isoforms predominant. Intracellular neurofibrillary tangles (i.e. tau deposits) in brains of subjects with Alzheimer’s disease are generally thought to contain both approximately equal amounts of 3R and 4R isoforms. Those tauopathies with tau deposits predominantly composed of tau with four MTBRs are referred to as “4R- tauopathies”. PSP, CBD, and AGD are non-limiting examples of 4R-tauopathies, as are some forms of FTLD. Notably, pathological tau deposits in brains of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, show a mix of 3R and 4R isoforms.

[0073] A clinical sign of a tauopathy may be aggregates of tau in the brain, including but not limited to neurofibrillary tangles. Methods for detecting and quantifying tau aggregates in the brain are known in the art (e.g., tau PET using tau-specific ligands such as [18F]THK5317, [18F]THK5351 , [18F]AV1451 , [11C]PBB3, [18F]MK-6240, [18F]RO-948, [18F]PI-2620, [18F]GTP1 , [18F]PM-PBB3, and [18F]JN J64349311 , [18F]JNJ-067), etc.).

[0074] The terms “treat,” “treating,” or “treatment” as used herein, refers to the provision of medical care by a trained and licensed professional to a subject in need thereof. The medical care may be a diagnostic test, a therapeutic treatment, and/or a prophylactic or preventative measure. The object of therapeutic and prophylactic treatments is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results of therapeutic or prophylactic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as com-pared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of disease.

II. Compositions

[0075] One aspect of the present disclosure encompasses a composition for modulating LRP1 activity. As described herein, modulation of LRP1 activity is useful in treating or preventing a viral infection. In addition, modulation of LRP1 activity is useful in treating or preventing a tauopathy. In some embodiments, LRP1 activity is modulated by reducing LRP1 activity. The term “low density lipoprotein receptor-related protein 1” or “LRP1” interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1 ) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a LRP1 nucleic acid (see, e.g., GenBank Accession No. NM_002332.2) or to an amino acid sequence of a LRP1 polypeptide (see, e.g., GenBank Accession No. NP_002323.2); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a LRP1 polypeptide (e.g., LRP1 polypeptides described herein); or an amino acid sequence encoded by a LRP1 nucleic acid (e.g., LRP1 polynucleotides described herein), and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a LRP1 protein, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a LRP1 nucleic acid (e.g., LRP1 polynucleotides, as described herein, and LRP1 polynucleotides that encode LRP1 polypeptides, as described herein).

[0076] In some embodiments, the present disclosure provides a recombinant LRP1 polypeptide or a fragment thereof as a decoy receptor. The structural organization of the LRP1 receptor, which is a type 1 transmembrane receptor consisting of a 515-kDa entirely extracellular a-chain non-covalently bound to an intracellular 85-kDa [3-chain. The a-chain, primarily responsible of the ligand-binding activity of LRP1 , includes four clusters of complement-like repeats (CCRs I-IV) and EGF- like domains. The [3-chain includes a tetra amino acidic YxxL motif, two NPxY motifs, which serve as docking sites for signaling adapter proteins, and numerous tyrosine residues, whose phosphorylation is necessary for LRP1 -mediated signal transduction. Accordingly, a fragment of LRP1 may be, in non-limiting examples, is an extracellular- domain, transmembrane-domain, or cytoplasmic-domain fragment. In an embodiment, a fragment thereof is one or more of the clusters of complement-like repeats. In a specific embodiment, an LRP1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

(MLTPPLLLLLPLLSALVAAAIDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSD EAPEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQDCMDGSDEGPHCRELQGN CSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGS FICGCVEGYLLQPDNRSCKAKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQT TAMDFSYANETVCWVHVGDSAAQTQLKCARMPGLKGFVDEHTINISLSLHHVEQMAID WLTGNFYFVDDIDDRIFVCNRNGDTCVTLLDLELYNPKGIALDPAMGKVFFTDYGQIPK VERCDMDGQNRTKLVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVVDYEGKGRQTIIQ GILIEHLYGLTVFENYLYATNSDNANAQQKTSVIRVNRFNSTEYQWTRVDKGGALHIY HQRRQPRVRSHACENDQYGKPGGCSDICLLANSHKARTCRCRSGFSLGSDGKSCKK PEHELFLVYGKGRPGIIRGMDMGAKVPDEHMIPIENLMNPRALDFHAETGFIYFADTTS YLIGRQKIDGTERETILKDGIHNVEGVAVDWMGDNLYWTDDGPKKTISVARLEKAAQT

RKTLIEGKMTHPRAIWDPLNGWMYWTDWEEDPKDSRRGRLERAWMDGSHRDIFVT SKTVLWPNGLSLDIPAGRLYVWDAFYDRIETILLNGTDRKIVYEGPELNHAFGLCHHGN YLFWTEYRSGSVYRLERGVGGAPPTVTLLRSERPPIFEIRMYDAQQQQVGTNKCRVN NGGCSSLCLATPGSRQCACAEDQVLDADGVTCLANPSYVPPPQCQPGEFACANSRCI QERWKCDGDNDCLDNSDEAPALCHQHTCPSDRFKCENNRCIPNRWLCDGDNDCGN SEDESNATCSARTCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPL TQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHWTCD GDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRWRCDGDTDCMD SSDEKSCEGVTHVCDPSVKFGCKDSARCISKA WCDGDNDCEDNSDEENCESLACR PPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNNGGCSHNCSVAPG

EGIVCSCPLGMELGPDNHTCQIQSYCAKHLKCSQKCDQNKFSVKCSCYEGWVLEPDG ESCRSLDPFKPFIIFSNRHEIRRIDLHKGDYSVLVPGLRNTIALDFHLSQSALYWTDWE DKIYRGKLLDNGALTSFEWIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGTLR TTLLAGDIEHPRAIALDPRDGILFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWP NGLTVDYLEKRILWIDARSDAIYSARYDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWT DWRTNTLAKANKWTGHNVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPC SHLCLINYNRTVSCACPHLMKLHKDNTTCYEFKKFLLYARQMEIRGVDLDAPYYNYIISF TVPDIDNVTVLDYDAREQRVYWSDVRTQAIKRAFINGTGVETVVSADLPNAHGLAVDW VSRNLFWTSYDTNKKQINVARLDGSFKNAWQGLEQPHGLWHPLRGKLYWTDGDNI SMANMDGSNRTLLFSGQKGPVGLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAM

RSQLGKATALAIMGDKLWWADQVSEKMGTCSKADGSGSWLRNSTTLVMHMKVYDE SIQLDHKGTNPCSVNNGDCSQLCLPTSETTRSCMCTAGYSLRSGQQACEGVGSFLLY SVHEGIRGIPLDPNDKSDALVPVSGTSLAVGIDFHAENDTIYWVDMGLSTISRAKRDQT WREDWTNGIGRVEGIAVDWIAGNIYWTDQGFDVIEVARLNGSFRYVVISQGLDKPRAI TVHPEKGYLFWTEWGQYPRIERSRLDGTERWLVNVSISWPNGISVDYQDGKLYWCD ARTDKIERIDLETGENREVVLSSNNMDMFSVSVFEDFIYWSDRTHANGSIKRGSKDNA TDSVPLRTGIGVQLKDIKVFNRDRQKGTNVCAVANGGCQQLCLYRGRGQRACACAH GMLAEDGASCREYAGYLLYSERTILKSIHLSDERNLNAPVQPFEDPEHMKNVIALAFDY RAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVENVGSVEGLAYHRGWDTLYWTSY TTSTITRHTVDQTRPGAFERETVITMSGDDHPRAFVLDECQNLMFWTNWNEQHPSIM RAALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDKIERCEYDGSHRYVILKSEPV HPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDIPQQPMGIIAVANDTNSC ELSPCRINNGGCQDLCLLTHQGHVNCSCRGGRILQDDLTCRAVNSSCRAQDEFECAN GECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVSNMLWCNGADD CGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEMNCSATDCSS YFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPRCPLNYFA CPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLCDGSDD CGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLY NSTCDDREFMCQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRC LSSRQWECDGENDCHDQSDEAPKNPHCTSQEHKCNASSQFLCSSGRCVAEALLCNG QDDCGDSSDERGCHINECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADV DECSTTFPCSQRCINTHGSYKCLCVEGYAPRGGDPHSCKAVTDEEPFLIFANRYYLRK LNLDGSNYTLLKQGLNNAVALDFDYREQMIYWTDVTTQGSMIRRMHLNGSNVQVLHR TGLSNPDGLAVDWVGGNLYWCDKGRDTIEVSKLNGAYRTVLVSSGLREPRALVVDVQ NGYLYWTDWGDHSLIGRIGMDGSSRSVIVDTKITWPNGLTLDYVTERIYWADAREDYI EFASLDGSNRHVVLSQDIPHIFALTLFEDYVYWTDWETKSINRAHKTTGTNKTLLISTLH RPMDLHVFHALRQPDVPNHPCKVNNGGCSNLCLLSPGGGHKCACPTNFYLGSDGRT CVSNCTASQFVCKNDKCIPFWWKCDTEDDCGDHSDEPPDCPEFKCRPGQFQCSTGI CTNPAFICDGDNDCQDNSDEANCDIHVCLPSQFKCTNTNRCIPGIFRCNGQDNCGDG EDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCVDGSDEPANCTQMTCGVDE FRCKDSGRCIPARWKCDGEDDCGDGSDEPKEECDERTCEPYQFRCKNNRCVPGRW QCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDCADGSDEKD CTPRCDMDQFQCKSGHCIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQCNN TLCKPLAWKCDGEDDCGDNSDENPEECARFVCPPNRPFRCKNDRVCLWIGRQCDGT DNCGDGTDEEDCEPPTAHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEED CSIDPKLTSCATNASICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLRFGT CSQLCNNTKGGHLCSCARNFMKTHNTCKAEGSEYQVLYIADDNEIRSLFPGHPHSAY EQAFQGDESVRIDAMDVHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDR GVTHLNISGLKMPRGIAIDWVAGNVYWTDSGRDVIEVAQMKGENRKTLISGMIDEPHAI WDPLRGTMYWSDWGNHPKIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWA DAKLSVIGSIRLNGTDPIVAADSKRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPL VNLTGGLSHASDWLYHQHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDN GTCVPVPSPTPPPDAPRPGTCNLQCFNGGSCFLNARRQPKCRCQPRYTGDKCELDQ CWEHCRNGGTCAASPSGMPTCRCPTGFTGPKCTQQVCAGYCANNSTCTVNQGNQP QCRCLPGFLGDRCQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRCEVNKCS RCLEGACWNKQSGDVTCNCTDGRVAPSCLTCVGHCSNGGSCTMNSKMMPECQCP PHMTGPRCEEHVFSQQQPGHIASILIPLLLLLLLVLVAGVVFWYKRRVQGAKGFQHQR MTNGAM NVEIGNPTYKMYEGGEPDDVGGLLDADFALDPDKPTNFTNPVYATLYMGG HGSRHSLASTDEKRELLGRGPEDEIGDPLA (SEQ ID NO: 1 )).

[0077] The terms “inhibiting,” “reducing,” “decreasing” with respect to

LRP1 function refers to inhibiting the function of LRP1 in a subject by a measurable amount using any method known in the art (e.g., binding and/or endocytosis; cellsignaling mediated downstream of LRP1 ; viral entry and/or replication; and/or tau internalization). The LRP1 function is inhibited, reduced or decreased if the measurable amount of LRP1 function, e.g., of ligand binding and/or downstream activity, is at least about 10%, 20%, 30%, 50%, 80%, or 100% reduced in comparison to the measurable amount of LRP1 function prior to administration of an inhibitor of LRP1 . In some embodiments, the LRP1 function is inhibited, reduced or decreased by at least about 1 - fold, 2-fold, 3-fold, 4-fold, or more in comparison to the LRP1 function prior to administration of the inhibitor of LRP1 .

[0078] The term “selective inhibition” or “selectively inhibit” as referred to a biologically active agent refers to the agent's ability to preferentially reduce the target activity as compared to off-target activity, via direct or indirect interaction with the target. In various embodiments, the inhibitory agent inhibits, reduces or prevents the binding between LRP1 and a viral Gn protein. In another embodiment, the inhibitory agent reduces or prevents the binding between Lrp1 and tau.

[0079] The term “candidate agent” refers to any molecule of any composition, including proteins, peptides, nucleic acids, lipids, carbohydrates, organic molecules, inorganic molecules, and/or combinations of molecules which are suspected to be capable of inhibiting a measured parameter (e.g., LRP1 activity, expression, signal transduction, binding between LRP1 and the viral glycoprotein, e.g., the binding between LRP1 and Gn) in a treated cell, tissue or subject in comparison to an untreated cell, tissue or subject.

[0080] In some embodiments, the expression of LRP1 is reduced indirectly by reducing the expression of GPC3, SNX17, GRP94 or RAP; or by increasing the expression of PCSK9. In another embodiment, a composition for modulating Lrp1 activity comprises a RAP polypeptide or fragment thereof (e.g., RAPDS) is administered to inhibit LRP1 activity. The terms “low density lipoprotein receptor-related protein associated protein 1”, “LRPAP1 ,” “alpha-2-macroglobulin receptor-associated protein,” and “RAP” interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1 ) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a RAP nucleic acid (see, e.g., GenBank Accession No. NM_002337.2) or to an amino acid sequence of a RAP polypeptide (see, e.g., GenBank Accession No. NP_002328.1 ); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a RAP polypeptide (e.g., RAP polypeptides described herein); or an amino acid sequence encoded by a RAP nucleic acid (e.g., RAP polynucleotides described herein), and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a RAP protein, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a RAP nucleic acid (e.g., RAP polynucleotides, as described herein, and RAP polynucleotides that encode RAP polypeptides, as described herein). In some embodiments, the inhibitor is a RAP peptide or fragment thereof. In a specific embodiment, the RAP peptide is the RAP D3 domain.

[0081 ] In still another embodiment, the LRP1 inhibitor can compete for ligand binding or prevent ligand receptor interaction. For example, the inhibitor can be a decoy receptor fusion protein or an engineered decoy viral inhibitors. As used herein a “decoy viral inhibitor” refers to a viral protein (e.g. Gn) which is recombinantly expressed and used to compete with binding to the host receptor. In another aspect, a decoy viral inhibitor of the disclosure may also be used as a therapeutic or imaging agent acting as a carrier to facilitates transfer across the blood-brain barrier (BBB). Rift Valley fever virus (RVFV) is an arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface. “Gn” as used herein refers to a structural protein of RVFV, which contains an extracellular domain and a C-terminal Golgi localization signal. The term “Gn” as used herein also includes fragment, derivatives or homologs thereof. As disclosed herein, the ectodomain of the Gn protein has a MW of approximately 54 kDa. Also, as part of the instant disclosure encompass modifications of the Gn sequence comprising the ectodomain of Gn that result in the glycosylation of that protein and/or linkage to a therapeutic agent. In a specific embodiment, the present disclosure provides a RVFV glycoprotein Gn ectodomain (amino acid 1 - 316; accession number 329 DQ380200) (PMID: 28827346). The present disclosure has found the Gn to bind directly to LRP1 thereby facilitating viral entry across the BBB. In some embodiments, a Gn polypeptide of the disclosure comprises the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

(MYVLLTILISVLVCEAVIRVSLSSTREETCFGDSTNPEMIEGAWDSLREEEMPEELSCSI SGIREVKTSSQELYRALKAIIAADGLNNITCHGKDPEDKISLIKGPPHKKRVGIVRCERRR DAKQIGRETMAGIAMTVLPALAVFALAPWFAEDPHLRNRPGKGHNYIDGMTQEDATC KPVTYAGACSSFDVLLEKGKFPLFQSYAHHRTLLEAVHDTIIAKADPPSCDLQSAHGNP CMKEKLVMKTHCPNDYQSAHYLNNDGKMASVKCPPKYGLTEDCNFCRQMTGASLKK GSYPLQDLFCQSSEDDGSKLKTKMKGVCEVGVQAHKKCDGQLSTAHEVVPFAVFKN SKKVYLDKLDLKTEENLLPDSFVCFEHKGQYKGTMDSGQTKRELKSFDISQCPKIGGH GSKKCTGDAAFCSAYECTAQYANAYCSHANGSGIVQIQVSGVWKKPLCVGYERVWK RELSAKPIQRVEPCTTCITKCEPHGLWRSTGFKISSAVACASGVCVTGSQSPSTEITLK YPGISQSSGGDIGVHMAHDDQSVSSKIVAHCPPQDPCLVHGCIVCAHGLINYQCHTAL SAFVWFVFSSIAIICLAVLYRVLKCLKIAPRKVLNPLMWITAFIRWIYKKMVARVAHNINQ VNREIGWMEGGQLVLGNPAPIPRHAPIPRYSTYLMLLLIVSYASACSELIQASSRITTCS TEGVNTKCRLSGTALIRAGSVGAEACLMLKGVKEDQTKFLKIKTVSSELSCREGQSYW TGSISPKCLSSRRCHLVGECHVNRCLSWRDNETSAEFSFVGESTTMRENKCFEQCGG WGCGCFNVNPSCLFVHTYLQSVRKEALRVFNCIDWVHKLTLEITDFDGSVSTIDLGAS SSRFTNWGSVSLSLDAEGISGSNSFSFIESPSKGYAIVDEPFSEIPRQGFLGEIRCNSES SVLSAHESCLRAPNLISYKPMIDQLECTTNLIDPFVVFERGSLPQTRNDKTFAASKGNR GVQAFSKGSVQADLTLMFDNFEVDFVGAAVSCDAAFLNLTGCYSCNAGARVCLSITST GTGSLSAHNKDGSLHIVLPSENGTKDQCQILHFTVPEVEEEFMYSCDGDERPLLVKGT LIAIDPFDDRREAGGESTWNPKSGSWNFFDWFSGLMSWFGGPLKLYSSFACMLHYQ LGSFSSLYILEEQASLKCGLLPLRRPHRSVRVKVIC (SEQ ID NO: 2))

[0082] It is appreciated that the present disclosure is directed to homologues, variants, derivatives, or fragments of LRP1 , RAP, or Gn, in other organisms/variants and is not limited to human LRP1 or RAP or RVFV Gn.

Homologues, variants, derivatives, or fragments can be found in other species by methods known in the art. In determining whether LRP1 , RAP, or Gn has significant homology or shares a certain percentage of sequence identity with a sequence of the invention, sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit. In particular, “percent identity” of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA

87:2264-2268, 1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches may be performed with the BLASTN program to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. Equally, BLAST protein searches may be per-formed with the BLASTX program to obtain amino acid sequences that are homologous to a polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) are employed. See ncbi.nlm.nih.gov for more details.

[0083] A homologue, variant, derivative, or fragment of LRP1 , RAP, or Gn may be at least 80%, 85%, 90%, or 95% homologous to human LRP1 , RAP, or RVFV Gn . In certain embodiments, a homologue, variant or derivative of LRP1 , RAP, or Gn may be at least 80%, 85%, 90%, or 95% homologous to human LRP1 , RAP, or RVFV Gn.

[0084] In an aspect, the present disclosure provides a LRP1 -, RAP-, or Gn-fusion. The fusion protein may comprise full-length Lrp1 protein or an extracellular domain of LRP1 (e.g., CLn or CLiv) fused to an IgG molecule. Non-limiting examples of suitable fusion domains include IgG, IgA, IgE and IgM Fc regions. In a specific embodiment, the target receptor ligand is an IgG Fc region. Non-limiting examples of suitable target receptors include the Fc receptors: FcRy, FcRa, FcRs, and FcR|i. FcRy belongs to the immunoglobulin superfamily and includes several members, FcRyl (CD64), FcRyllA (CD32), FcRyllB (CD32), FcRylllA (CD16a), and FcRylllB (CD16b). In a specific embodiment, the target receptor is FcRyl (CD64). Fc receptors are cellsurface receptors that recognize the Fc region of an antibody. Non-limiting examples of target receptor ligands for an Fc receptor are IgG, IgA, IgE and IgM Fc regions. In a specific embodiment, the target receptor ligand is an IgG Fc region. In another embodiment, a targeting moiety may comprise an antibody capable of specifically binding to an antigenic determinant on a target site, or a fragment thereof that retains specific binding to the antigenic determinant. [0085] In another embodiment, the decoy receptor can comprise one or more full length LRP1 , RAP, or Gn peptides or one or more extracellular domains of Lrp 1 , for example at least one CLn domain(s) and at least one CLiv domain(s). In some embodiments, the decoy receptor(s) may be directly or indirectly conjugated to a nanoparticle. In certain embodiments, the SpyTag/SpyCatcher system is used. The SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins (e.g. Lrp1 decoy receptors). The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair. DNA sequence encoding either SpyTag or SpyCatcher can be recombinantly introduced into the DNA sequence encoding a protein of interest, forming a fusion protein. These fusion proteins can be covalently linked when mixed in a reaction through the SpyTag/SpyCatcher system. Using the Tag/Catcher pair, bioconjugation can be achieved between two recombinant proteins that would otherwise be restrictive or impossible with traditional direct genetic fusion between the two proteins. For example, issues regarding protein folding, suboptimal expression host, and specialized post-translational modifications can be alleviated by separating the production of the proteins with the modularity of the Tag/Catcher system.

[0086] As noted above, the present disclosure has found the Gn to bind directly to LRP1 thereby facilitating viral entry across the BBB. The present disclosure utilizes this interaction to facilitate the transfer of a therapeutic agent or imaging agent which is covalently or non-covalently attached to a Gn polypeptide. Thus, the Gn polypeptide may be conjugated to a payload, such as a therapeutic agent, a detectable, and/or a delivery device (including, but not limited to, a liposome or a nanoparticle) containing the drug or detectable label. Methods of conjugating a Gn protein to a therapeutic agent, a detectable label, a liposome, a nanoparticle or other delivery device are known in the art. Generally speaking, the conjugation should not interfere with the transfer of the Gn polypeptide to cross the BBB. In some instances, a Gn polypeptide may be generated with a cleavable linkage between the Gn polypeptide and the payload. Such a linker may allow release of the payload at a specific cellular location. Suitable linkers include, but are not limited to, amino acid chains and alkyl chains functionalized with reactive groups for conjugating to both the Gn polypeptide of the disclosure and the detectable label and/or therapeutic agent.

[0087] In an aspect, a Gn polypeptide of the disclosure may be conjugated to a detectable label. A detectable label may be directly conjugated to a Gn polypeptide of the disclosure or may be indirectly conjugated to a Gn polypeptide of the disclosure. In an embodiment, a detectable label may be complexed with a chelating agent that is conjugated to a Gn polypeptide of the disclosure. In another embodiment, a detectable label may be complexed with a chelating agent that is conjugated to a linker that is conjugated to a Gn polypeptide of the disclosure. In still another embodiment, a detectable label may be conjugated to a linker that is conjugated to a Gn polypeptide of the disclosure. In still yet another embodiment, a detectable label may be indirectly attached to a Gn polypeptide of the disclosure by the ability of the label to be specifically bound by a second molecule. One example of this type of an indirectly attached label is a biotin label that can be specifically bound by the second molecule, streptavidin or other biotin binding protein. Single, dual or multiple labeling may be advantageous. An isolated a Gn polypeptide of the disclosure may be conjugated to one, two, three, four, or five types of detectable labels.

[0088] As used herein, a “detectable label” is any type of label which, when attached to a Gn polypeptide of the disclosure renders the Gn polypeptide detectable. A detectable label may also be toxic to cells or cytotoxic. Accordingly, a detectable label may also be a therapeutic agent or cytotoxic agent. In general, detectable labels may include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioisotopes, radionuclides, cintillants, massive labels such as a metal atom (for detection via mass changes), biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, Grb2, polyhistidine, Ni²⁺, Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates. The skilled artisan would readily recognize other useful labels that are not mentioned above, which may be employed in the operation of the present disclosure. [0089] A detectable label emits a signal that can be detected by a signal transducing machine. In some cases, the detectable label can emit a signal spontaneously, such as when the detectable label is a radionuclide. In other cases the detectable label emits a signal as a result of being stimulated by an external field such as when the detectable label is a relaxivity metal. Examples of signals include, without limitation, gamma rays, X-rays, visible light, infrared energy, and radiowaves. Examples of signal transducing machines include, without limitation, gamma cameras including SPECT/CT devices, PET scanners, fluorimeters, and Magnetic Resonance Imaging (MRI) machines. As such, the detectable label comprises a label that can be detected using magnetic resonance imaging, scintigraphic imaging, ultrasound, or fluorescence. In a specific embodiment, the detectable label comprises a label that can be detected using positron emission tomography, single photon emission computed tomography, gamma camera imaging, or rectilinear scanning.

[0090] Suitable fluorophores include, but are not limited to, fluorescein isothiocyante (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes. B12 or an analog thereof can be labeled for fluorescence detection by labeling the agent with a fluorophore using techniques well known in the art (see, e.g., Lohse et al., Bioconj Chem 8:503-509 (1997)). For example, many known dyes are capable of being coupled to NH2-terminal groups. Alternatively, a fluorochrome such as fluorescein may be bound to a lysine residue of a peptide linker. In a specific embodiment, an alkyne modified dye, such an Alexa Fluor dye, may be clicked to an azido modified B12 using, for example, Sharpless click chemistry (Kolb et al., Angew Chem Int Ed 2001 ; 40: 2004- 2021 , which incorporated by reference in its entirety).

[0091 ] A radionuclide may be a y-em itting radionuclide, Auger-emitting radionuclide, [3-emitting radionuclide, an a-emitting radionuclide, or a positron-emitting radionuclide. A radionuclide may be a detectable label and/or a therapeutic agent. Nonlimiting examples of suitable radionuclides may include carbon-11 , nitrogen-13, oxygen- 15, fluorine-18, fluorodeoxyglucose-18, phosphorous-32, scandium-47, copper-64, 65 and 67, gallium-67 and 68, bromine-75, 77 and 80m, rubidium-82, strontium-89, zirconium-89, yttrium-86 and 90, ruthenium-95, 97,103 and 105, rhenium-99m, 101 , 105, 186 and 188, technetium-99m, rhodium-105, mercury-107, palladium-109, indium- 111 , silver-111 , indium-113m, lanthanide-114m, tin-117m, tellurium-121 m, 122m and 125m, iodine-122, 123, 124, 125, 126, 131 and 133, praseodymium-1 2, promethium- 149, samarium-153, gadolinium-159, thulium-165, 167 and 168, dysprosium-165, holmium-166, lutetium-177, rhenium-186 and 188, iridium-192, platinum-193 and 195m, gold-199, thallium-201 , titanium-201 , astatine-211 , bismuth-212 and 213, lead-212, radium-223, actinium-225, and nitride or oxide forms derived there from. In a specific embodiment, a radionuclide is selected from the group consisting of copper-64, zirconium-89, yttrium-86, yttrium-90, technetium-99m, iodine-125, iodine- 131 , lutetium- 177, rhenium-186 and rhenium-188.

[0092] A variety of metal atoms may be used as a detectable label. The metal atom may generally be selected from the group of metal atoms comprised of metals with an atomic number of twenty or greater. For instance, the metal atoms may be calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms. In some embodiments, the metal atoms may be selected from the group comprising alkali metals with an atomic number greater than twenty. In other embodiments, the metal atoms may be selected from the group comprising alkaline earth metals with an atomic number greater than twenty. In one embodiment, the metal atoms may be selected from the group of metals comprising the lanthanides. In another embodiment, the metal atoms may be selected from the group of metals comprising the actinides. In still another embodiment, the metal atoms may be selected from the group of metals comprising the transition metals. In yet another embodiment, the metal atoms may be selected from the group of metals comprising the poor metals. In other embodiments, the metal atoms may be selected from the group comprising gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms. In preferred embodiments, the metal atoms may be selected from the group comprising metals with an atomic number of 53 (i.e. iodine) to 83 (i.e. bismuth). In an alternative embodiment, the metal atoms may be atoms suitable for magnetic resonance imaging. In another alternative embodiment, the metal atoms may be selected from the group consisting of metals that have a K-edge in the x-ray energy band of CT. Preferred metal atoms include, but are not limited to, manganese, iron, gadolinium, gold, and iodine.

[0093] The metal atoms may be metal ions in the form of +1 , +2, or +3 oxidation states. For instance, non-limiting examples include Ba²⁺, Bi³⁺, Cs⁺, Ca²⁺, Cr²⁺, Cr³⁺, Cr⁵⁺, Co²⁺, Co³⁺, Cu⁺, Cu²⁺, Cu³⁺, Ga³⁺, Gd³⁺, Au⁺, Au³⁺, Fe²⁺, Fe³⁺, F³⁺, Pb²⁺, Mn²⁺, Mn³⁺, Mn⁴⁺, Mn⁷⁺, Hg²⁺, Ni²⁺, Ni³⁺, Ag⁺, Sr²⁺, Sn²⁺, Sn4⁺, and Zn²⁺. The metal atoms may comprise a metal oxide. For instance, non-limiting examples of metal oxides may include iron oxide, manganese oxide, or gadolinium oxide. Additional examples may include magnetite, maghemite, or a combination thereof.

[0094] In an embodiment where a Gn polypeptide of the disclosure is conjugated to a non-radioactive isotope, it may be used in neutron capture therapy (NCT). Neutron capture therapy (NCT) is a noninvasive therapeutic modality for treating locally invasive malignant tumors. NCT is a two-step procedure: first, the subject is injected with a tumor localizing drug containing a non-radioactive isotope that has a high propensity or cross section (a) to capture slow neutrons. The cross section of the capture agent is many times greater than that of the other elements present in tissues such as hydrogen, oxygen, and nitrogen. In the second step, the subject is radiated with epithermal neutrons, which after losing energy as they penetrate tissue, are absorbed by the capture agent, which subsequently emits high-energy charged particles, thereby resulting in a biologically destructive nuclear reaction. In certain embodiments, the nonradioactive isotope may be boron-10 or gadolinium.

[0095] In an aspect, a Gn polypeptide of the disclosure may be conjugated to a therapeutic agent, such that the therapeutic agent can be selectively targeted to the CNS. In a specific embodiment, the therapeutic agent can be selectively targeted to a cell in the CNS. The therapeutic agent may be directly conjugated to a Gn polypeptide of the disclosure or may be indirectly conjugated to a Gn polypeptide of the disclosure. In an embodiment, the therapeutic agent may be complexed with a chelating agent that is conjugated to a Gn polypeptide of the disclosure. In another embodiment, the therapeutic agent may be complexed with a chelating agent that is conjugated to a linker that is conjugated to a Gn polypeptide of the disclosure. In still another embodiment, the therapeutic agent may be conjugated to a linker that is conjugated to a Gn polypeptide of the disclosure. In still yet another embodiment, the therapeutic agent may be conjugated to a linker that is conjugated to a chelating agent that is complexed with a detectable label and conjugated to a Gn polypeptide of the disclosure.

[0096] A “therapeutic agent” is any compound known in the art that is used in the detection, diagnosis, or treatment of a condition or disease. Such compounds may be naturally-occurring, modified, or synthetic. Non-limiting examples of therapeutic agents may include drugs, therapeutic compounds, toxins, genetic materials, metals (such as radioactive isotopes), proteins, peptides, carbohydrates, lipids, steroids, nucleic acid based materials, or derivatives, analogues, or combinations thereof in their native form or derivatized with hydrophobic or charged moieties to enhance incorporation or adsorption into a cell. Such therapeutic agents may be water soluble or may be hydrophobic. Non-limiting examples of therapeutic agents may include immune- related agents, thyroid agents, respiratory products, antineoplastic agents, antihelm intics, anti-malarials, mitotic inhibitors, hormones, toxins, anti-protozoans, anti- tuberculars, cardiovascular products, blood products, biological response modifiers, anti-fungal agents, vitamins, peptides, anti-allergic agents, anti-coagulation agents, circulatory drugs, metabolic potentiators, anti-virals, anti-anginals, antibiotics, anti- inflammatories, anti-rheumatics, narcotics, cardiac glycosides, neuromuscular blockers, sedatives, local anesthetics, general anesthetics, or radioactive atoms or ions. Nonlimiting examples of therapeutic agents are described below. In a specific embodiment, a therapeutic agent may be a compound used in the detection diagnosis or treatment of cancer. The therapeutic agent preferably reduces or interferes with tumor growth or otherwise reduces the effect of the tumor within the body or organism. A therapeutic agent that reduces the symptoms produced by the tumor or reduces tumor growth is suitable for the present disclosure. Additionally, any therapeutic agent that reduces the symptoms associated with tumor cell growth will work for purposes of the present disclosure.

[0097] A Gn polypeptide of the disclosure may be conjugated to one, two, three, four, or five therapeutic agents. A linker may or may not be used to conjugate a therapeutic agent to a Gn polypeptide of the disclosure. Generally speaking, the conjugation should not interfere with the Gn polypeptide binding to transfer across the BBB. In some instances, a Gn polypeptide of the disclosure may be generated with a cleavable linkage between the Gn polypeptide and therapeutic agent. Such a linker may allow release of the therapeutic agent at a specific cellular location. In other instances, a Gn polypeptide of the disclosure may be generated with an enzyme linked to it to create a prodrug. For example, cytidine deaminase may be linked to a Gn polypeptide of the disclosure. The cytidine deaminase then cleaves the prodrug to create a cytotoxic drug.

[0098] A therapeutic agent of the disclosure may be a toxin. The term "toxin" means the toxic material or product of plants, animals, microorganisms (including, but not limited to, bacteria, viruses, fungi, rickettsiae or protozoa), or infectious substances, or a recombinant or synthesized molecule, whatever their origin and method of production. A toxin may be a small molecule, peptide, or protein that is capable of causing disease on contact with or absorption by body tissues interacting with biological macromolecules such as enzymes or cellular receptors. A toxin may be a “biotoxin” which is used to explicitly identify the toxin as from biological origin. Biotoxins may be further classified into fungal biotoxins, or short mycotoxins, microbial biotoxins, plant biotoxins, short phytotoxins and animal biotoxins. Non-limiting examples of biotoxins include: endotoxins produced by bacteria, such as Pseudomonas endotoxin; cyanotoxins produced by cyanobacteria, such as microcystins, nodularins, anatoxin-a, cylindrospermopsins, lyngbyatoxin-a, saxitoxin, lipopolysaccharides, aplysiatoxins, BMAA; dinotoxins produced by dinoflagellates, such as saxitoxins and gonyautoxins; necrotoxins produced by, for example, the brown recluse or "fiddle back" spider, most rattlesnakes and vipers, the puff adder, Streptococcus pyogenes; neurotoxins produced by, for example, the black widow spider, most scorpions, the box jellyfish, elapid snakes, the cone snail, the Blue-ringed octopus, venomous fish, frogs, palythoa coral, various different types of algae, cyanobacteria and dinoflagellates, such as botulinum toxin (e.g. Botox), tetanus toxin, tetrodotoxin, chlorotoxin, conotoxin, anatoxin-a, bungarotoxin, caramboxin, curare; myotoxins, found in, for example, snake and lizard venoms; and cytotoxins such as ricin, from castor beans, apitoxin, from honey bees, and T-2 mycotoxin, from certain toxic mushrooms. In certain embodiments, a toxin is a cytotoxin. In an embodiment, a cytotoxin is an endotoxin from Pseudomonas.

[0099] A therapeutic agent of the disclosure may be a small molecule therapeutic, a therapeutic antibody, a therapeutic nucleic acid, or a chemotherapeutic agent. Non-limiting examples of therapeutic antibodies may include muromomab, abciximab, rituximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, etanercept, gemtuzumab, alemtuzumab, ibritomomab, adalimumab, alefacept, omalizumab, tositumomab, efalizumab, cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, and certolizumab. A representative therapeutic nucleic acid may encode a polypeptide having an ability to induce an immune response and/or an anti-angiogenic response in vivo. Representative therapeutic proteins with immunostimulatory effects include but are not limited to cytokines (e.g., an interleukin (IL) such as IL2, IL4, IL7, IL12, interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-a)), immunomodulatory cell surface proteins (e.g., human leukocyte antigen (HLA proteins), co-stimulatory molecules, and tumor-associated antigens. See Kirk & Mule, 2000; Mackensen et al., 1997; Walther & Stein, 1999; and references cited therein. Representative proteins with anti-angiogenic activities that can be used in accordance with the presently disclosed subject matter include: thrombospondin I (Kosfeld & Frazier, 1993; Tolsma et al., 1993; Dameron et al., 1994), metallospondin proteins (Carpizo & Iruela-Arispe, 2000), class I interferons (Albini et al., 2000), IL12 (Voest et al., 1995), protamine (Ingber et al., 1990), angiostatin (O'Reilly et al., 1994), laminin (Sakamoto et al., 1991 ), endostatin (O'Reilly et al., 1997), and a prolactin fragment (Clapp et al., 1993). In addition, several anti-angiogenic peptides have been isolated from these proteins (Maione et al., 1990; Eijan et al., 1991 ; Woltering et al., 1991 ). Representative proteins with both immunostimulatory and anti- angiogenic activities may include IL12, interferon-y, or a chemokine. Other therapeutic nucleic acids that may be useful for cancer therapy include but are not limited to nucleic acid sequences encoding tumor suppressor gene products/antigens, antimetabolites, suicide gene products, and combinations thereof.

[00100] A chemotherapeutic agent refers to a chemical compound that is useful in the treatment of cancer. The compound may be a cytotoxic agent that affects rapidly dividing cells in general, or it may be a targeted therapeutic agent that affects the deregulated proteins of cancer cells. A cytotoxic agent is any naturally-occurring, modified, or synthetic compound that is toxic to tumor cells. Such agents are useful in the treatment of neoplasms, and in the treatment of other symptoms or diseases characterized by cell proliferation or a hyperactive cell population. The chemotherapeutic agent may be an alkylating agent, an anti-metabolite, an anti-tumor antibiotic, an anti-cytoskeletal agent, a topoisomerase inhibitor, an anti-hormonal agent, a targeted therapeutic agent, a photodynamic therapeutic agent, or a combination thereof. In an exemplary embodiment, the chemotherapeutic agent is selected from the group consisting of liposomal doxorubicin and nanoparticle albumin docetaxel.

[00101 ] Non-limiting examples of suitable alkylating agents may include altretamine, benzodopa, busulfan, carboplatin, carboquone, carmustine (BCNll), chlorambucil, chlornaphazine, cholophosphamide, chlorozotocin, cisplatin, cyclosphosphamide, dacarbazine (DTIC), estramustine, fotemustine, ifosfamide, improsulfan, lipoplatin, lomustine (CCNU), mafosfamide, mannosulfan, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, meturedopa, mustine (mechlorethamine), mitobronitol, nimustine, novembichin, oxaliplatin, phenesterine, piposulfan, prednimustine, ranimustine, satraplatin, semustine, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, triethylenephosphoramide (TEPA), triethylenethiophosphaoramide (thiotepa), trimethylolomelamine, trofosfamide, uracil mustard and uredopa.

[00102] Suitable anti-metabolites may include, but are not limited to aminopterin, ancitabine, azacitidine, 8-azaguanine, 6-azauridine, capecitabine, carmofur (1-hexylcarbomoyl-5-fluorouracil), cladribine, clofarabine, cytarabine (cytosine arabinoside (Ara-C)), decitabine, denopterin, dideoxyuridine, doxifluridine, enocitabine, floxuridine, fludarabine, 5-fluorouracil, gemcitabine, hydroxyurea (hydroxycarbamide), leucovorin (folinic acid), 6-mercaptopurine, methotrexate, nafoxidine, nelarabine, oblimersen, pemetrexed, pteropterin, raltitrexed, tegofur, tiazofurin, thiamiprine, tioguanine (thioguanine), and trimetrexate.

[00103] Non-limiting examples of suitable anti-tumor antibiotics may include aclacinomysin, aclarubicin, actinomycins, adriamycin, aurostatin (for example, monomethyl auristatin E), authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, epoxomicin, esorubicin, idarubicin, marcellomycin, mitomycins, mithramycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, plicamycin, potfiromycin, puromycin, quelamycin, rodorubicin, sparsomycin, streptonigrin, streptozocin, tubercidin, valrubicin, ubenimex, zinostatin, and zorubicin.

[00104] Non-limiting examples of suitable anti-cytoskeletal agents may include cabazitaxel, colchicines, demecolcine, docetaxel, epothilones, ixabepilone, macromycin, omacetaxine mepesuccinate, ortataxel, paclitaxel (for example, DHA- paclitaxel), taxane, tesetaxel, vinblastine, vincristine, vindesine, and vinorelbine.

[00105] [Suitable topoisomerase inhibitors may include, but are not limited to, amsacrine, etoposide (VP-16), irinotecan, mitoxantrone, RFS 2000, teniposide, and topotecan.

[00106] Non-limiting examples of suitable anti-hormonal agents may include aminoglutethimide, antiestrogen, aromatase inhibiting 4(5)-imidazoles, bicalutamide, finasteride, flutamide, fluvestrant, goserelin, 4-hydroxytamoxifen, keoxifene, leuprolide, LY117018, mitotane, nilutamide, onapristone, raloxifene, tamoxifen, toremifene, and trilostane. [00107] Examples of targeted therapeutic agents may include, without limit, monoclonal antibodies such as alemtuzumab, cartumaxomab, edrecolomab, epratuzumab, gemtuzumab, gemtuzumab ozogamicin, glembatumumab vedotin, ibritumomab tiuxetan, reditux, rituximab, tositumomab, and trastuzumab; protein kinase inhibitors such as bevacizumab, cetuximab, crizonib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, mubritinib, nilotinib, panitumumab, pazopanib, sorafenib, sunitinib, toceranib, and vandetanib.

[00108] Non limiting examples of angiogeneisis inhibitors may include angiostatin, bevacizumab, denileukin diftitox, endostatin, everolimus, genistein, interferon alpha, interleukin-2, interleukin-12, pazopanib, pegaptanib, ranibizumab, rapamycin (sirolimus), temsirolimus, and thalidomide.

[00109] Non limiting examples of growth inhibitory polypeptides may include bortazomib, erythropoietin, interleukins (e.g., IL-1 , IL-2, IL-3, IL-6), leukemia inhibitory factor, interferons, romidepsin, thrombopoietin, TNF-a, CD30 ligand, 4-1 BB ligand, and Apo-1 ligand.

[00110] Non-limiting examples of photodynamic therapeutic agents may include aminolevulinic acid, methyl aminolevulinate, retinoids (alitretinon, tamibarotene, tretinoin), and temoporfin.

[00111 ] Other antineoplastic agents may include anagrelide, arsenic trioxide, asparaginase, bexarotene, bropirimine, celecoxib, chemically linked Fab, efaproxiral, etoglucid, ferruginol, lonidamide, masoprocol, miltefosine, mitoguazone, talapanel, trabectedin, and vorinostat.

[00112] Also included are pharmaceutically acceptable salts, acids, or derivatives of any of the above listed agents. The dose of the chemotherapeutic agent can and will vary depending upon the agent and the type of tumor or neoplasm. A skilled practitioner will be able to determine the appropriate dose of the chemotherapeutic agent.

[00113] Other therapeutic agents may comprise a virus or a viral genome such as an oncolytic virus. An oncolytic virus comprises a naturally occurring virus that is capable of killing a cell in the target tissue (for example, by lysis) when it enters such a cell. [00114] A Gn polypeptide of the disclosure may be conjugated to a vehicle for cellular delivery. In these embodiments, typically an antibody of the disclosure, which may or may not be conjugated to a detectable label and/or therapeutic agent, is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the antibody, or to minimize potential toxicity of the Gn polypeptide. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a Gn polypeptide of the disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating antibodies into delivery vehicles are known in the art. Although various embodiments are presented below, it will be appreciate that other methods known in the art to incorporate a Gn polypeptide of the disclosure into a delivery vehicle are contemplated.

[00115] In one alternative embodiment, a liposome delivery vehicle may be utilized. Liposomes, depending upon the embodiment, are suitable for delivery of the therapeutic agent where the liposome is labeled with a Gn polypeptide of the disclosure in view of their structural and chemical properties. Generally speaking, liposomes are spherical vesicles with a phospholipid bilayer membrane. The lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells. In this manner, the Gn polypeptide of the disclosure may be used to label the liposome facilitating transfer across the BBB thereby selectively delivering the liposome to the CNS.

[00116] Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths. Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated. Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n- tretradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9, 12- octadecandienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolenate), and all cis-5,8, 11 ,14-eicosatetraenoate (arachidonate). The two fatty acid chains of a phospholipid may be identical or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.

[00117] The phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids. For example, egg yolk is rich in PC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brain or spinal cord is enriched in PS. Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties. The above mentioned phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1 -(2,3-dioleolyoxy)propyl)-N, N,N- trimethyl ammonium chloride, 1 ,T-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchloarate, 3,3’-deheptyloxacarbocyanine iodide, 1 ,T-dedodecyl-3,3,3’,3’- tetramethylindocarbocyanine perchloarate, 1 ,T-dioleyl-3,3,3’,3’-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1 ,1 ,-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine perchloarate.

[00118] Liposomes may optionally comprise sphingolipids, in which spingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes. Liposomes may optionally, contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.

[00119] Liposomes may further comprise a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof. [00120] Liposomes of the disclosure may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos. 4,241 ,046, 4,394,448, 4,529,561 , 4,755,388, 4,828,837, 4,925,661 , 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety. For example, liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing. In a preferred embodiment the liposomes are formed by sonication. The liposomes may be multilamellar, which have many layers like an onion, or unilamellar. The liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar liposomes.

[00121 ] As would be apparent to one of ordinary skill, all of the parameters that govern liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.

[00122] In another embodiment, a Gn polypeptide of the disclosure may be part of a microemulsion. Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and “oil." The "oil" in this case, is the supercritical fluid phase. The surfactant rests at the oil-water interface. Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art. The aqueous microdomains suitable for use in the invention generally will have characteristic structural dimensions from about 5 nm to about 100 nm. Aggregates of this size are poor scatterers of visible light and hence, these solutions are optically clear. As will be appreciated by a skilled artisan, microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates. In one embodiment, the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil. In an alternative embodiment, the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant. The “oil” of microemulsions optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions. The Gn polypeptide of the disclosure may be encapsulated in a microemulsion by any method generally known in the art.

[00123] In yet another embodiment, a Gn polypeptide of the disclosure may be delivered in a dendritic macromolecule, or a dendrimer. Generally speaking, a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe. Generally, the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer. Alternatively, phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate a Gn polypeptide of the disclosure therein. For example, dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape. Furthermore, the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for use in the invention. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.

[00124] The nucleic acid molecules of the invention may be inserted into vectors and used as gene therapy vectors. Gene therapy vectors may be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. [00125] The gene therapy vectors of the invention may be either viral or non-viral. Examples of plasmid-based, non-viral vectors are discussed in Huang et al. (1999) Nonviral Vectors for Gene Therapy. A modified plasmid is one example of a non-viral gene delivery system. Peptides, proteins (including antibodies), and oligonucleotides may be stably conjugated to plasmid DNA by methods that do not interfere with the transcriptional activity of the plasmid (Zelphati et al. (2000) BioTechniques 28:304-315). The attachment of proteins and/or oligonucleotides may influence the delivery and trafficking of the plasmid and thus render it a more effective pharmaceutical composition.

[00126] Another aspect of the present disclosure provides nucleic acids encoding any of the decoy receptor or decoy viral inhibitor described above. The nucleic acid can be DNA or RNA. In one embodiment the DNA can be present in a vector. The nucleic acid sequences which encode the dominant negative molecule of the invention can be operatively linked to expression control sequences. “Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. An expression control sequence operatively linked to a coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e. , ATG) in front of a protein-encoding gene, splicing signals for introns, and maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter. [00127] In one aspect, the present disclosure provides for a vector comprising a nucleic acid sequence encoding for a decoy receptor or decoy viral inhibitor. In one aspect, the present disclosure is predicated, at least in part, on the ability of adeno-associated virus (AAV) vectors to be safely administered to humans and to provide persistent expression of a decoy receptor or a decoy viral inhibitor. The invention provides an adeno-associated virus (AAV) vector which comprises, consists essentially of, or consists of a nucleic acid sequence encoding a decoy receptor or a decoy viral inhibitor. When the AAV vector consists essentially of a nucleic acid sequence encoding decoy receptor or decoy viral inhibitor polypeptide, additional components can be included that do not materially affect the AAV vector (e.g., genetic elements such as poly(A) sequences or restriction enzyme sites that facilitate manipulation of the vector in vitro). When the AAV vector consists of a nucleic acid sequence encoding decoy receptor or decoy viral inhibitor, the AAV vector does not comprise any additional components (i.e. , components that are not endogenous to AAV and are not required to effect expression of the nucleic acid sequence to thereby provide the decoy receptor or decoy viral inhibitor).

[00128] Adeno-associated virus is a member of the Parvoviridae family and comprises a linear, single-stranded DNA genome of less than about 5,000 nucleotides. AAV re-quires c-infection with a helper virus (i.e., an adenovirus or a herpes virus), or expression of helper genes, for efficient replication. AAV vectors used for administration of therapeutic nucleic acids typically have approximately 96% of the parental genome deleted, such that only the terminal repeats ( ITRs), which contain recognition signals for DNA replication and packaging, remain. This eliminates immunologic or toxic side effects due to expression of viral genes. In addition, delivering specific AAV proteins to producing cells enables integration of the AAV vector comprising AAV ITRs into a specific region of the cellular genome, if desired (see, e.g., U.S. Pat. Nos. 6,342,390 and 6,821 ,511 ). Host cells comprising an integrated AAV genome show no change in cell growth or morphology (see, for example, U.S. Pat. No. 4,797,368).

[00129] The AAV ITRs flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)). The terminal 145 nucleotides are self- complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication by serving as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the p19 promoter. The Rep78 and Rep68 proteins are multifunctional DNA binding proteins that perform helicase and nickase functions during productive replication to allow for the resolution of AAV termini (see, e.g., Im et al., Cell, 61 : 447-57 (1990)). These proteins also regulate transcription from endogenous AAV promoters and promoters within helper viruses (see, e.g., Pereira et al., J. Virol., 71 : 1079-1088 (1997)). The other Rep proteins modify the function of Rep78 and Rep68. The cap genes encode the capsid proteins VP1 , VP2, and VP3. The cap genes are transcribed from the p40 promoter. In a particular embodiment, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression (e.g. hepatocytes) operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus (e.g. an arginine-degrading enzyme). Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No. 6,261 ,834 is herein incorporated by reference in its entirety for material related to the AAV vector.

[00130] As used herein, the term "AAV vector" means a vector derived from an adeno-associated virus serotype. In non-limitation examples AAV vectors include, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. De-spite the high degree of homology, the different serotypes have tropisms for different tissues. In an exemplary embodiment, the AAV vector is AAV9.

[00131 ] An AAV vector, as disclosed herein, can be generated using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or nonhuman primate tissues (reviewed in, e.g., Wu et al., Molecular Therapy, 14(3): 316-327 (2006)). Generally, the AAV serotypes have genomic sequences of significant homology at the nucleic acid sequence and amino acid sequence levels, such that different serotypes have an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. AAV serotypes 1 -6 and 7-9 are defined as "true" serotypes, in that they do not efficiently cross-react with neutralizing sera specific for all other existing and characterized serotypes. In contrast, AAV serotypes 6, 10 (also referred to as Rh10), and 11 are considered "variant" serotypes as they do not adhere to the definition of a "true" serotype. AAV serotype 2 (AAV2) has been used extensively for gene therapy applications due to its lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression (see, e.g., Carter, B. J., Hum. Gene Then, 16: 541 -550 (2005); and Wu et al., supra). Genome sequences of various AAV serotypes and comparisons thereof are disclosed in, for example, GenBank Accession numbers U89790, J01901 , AF043303, and AF085716; Chiorini et al., J. Virol., 71 : 6823-33 (1997); Srivastava et al., J. Virol., 45: 555-64 (1983); Chiorini et al., J. Virol., 73: 1309- 1319 (1999); Rutledge et al., J. Vi-rol., 72: 309-319 (1998); and Wu et al., J. Virol., 74: 8635-47 (2000)).

[00132] AAV rep and ITR sequences are particularly conserved across most AAV serotypes. For example, the Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4, and AAV6 are reportedly about 89-93% identical (see Bantel-Schaal et al., J. Virol., 73(2): 939-947 (1999)). It has been reported that AAV serotypes 2, 3A, 3B, and 6 share about 82% total nucleotide sequence identity at the genome level (Bantel-Schaal et al., supra). Moreover, the rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally substitute) corresponding sequences from other serotypes during production of AAV particles in mammalian cells.

[00133] Generally, the cap proteins, which determine the cellular tropicity of the AAV particle, and related cap protein-encoding sequences, are significantly less conserved than Rep genes across different AAV serotypes. In view of the ability Rep and ITR sequences to cross-complement corresponding sequences of other serotypes, the AAV vector can comprise a mixture of serotypes and thereby be a "chimeric" or "pseudo-typed" AAV vector. A chimeric AAV vector typically comprises AAV capsid proteins de-rived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes. In contrast, a pseudo-typed AAV vector comprises one or more ITRs of one AAV serotype packaged into a capsid of another AAV serotype. Chimeric and pseudotyped AAV vectors are further described in, for example, U.S. Pat. No. 6,723,551 ; Flotte, Mol. Then, 13(1 ): 1-2 (2006); Gao et al., J. Virol., 78: 6381 -6388 (2004); Gao et al., Proc. Natl. Acad. Sci. USA, 99: 11854-11859 (2002); De et al., Mol. Then, 13: 67-76 (2006); and Gao et al., Mol. Then, 13: 77-87 (2006).

[00134] In one embodiment, the AAV vector is generated using an AAV that infects hu-mans (e.g., AAV2). Alternatively, the AAV vector is generated using an AAV that infects non-human primates, such as, for example, the great apes (e.g., chimpanzees), Old World monkeys (e.g., macaques), and New World monkeys (e.g., marmosets). Preferably, the AAV vector is generated using an AAV that infects a non- human primate pseudotyped with an AAV that infects humans. Examples of such pseudotyped AAV vectors are disclosed in, e.g., Cearley et al., Molecular Therapy, 13: 528-537 (2006). In one embodiment, an AAV vector can be generated which comprises a capsid protein from an AAV that infects rhesus macaques pseudotyped with AAV2 inverted terminal repeats (ITRs). In a particularly preferred embodiment, the inventive AAV vector comprises a capsid protein from AAV10 (also referred to as "AAVrh.10"), which infects rhesus macaques pseudotyped with AAV2 ITRs (see, e.g., Watanabe et al., Gene Then, 17(8): 1042-1051 (2010); and Mao et al., Hum. Gene Therapy, 22: 1525-1535 (2011 )).

[00135] An AAV vector, as disclosed herein, comprises a nucleic acid sequence encoding an arginine-degrading enzyme polypeptide. "Nucleic acid sequence" is intended to encompass a polymer of DNA or RNA, i.e. , a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms "nucleic acid" and "polynucleotide" as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides.

[00136] In some embodiments, a vector comprising a nucleic acid sequence encoding a decoy receptor or decoy viral inhibitor can be a plasmid, cosmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), viral vector or bacteriophage. The vectors can provide for replication of decoy receptor or decoy viral inhibitor nucleic acids, expression of a decoy receptor or decoy viral inhibitor or integration of decoy receptor or decoy viral inhibitor into the chromosome of a host cell. The choice of vector is dependent on the desired purpose. Certain cloning vectors are useful for cloning, mutation and manipulation of the decoy receptor or decoy viral inhibitor encoding nucleic ac-id. Other vectors are useful for expression of a decoy receptor or decoy viral inhibitor. The vector can also be chosen on the basis of the host cell, e.g., to facilitate expression in bacteria, mammalian cells, insect cells, fish cell (e.g., zebrafish) and/or amphibian cells. The choice of matching vector to host cell is apparent to one of skill in the art, and the types of host cells are discussed below. Many vectors or vector systems are available commercially, for example, the pET bacterial expression system (InvitrogenTM, Carlsbad Calif.).

[00137] The vectors disclosed herein can be viral or non-viral vectors. For example, as discussed above the disclosed vectors can be viral vectors. There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physicomechanical methods such as electroporation and direct diffusion of DNA, are described by, for ex-ample, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991 ). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain neurodegenerative diseases or disorders and cell populations by using the targeting characteristics of the carrier.

[00138] Vectors can include various components including, but not limited to, an origin of replication, one or more marker or selectable genes (e.g. GFP, neo), promoters, enhancers, terminators, poly-adenylation sequences, repressors or activators. Such elements are provided in the vector so as to be operably linked to the coding region of a decoy receptor or decoy viral inhibitor -encoding nucleic acid, thereby facilitating expression in a host cell of interest. Cloning and expression vectors can contain an origin of replication which allows the vector to replicate in the host cells. Vectors can also include a selectable marker, e.g., to confer a resistance to a drug or compliment deficiencies in growth. Examples of drug resistance markers include, but are not limited to, ampicillin, tetracycline, neomycin or methotrexate. Examples of other marker genes can be the fluorescent polypeptides such one of the members of the fluorescent family of proteins, for example, GFP, YFP, BFP, RFP etc. These markers can be contained on the same vector as the gene of interest or can be on separate vectors and co-transfected with the vector containing the gene of interest.

[00139] The vector can contain a promoter that is suitable for expression of the decoy receptor or decoy viral inhibitor in mammalian cells, which promoter can be operably linked to provide for inducible or constitutive expression of an argininedegrading enzyme polypeptide. Exemplary inducible promoters include, for example, the metallothionine promoter or an ecdysone-responsive promoter. Exemplary constitutive promoters include, for example, the viral promoters from cytomegalovirus (CMV), Rous Sarcoma virus (RSV), Simian virus 40 (SV40), avian sarcoma virus, the beta-actin promoter and the heat-shock promoters. The promoter can be chosen for its tissue specificity. Certain promoters only express in certain tissues, and when it is desirable to express the polypeptide of interest only in a selected tissue, one of these promoters can be used. The choice of promoter will be apparent to one of skill in the art for the desired host cell system. [00140] The vector encoding an arginine-degrading enzyme can be a viral vector. Examples of viral vectors include retroviral vectors, such as: adenovirus, simian virus 40 (SV40), cytomegalovirus (CMV), Moloney murine leukemia virus (MoMuLv), Rous Sar-coma Virus (RSV), lentivirus, herpesvirus, poxvirus and vaccinia virus. A viral vector can be used to facilitate expression in a target cell, e.g. , for production of decoy receptor or decoy viral inhibitor or for use in therapy (e.g., to deliver an decoy receptor or decoy viral inhibitor to a subject by expression from the vector). Where used for therapy, decoy receptor or decoy viral inhibitor-encoding vectors (e.g, viral vectors), can be administered directly to the patient via an appropriate route or can be administered using an ex vivo strategy using subject cells (autologous) or allogeneic cells, which are suitable for administration to the patient to be treated.

[00141 ] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as a nucleic acid sequence capable of encoding one or more of the disclosed peptides into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some embodiments the nucleic acid sequences disclosed herein are derived from any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that ex-press the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e. , a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. The viral vectors may be formulated in pharmaceutical compositions as those described above

[00142] Retroviral vectors, in general, are described by Verma, I. M., Retroviral vectors for gene transfer. In Microbiology, Amer. Soc. for Microbiology, pp. 229-232, Washington, (1985), which is hereby incorporated by reference in its entirety. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy.

[00143] Other useful systems include, for example, replicating and host- restricted non-replicating vaccinia virus vectors. In addition, the disclosed nucleic acid sequences can be delivered to a target cell in a non-nucleic acid based system. For example, the disclosed polynucleotides can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for ex-ample in vivo or in vitro.

[00144] Thus, the compositions can comprise, in addition to the disclosed expression vectors, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a peptide and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respiratory tract. For example, a composition comprising a peptide or nucleic acid sequence described herein and a cationic liposome can be administered to a subjects lung cells. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the com-pound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[00145] Exemplary host cells include bacteria, yeast, mammalian cells (e.g., human cells or cell lines), insect cells, and the like. Examples of bacterial host cells include E. coli and other bacteria which can find use in cloning, manipulation and production of decoy receptor or decoy viral inhibitor nucleic acids or the production of decoy receptor or decoy viral inhibitor polypeptide. Examples of mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, HEK 293 cells, human cervical carcinoma cells (Hela), ca-nine kidney cells (MDCK), human liver cells (HepG2), baby hamster kidney cells (BHK), and monkey kidney cells (CV1 ).

[00146] The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of LRP1 . Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of LRP1 . Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of LRP1 and one or more additional active compounds.

[00147] A pharmaceutical composition of the invention may be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00148] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ex-temporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, a composition may be sterile and may be fluid to the extent that easy syringeability exists. A composition may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The 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. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00149] Sterile injectable solutions may be prepared by incorporating the active com-pound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a 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, which yields a powder of the active ingredient plus any additional de-sired ingredient from a previously sterile-f iltered solution thereof.

[00150] Oral compositions generally may include an inert diluent or an edible carrier. Oral compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches, and the like, may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00151 ] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and may include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00152] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, poly-anhydrides, polyg lycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00153] The composition can be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient. Such compositions can be administered orally (e.g. inhalation), or parenterally in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, or intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Gennaro, A. R., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980). In a specific embodiment, a composition may be a food supplement or a composition may be a cosmetic.

[00154] For parenteral administration (including subcutaneous, intraocular, intradermal, intravenous, intramuscular, intra-articular and intraperitoneal), the preparation may be an aqueous or an oil-based solution. Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[00155] Generally, a safe and effective amount of a nanoparticle composition is administered, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a nanoparticle composition described herein can substantially reduce viral infectivity in a subject suffering from a viral infection. In some embodiments, an effective amount is an amount capable of treating a respiratory viral infection. In some embodiments, an effective amount is an amount capable of treating one or more symptoms associated with a respiratory viral infection.

[00156] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[00157] The concentration of the nanoparticle of the present disclosure in the fluid pharmaceutical formulations can vary widely, i.e., from less than about 0.05% usually or at least about 2-10% to as much as 30 to 50% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. The amount of nanoparticle pharmaceutical composition administered will depend upon the particular therapeutic entity entrapped inside the nanoparticle, the type of nanoparticle being used, and the judgment of the clinician. Generally the amount of nanoparticle pharmaceutical composition administered will be sufficient to deliver a therapeutically effective dose of the particular therapeutic entity.

[00158] The quantity of nanoparticle pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods, common in the art of drug testing. See, for example, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages for various therapeutic entities are well known to those of skill in the art; and according to the present disclosure a therapeutic entity delivered via the pharmaceutical liposome composition of the present invention provides at least the same, or 2-fold, 4-fold, or 10-fold higher activity than the activity obtained by administering the same amount of the therapeutic entity in its routine non-liposome formulation. Typically the dosages for the nanoparticle pharmaceutical composition of the present disclosure range between about 0.005 and about 500 mg of the therapeutic entity per kilogram of body weight, most often, between about 0.1 and about 100 mg therapeutic entity/kg of body weight.

[00159] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the EDso, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[00160] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[00161 ] Administration of a nanoparticle composition can occur as a single event or over a time course of treatment. For example, one or more of a nanoparticle composition can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[00162] Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a respiratory virus.

[00163] The present disclosure encompasses pharmaceutical compositions comprising compounds as disclosed above, so as to facilitate administration and promote stability of the active agent. For example, a compound of this disclosure may be admixed with at least one pharmaceutically acceptable carrier or excipient resulting in a pharmaceutical composition which is capably and effectively administered (given) to a living subject, such as to a suitable subject (i.e. “a subject in need of treatment” or “a subject in need thereof”). For the purposes of the aspects and embodiments of the invention, the subject may be a human or any other animal.

III. METHODS

[00164] Disclosed herein are compositions, methods, and treatment plans for treating an individual who is at risk of having a viral infection, has symptoms of a viral infection, or is at risk of a viral infection. A composition of the present disclosure comprising a receptor decoy or decoy viral inhibitor composition disclosed herein (e.g. compositions disclosed in section II and the Examples and incorporated into this section by reference) may be used to treat, prevent, or reduce the infectivity of a viral infection (e.g., a bunyaviral infection). A treatment plan may comprise administering a composition (e.g., a composition comprising a liposome or nanoparticle or fusion protein composition of the disclosure) to an individual at risk of having a viral infection or who has a viral infection, thereby preventing or treating the viral infection. In some embodiments, a viral infection may be prevented by reducing the amount of virus capable of binding to a host cell or tissue. For example, a composition of the present disclosure may comprise a decoy receptor LRP1 polypeptide or fragment thereof; or a decoy viral inhibitor Gn polypeptide or fragment thereof. In some embodiments, a decoy receptor binds to the virus and at the same time prevents or reduces viral binding to host cells and tissues. In some embodiments, a decoy viral inhibitor binds to the host receptor and at the same time prevents or reduces viral binding to host cells and tissues. In some embodiments, a viral infection may be prevented by disrupting interactions between a viral surface proteins and host cell proteins that activate or enhance insertion of the viral genetic material into the host cell. For example, interactions between Gn, and a host cell LRP1 .

[00165] In some embodiments, the methods and compositions provided herein may prevent or reduce the infectivity of a viral infection by preventing internalization of a virus into a cell of the subject or by preventing internalization of a viral genome into a cell of the subject. In some embodiments, the methods and compositions provided herein may disrupt or prevent an interaction between a viral surface protein (e.g., Gn) and a host receptor protein (e.g., LRP1 ). For example, the methods and compositions provided herein may block internalization of a bunyavirus into a cell of a subject by blocking or disrupting interactions between a bunyavirus Gn protein and a host receptor protein or sequestering the virus in vivo allowing for the virus bound to the composition to be eliminated by immune cells. Administering a composition of the disclosure to a subject at risk for a viral infection may reduce the risk of bunyavirus infection in the subject.

[00166] In other embodiments, the present disclosure provides methods to treat, prevent, or reduce the infectivity of a bunyaviral infection. The bunyavirus may be RVFV, LACV, or OROV. A subject at risk for a bunyavirus infection may come in contact with an asymptomatic carrier of the bunyavirus infection, thereby unknowingly contracting the coronavirus infection.

[00167] Another aspect of the present disclosure is a method for treating a tauopathy in a subject having or suspected of having tau pathology. The method generally comprises administering to the subject a therapeutically effective amount of a composition comprising decoy receptor or decoy viral inhibitor as disclosed herein. The cell-to-cell spread of pathogenic tau is a major contributor to the progression of neurodegeneration. In these events, pathogenic misfolded tau is internalized by healthy neurons, providing a template upon which normal cellular tau assembles and then misfolds, thus propagating pathology across a neural network. LRP1 plays a primary role in this process by mediating cellular internalization of tau. The compositions and methods disclosed herein block this interaction to inhibit cellular propagation of and progressive neurodegeneration caused by pathogenic tau. In some embodiments, administration of RVFV Gn or a fragment thereof potently blocks the ability of LRP1 to engage tau thereby reducing or preventing cell-to-cell spread of pathogenic tau.

[00168] A disease associated with tau deposition in the brain may be referred to as a "tauopathy". Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy, dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, LyticoBodig disease, Parkinson-dementia complex of Guam, tangle- predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic grain disease (AGD), Frontotemporal lobar degeneration, Alzheimer's Disease, and frontotemporal dementia. In a specific embodiment, the tauopathy is Alzheimer’s Disease.

[00169] Another aspect of the present disclosure is a method for treating a subject in need thereof comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a Gn polypeptide of the disclosure conjugated to a therapeutic agent. In some embodiments, the methods include increasing the transfer of a therapeutic or imaging agent across the blood-brain- barrier. In some embodiments, the amount of therapeutic agent in the CNS is increased when administered with the Gn polypeptide relative to the amount of therapeutic agent or imaging agent administered without the Gn polypeptide. In some embodiments, the disease or disorder to be treated can be any aliment of the CNS including neurodegenerative diseases, cancers or tumors of the CNS, inflammatory diseases of the CNS, autoimmune diseases of the CNS, traumatic brain injury, and the like.

[00170] The terms “treat,” "treating," or "treatment" as used herein, refers to the provision of medical care by a trained and licensed professional to a subject in need thereof. The medical care may be a diagnostic test, a therapeutic treatment, and/or a prophylactic or preventative measure. The object of therapeutic and prophylactic treatments is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results of therapeutic or prophylactic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented. In some embodiments, a subject receiving treatment is asymptomatic. An “asymptomatic subject,” as used herein, refers to a subject that does not show any signs or symptoms of a central nervous system tumor. In other embodiments, a subject may exhibit signs or symptoms of central nervous system tumor (e.g., memory loss, changes in mood or behavior, pain, etc,).

[00171 ] The terms “treating” and “treatment” and variants thereof refer to delaying the onset of, retarding or reversing the progress of, alleviating or preventing either the disease or condition to which the term applies (injury or damage to the CNS, e.g., resulting from surgical resection, spinal cord injury or traumatic brain injury), or one or more symptoms of such disease or condition. Treating and treatment also refers to increasing, enhancing and promoting neuron regeneration and/or nerve growth in the presence of injury to the CNS. Treating and treatment encompass both therapeutic and prophylactic treatment regimens.

[00172] The term “effective amount” or “therapeutically effective amount” refers to the amount of an active agent sufficient to induce a desired biological result. That result may be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The term “therapeutically effective amount” is used herein to denote any amount of the formulation which causes a substantial improvement in a disease condition when applied to the affected areas repeatedly over a period of time. The amount will vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied. Appropriate amounts in any given instance will be readily apparent to those skilled in the art or capable of determination by routine experimentation.

[00173] A “therapeutic effect,” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[00174] In certain aspects, a therapeutically effective amount of a pharmaceutical composition may be administered to a subject. Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system. Peripheral administration includes but is not limited to oral, inhalation, intravenous, intraperitoneal, intra-articular, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation. The route of administration may be dictated by the disease or condition to be treated.

[00175] Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, and the like are used as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners.

[00176] In each of the above embodiments, a pharmaceutical composition may comprise an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g. fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radionuclide- labeled antibodies, etc.).

[00177] In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.

[00178] The frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms. The timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Treatment could begin immediately, such as at the site of the injury as administered by emergency medical personnel. Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic. Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.

[00179] Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.

[00180] A subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In still another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the subject is a human.

III. Kits

[00181 ] Also provided are kits. Such kits can include a composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the compositions as disclosed herein (see, e.g., section II) can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to systems, assays, primers, or software. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[00182] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[00183] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

General Techniques [00184] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991 ); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.l. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[00185] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

[00186] The term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ± 5%, but can also be ± 4%, 3%, 2%, 1 %, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[00187] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[00188] The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[00189] As used herein, the term “subject” refers to a mammal, preferably a human. The mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.

[00190] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[00191 ] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, a-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[00192] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[00193] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[00194] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[00195] The following eight groups each contain amino acids that are conservative substitutions for one another:

1 ) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and

7) Serine (S), Threonine (T) (see, e.g., Creighton, Proteins (1984)).

[00196] A “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases (“kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term “base pairs”. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired.

[00197] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e. , share at least about 80% identity, for example, at least about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region to a reference sequence, e.g., a LRP1 polynucleotide or polypeptide sequence or fragment thereof, a RAP polynucleotide or polypeptide sequence or fragment thereof, a Gn polynucleotide or polypeptide sequence or fragment thereof, or a Grp94 polynucleotide or polypeptide sequence or fragment thereof as described herein, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50-100 amino acids or nucleotides in length, or over the full-length of a reference sequence.

[00198] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to the nucleic acids and proteins as disclosed herein, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used. [00199] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology (1995 supplement)).

[00200] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1977), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1 , N=— 2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[00201 ] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001 .

[00202] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[00203] The term “individual,” “patient,”, “subject” interchangeably refer to a mammal, for example, a human, a non-human primate, a domesticated mammal (e.g., a canine or a feline), an agricultural mammal (e.g., equine, bovine, ovine, porcine), or a laboratory mammal (e.g., rattus, murine, lagomorpha, hamster).

[00204] 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 multi-specific antibodies. The term “antibody” also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatised 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 (scFvs), Fab fragments, F(ab') 2 fragments, disulfide-linked Fvs (sdFv) fragments, for example, as produced by a Fab expression library, anti-idiotypic (anti-ld) antibodies, intrabodies, nanobodies, synthetic antibodies, and epitope-binding fragments of any of the above.

[00205] The terms “systemic administration” and “systemically administered” refer to a method of administering a composition as disclosed herein to a mammal so that the inhibitor is delivered to sites in the body, including the targeted site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (i.e. , other than through the alimentary tract, such as intramuscular, intravenous, intra-arterial, transdermal and subcutaneous) administration.

[00206] The term “co-administer” and “co-administering” and variants thereof refer to the simultaneous presence of two or more active agents in the blood of an individual. The active agents that are co-administered can be concurrently or sequentially delivered. As used herein, inhibitors of LRP1 , Gn carrier protein, or Gn polypeptide can be co-administered with another active agent efficacious in promoting a therapeutic effect in the CNS.

[00207] The phrase “cause to be administered” refers to the actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a subject, that control and/or permit the administration of the agent(s)/compound(s) at issue to the subject. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.

[00208] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[00209] As various changes could be made in the above-described materials and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

EXAMPLES

[00210] The following examples are included to demonstrate various embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 : Lrp1 is a host entry factor for Rift Valley fever virus

[00211 ] Rift Valley Fever Virus (RVFV) is a zoonotic pathogen with a pandemic potential that can impact human and animal health. RVFV entry is mediated by the viral glycoprotein (Gn); however, the host proteins involved in this process are not well understood. A genome-scale CRISPR loss of function screen in murine cells was conducted with the pathogenic RVFV ZH501 strain, which identified the low-density lipoprotein receptor-related protein 1 (Lrp1 ), as well as Grp94 and receptor associated protein (RAP), as host factors important for RVFV infection. Deletion of Lrp1 limited RVFV infection. Cells lacking Grp94 or RAP showed decreased Lrp1 expression and correspondingly lower RVFV infection. Consistent with a role for Lrp 1 in RVFV infection, the RVFV glycoprotein Gn directly binds to LRP1 cluster II (CLH) and cluster IV (CLiv) domains. Exogenous addition of murine RAP domain 3 (mRAPos), which has an overlapping binding site with RVFV Gn on Lrp1 , inhibits RVFV infection in multiple evolutionarily distinct cell lines, including those derived from humans and ruminants that are part of the zoonotic transmission chain. This Example demonstrates a critical role for Lrp1 as an essential host factor for RVFV infection. Viral hijacking of an evolutionarily conserved host factor, Lrp1 , provides a therapeutic target to limit RVFV infections.

[00212] Emerging viral diseases can be unpredictable and have a devastating impact on human health and the global economy, as evidenced by the recent outbreaks of Ebola virus (2014-15, 2018, and 2020), Zika virus (2015-16), and the ongoing severe acute respiratory syndrome-related coronavirus 2 pandemic. Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that belongs to the Phenuiviridae family (formerly Bunyaviridae) of negative-sense RNA viruses. The geographic range of RVFV includes most of Africa, Madagascar, and the Saudi Arabian peninsula. Importantly, competent mosquito species are found in North America and Europe, and climate change is rapidly altering the natural habitat of RVFV-com petent mosquito and reservoir species. RVFV causes severe disease in livestock, including sheep and cattle, dramatically impacting the socio-economic framework in endemic areas. RVFV is zoonotically transmitted from animals to people, and human infections can result in severe health consequences, including hepatitis, hemorrhagic fever, encephalitis, and retinal vasculitis. The World Health Organization prioritized research on RVFV because of its public health risk and epidemic potential (World Health Organization, 2018). Despite its significance to human health and the potential to negatively impact the economic landscape, there are no safe and efficacious prophylactic or therapeutic treatment options for human use. This gap is in part due to our lack of knowledge of host factors that contribute to cellular RVFV infection.

[00213] RVFV is an enveloped virus with a tripartite genome: L (large) segment, M (medium) segment, and S (small) segment. L encodes the viral RNA- dependent RNA polymerase (RdRp). M encodes the glycoprotein precursor (GPC) and the nonstructural protein NSm. S encodes for the nucleocapsid protein N and nonstructural protein NSs. GPC is post-translationally cleaved into Gn and Gc. Gn forms the glycoprotein spikes, and Gc is a class II fusion protein that remains oriented away from the viral membrane. Gn and Gc together form an icosahedral lattice on the virion surface. The viral Gn/Gc complexes mediate cell entry and fusion. However, the host proteins involved in the process of entry are not well understood.

[00214] RVFV infects the liver in animals including livestock, humans, and laboratory rodents. Cellular tropism of RVFV is very broad, and most cell types can become infected by RVFV, including neurons, epithelial cells, macrophages, granulocytes, pancreatic islet cells, adrenal glands, ovaries, testes, and placenta. Early studies identified the lectin DC-SIGN on skin dendritic cells (DCs) as a factor for the internalization of RVFV through an incompletely defined mechanism. Lectin molecules closely related to DC-SIGN, such as L-SIGN and DC-SIGNR, are found on RVFV- permissive cells, including hepatocytes. Although prior studies show that DC-SIGN is important for RVFV internalization, deglycosylation of the virus did not reduce infectivity, suggesting that attachment and entry may require distinct proteins. Another study identified heparan sulfate as a potential attachment factor for RVFV; however, removal of heparan sulfate reduced but did not eliminate RVFV infection. Although these studies implicate several host factors in RVFV infection, definitive insights into host proteins that are essential for RVFV infection in different cell types and across taxonomically diverse species have yet to be described. [00215] To address this, the present example provides an unbiased genome-wide CRISPR/Cas9 screen where infection by the virulent ZH501 strain of RVFV was used to identify host factors that are either pro-viral or anti-viral. The surface receptor low-density lipoprotein (LDL) receptor-related protein 1 (Lrp1 in mice and LRP1 in humans) was identified as an essential host factor. The screen also identified RAP and GRP94, proteins that modulate Lrp1 surface presentation and function. Clonal knockout (KO) cells lacking Lrp1 , RAP, or GRP94 show significantly reduced infection by both the pathogenic RVFV ZH501 strain and an attenuated vaccine strain, RVFV MP12^GFP . Inhibition of the interaction between RVFV Gn glycoprotein and Lrp1 domain clusters using recombinantly purified mouse RAP domain 3 (mRAPos) protein or anti- Lrp 1 antibodies blocked RVFV entry to target cells from a range of host species. The significance of the Gn-Lrp1 interaction was evaluated in vivo in a mouse model, whereby intracranial infection (IC) of RVFV ZH501 with co-injection with mRAPos resulted in significant protection from disease and death. A mutant mRAPos has diminished binding to two of the four clusters within Lrp1 termed cluster II (CLI I) and cluster IV (CLIV) and shows a correspondingly diminished ability to inhibit RVFV infection in cell culture and in mice. These results demonstrate that Lrp1 is an essential host factor for RVFV infection and that a direct interaction between Lrp1 and the viral Gn protein plays a key role in the infection process.

[00216] CRISPR/Cas 9 screen identified multiple host factors essential for RVFV infection: To identify host factors critical for RVFV infection, an unbiased CRISPR/Cas9 library in a murine microglial BV2 cell line (FIG. 1A) was used. Using the pathogenic BSL-3 strain RVFV ZH501 , the cytopathic effect (CPE) of RVFV infection in BV2 cells expressing Cas9 protein was assessed. Near 100% CPE was achieved within 48 h post-infection (hpi) using multiplicities of infection (MOI) between 0.01-1.0. The BV2 library, consisting of 40 million cells transduced with lentiviruses expressing single guide RNAs (sgRNA) targeting 20,000 unique genes (4 guides/gene), was screened at an average of 5003 redundancy. For the screen, initial infection of the library at MOI 0.1 and 0.01 resulted in significant CPE (FIG. 1 B). At around 8 days post-infection (dpi), survivor clusters (colonies) were observed in transduced but not control cells. Surviving cells were resistant to re-infection with RVFV (FIG. 1 C), suggesting that the resistant cells lacked pro-viral factor(s) required for RVFV infection.

[00217] Cells from the initial infections (MOI 0.1 or 0.01 ) and reinfections were subjected to next-generation sequencing (NGS) and analysis (FIG. 1 D). Among the candidate pro-viral genes, the low-density lipoprotein (LDL) receptor-related protein 1 (Lrp1 ), lipoprotein receptor-related protein-associated protein 1 was identified (Lrpapi ; herein RAP), and endoplasmin (Hsp90b1 ; herein Grp94). other known regulators of Lrp1 expression such as Pcsk9 was also identified. Together, the connectivity of the genes from the screen suggests a key role for LRP1 and its regulators in RVFV infection. Lrp1 is a member of the LDL receptor family (LDLR). LDLRs are highly conserved across species and play roles in lipid metabolism, clearance of circulating lipoproteins including LDL, and in a variety of endocytic and inflammatory signaling processes relevant to lipid metabolism, atherosclerosis, and neurohomeostasis. Lrp1 is ubiquitously expressed, with higher levels of expression in the liver, placenta, and brain. Lrp1 is an essential gene as null mutations in the gene are embryonically lethal in mice. RAP is an important molecular chaperone of Lrp1 that universally prevents ligand interaction to ensure passage of Lrp1 from the ER to the cell surface. Grp94 is an endoplasmic reticulum resident chaperone that controls Lrp1 expression by inhibiting degradation (FIG. 1E).

[00218] RVFV infection is reduced in cells lacking Lrp1: To determine the role of LRP1 in RVFV infection, clonal BV2 cells with knockout alleles of Lrp1 (Lrp1 ^K0 C3 and R1 -R6) were generated by deleting 10-kb sequences containing exons 1 and 2 (FIG. 2A). Clone R3 displayed hypomorphic Lrp 1 expression from an allele with an 8-kb deletion, leaving exon 1 intact and maintained the reading frame of the transcript whereas the other cell lines lacked detectable Lrp1 protein expression (FIG. 2A). RVFV ZH501 infection of these Lrp1 -deficient clones resulted in diminished viral RNA and Gn protein expression at 18 hpi (FIG. 2B). Based on these results, Lrp1 ^PK0 R3 and Lrp1^K0 R4 clones were selected for further characterization. Infection of these cells with the vaccine strain MP12 expressing GFP in place of NSs (MP12^GFP) resulted in decreased infection as assayed by flow cytometry for GFP (FIG. 2D and FIG. 2E). At earlier time points (3 and 6 hpi), MP12^GFP infection was highly reduced in Lrp1 ^K0 R4 cells compared to BV2 wild-type ( VT) as determined by examination of GFP expression by microscopy (FIG. 2C) or viral RNA levels by RT-qPCR analysis. To control for non-specific effects of Lrp1 mutation on viral infection in general, Lrp1 ^K0 R4 cells were infected with influenza A (IAV) PR8 strain and measured viral RNA levels by RT-qPCR at 6 hpi. There was no significant effect of mutation of Lrp1 on IAV infection indicating that Lrp1 is a critical host factor specific for RVFV infection.

[00219] Primary murine cells deficient for Lrp 1 have reduced RVFV infection-. To test if Lrp1 is important in the infection of primary cells, mouse embryonic fibroblasts (MEFs) from mice with floxed Lrp1 alleles were generated. Infection of floxed MEFs (Lrp1 ^F/F) with adenovirus expressing Cre recombinase (Ad^Cre) resulted in a reduction in Lrp1 protein expression by western blot (FIG. 2F). Infection of these Lrp1 - deficient MEFs with RVFV MP12^GFP resulted in >90% reduction in GFP levels, corresponding to RVFV infection (FIG. 2G and FIG. 2H). IAV did not infect MEFs, and therefore as an alternative control, infection of Lrp1 -deficient MEFs was tested with respiratory syncytial virus (RSV), which can readily infect MEFs. Resulting data showed no significant decrease in infection compared to non-floxed MEFs infected with Ad^Cre, suggesting that Cre-dependent deletion of Lrp1 contributed to reduced infection by RVFV MP12^GFP , further supporting the significance of Lrp1 for RVFV infection.

[00220] Host proteins that regulate Lrp 1 surface expression are also important for RVFV infection-, the roles of RAP and Grp94 in RVFV infection was assessed next. RAP is a critical chaperone for members of the LDL receptor family. Two BV2 RAP^K0 clones were generated; RAP^K0 clone A3 was hypomorphic and retained partial Lrp 1 expression, whereas clone RAP^K0 A7 displayed near complete loss of Lrp 1 expression (FIG. 3A). Infection of RAPP^K0 A3 and RAP^K0 A7 clones with RVFV MP12^GFP resulted in 30% and 80% reduction in infectivity, respectively (FIG. 3B and FIG. 3C). These results highlight a correlation between reduced RAP and Lrp1 expression with a reduction in RVFV infection. Loss of Grp94 expression and the concomitant enhancement of proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression is known to increase the degradation of LDL receptors, including Lrp 1 . Two clonal BV2 Grp94 KO lines were generated; Grp94^KO A8 and Grp94^KO B7, which also lacked Lrp1 expression (FIG. 3D). Infection of Grp94 KO lines with RVFV MP12^GFP showed an 95% reduction of infectivity (FIG. 3E and FIG. 3F). Both RAP^K0 A7 and Grp94^KO A8 were as permissive as the WT BV2 cells to IAV infection. Together, these results suggests that multiple host factors regulating Lrp1 surface expression, including RAP and Grp94, are important for RVFV entry.

[00221 ] LRP1 ligand-binding clusters are essential for RVFV Infection'.

LRP1 is a large multidomain protein that consists of two chains, a 515-kDa extracellular alpha chain and an intracellular beta chain connected by an 85-kDa transmembrane domain. Within the alpha chain, there are four ligand-binding active regions with complement-like repeat clusters (CL; termed CLi, CLn, CLm, and CLiv) that are separated by epidermal growth factor (EGF) repeats and b-propeller (YWTD) domains (FIG. 4A). To determine the relative significance of LRP1 clusters for RVFV infection, Lrp1 ^K0 R4 cells were transcomplemented with each of the LRP1 mini-clusters. Infection of transduced Lrp1 ^K0 R4 cells expressing an individual LRP1 CL with RVFV MP12^GFP revealed that LRP1 CLn and CLiv partially restored RVFV infection (FIG. 4B). These results also identified LRP1 CLiv as making a greater contribution to infection, 50% relative to WT. The expression of LRP1 CL in transduced Lrp1^K0 R4 was confirmed by flow cytometry and western blot, where all 4 clusters were detected via staining for HA antigen (FIG. 4C). Because the RVFV glycoprotein Gn mediates viral entry, it was next determined whether Gn interacts with one or more of the LRP1 clusters through direct protein-protein interactions or if Gn-mediated entry requires additional host factors. Coimmunoprecipitation (coIP) assays with LRP1 CL proteins expressed as Fc fusions (termed CL-Fc) (FIG. 4A) demonstrated that recombinant RVFV Gn binds with LRP1 CLiv-Fc and CLn-Fc with high affinity, but not with CLm-Fc or control Fc. These results suggest that LRP1 CLn and CLiv contain binding regions for RVFV Gn protein, consistent with the transcomplementation data (FIG. 4B). The interaction between Gn and LRP1 domain clusters were further characterized by biolayer interferometry (BLI), which revealed preferential binding of RVFV Gn to LRP1 CLiv-Fc, relative to Fc only, CLn-Fc (FIG. 4D-4F), or CLm-Fc. The measured binding constant, KD of 96 ± 16 nM, was obtained with the steady-state BLI data in the association phase. Compared to LRP1 CLiv-Fc binding, RVFV Gn displayed weaker binding to LRP1 CLn-Fc with a KD of 485 ± 139 nM. Next, it was tested if exogenously added LRP1 CLn-Fc, CLm-Fc, CLiv-Fc, and control Fc can inhibit RVFV MP12^GFP infection. All three LRP1 clusters showed measurable neutralization (FIG. 4G-4I), with LRP1 CLiv-Fc resulting in the most significant neutralization. Altogether, the data support a dominant role for LRP1 CLiv in MP12^GFP infection through direct engagement of the viral Gn protein.

[00222] Although the data for LRP1 cluster binding to RVFV Gn or RVFV MP12^GFP neutralization were consistent with a role for CLn-Fc and CLiv-Fc, previous reports have shown that glycosylation alone may also provide viral attachment. Moreover, cluster-Fc binding analysis revealed slower off rates in the BLI studies (FIG. 4E and FIG. 4F), potentially implicating avidity in the interaction between Lrp1 clusters and RVFV Gn proteins. To address these observations, the CLn-Fc and CLiv-Fc fusion proteins were further characterized by reducing and nonreducing SDS-PAGE, which confirmed conformational changes due to the reducing agent, and upon deglycosylation. In each protein, a change in the SDS-PAGE mobility was observed that is consistent with a loss of mass due to deglycosylation. Corresponding size exclusion chromatography further revealed that glycosylated and deglycosylated LRP1 CLn-Fc and CLiv-Fc were not misfolded or aggregated and eluted within the included volume of the column. Because cluster-Fc binding showed slower off rates in the BLI studies, the CLn-Fc and CLiv-Fc fusion proteins were further characterized by mass spectrometry (MS). Based on MS analysis under denaturing and native conditions, glycosylated CLn- Fc and CLiv-Fc proteins have a mass higher than the expected molecular weight from the amino acid sequence and is consistent with additional mass contributions from glycosylation, whereas deglycosylated CLn-Fc and CLiv-Fc proteins resulted in a mass consistent with a CL-Fc dimer. Further analysis of the deglycosylated CLn-Fc and CLi v- Fc proteins under strong reducing conditions resulted in measurements of 68.1 and 76.7 kDa, respectively for CLn-Fc and CLiv-Fc, which are in agreement with the predicted masses of both the proteins in their monomeric forms.

[00223] Because glycosylation can impact binding between LRP1 clusters and RVFV Gn, LRP1 CLn-Fc before and after deglycosylation were further tested in a pull-down assay with RVFV Gn. A similar assay was carried out for LRP1 CLiv-Fc before and after deglycosylation with RVFV Gn. In each assay, interaction between LRP1 clusters were observed regardless of the glycosylation state, whereas the control Fc-only protein did not appear to interact with RVFV Gn. Together, these results show that although LRP1 proteins were glycosylated, the interaction between RVFV Gn and LRP1 clusters was glycosylation independent.

[00224] RVFV entry is reduced in Lrp 1 -deficient cells: T o determ ine if RVFV attachment and internalization are compromised in Lrp1^K0 cells, BV2 WT and BV2 Lrp1 ^K0 R4 cells were incubated with RVFV-MP12^GFP at 4 C and 37 C. RVFV virions bound to the cells (4 C) or internalized (37 C) were subjected to RT-qPCR analysis for quantification. The results show a significant reduction in binding and internalization of the virus particles in BV2 Lrp1 ^K0 R4 cells, compared to BV2 WT cells (FIG. 5A and FIG. 5B)

[00225] Next, recombinant vesicular stomatitis virus (VSV) were engineered by replacing VSV glycoprotein with RVFV glycoproteins (VSV-RVFV) because glycoprotein spikes facilitate the virus attachment and internalization. VSV and VSV- RVFV were labeled with Alexa-flour 647 and Alexa-flour 588, respectively. Upon incubation of the labeled viruses with BV2 WT and Lrp1 ^K0 R4 cells at 4 C and 37 C, reduced binding occurred for VSV-RVFV to BV2 Lrp1 ^K0 R4 cells as compared to VSV, with the VSV glycoprotein on the viral surface (FIG. 5C and FIG. 5D). Taken together, the results demonstrate that Lrp 1 is a critical host factor for RVFV attachment and entry, and this interaction is dependent on the RVFV Gn protein.

[00226] RVFV Gn glycosylation and host glycosaminoglycans (GAGs) are dispensable for RVFV infection: Viral glycoproteins are highly glycosylated, and previous studies revealed that the lectin DC-SIGN promotes RVFV internalization in dermal dendritic cells. Related lectin molecules, such as L-SIGN and DC-SIGNR, are also found on RVFV-permissive cells, including hepatocytes. However, deglycosylating the virus did not reduce the infectivity, and interaction with lectin molecules was dependent on the glycosylation of Gn. Here, several binding assays were performed with purified bacterial recombinant RVFV Gn protein, which lacks any glycosylation (FIG. 4D-4F), and tested the ability to inhibit RVFV infection. Cells that were pre-treated with non-glycosylated Gn displayed dose-dependent inhibition of RVFV MP12^GFP infection, suggesting that viral Gn glycosylation is not critical for RVFV entry. [00227] Host glycosaminoglycans (GAGs) such as heparan sulfate were also reported to play a role in RVFV infection, but removal of heparan sulfate did not eliminate RVFV infection. Consistent with this observation, the screen identified Ext2 encoding Exostosin-2, a key protein in the heparan sulfate biosynthesis pathway, as a host factor for RVFV entry. However, it was found that deletion of Ext1 or Ext2 did not significantly impact virus infection. Furthermore, pretreatment with surfen, a GAG inhibitor, did not result in a substantial change in virus infection. Taken together, viral Gn glycosylation and host GAGs are not essential factors for RVFV infection.

[00228] CLn and CLiv-specific Abs reduce infection by R VFV: T o further evaluate the significance of Lrp1 as a potential receptor for RVFV, a phage-displayed library of synthetic human antigen-binding fragments (Fabs) was used to identify Fabs that specifically recognized Lrp1 CLn or CLiv. These efforts led to the identification of many unique Fabs with high affinity for Lrp1 CLn and for CLiv. For each set of Fabs, their cross reactivity to CLn and CLiv were also evaluated. From these results, four distinct Fab sequences were identified for further evaluation in the full-length immunoglobulin (IgG) format, and their specificities for Lrp1 CLn-Fc and Lrp1 CLiv-Fc were evaluated (15408, 15409, 15430, and 15438). Fab15409 bound with an affinity and specificity to CLn (KD CLn = 2.3 nM and KDCLiv = not determined [ND]), whereas Fab 15408 bound with high affinity to CLn and moderate affinity to CLiv (KD CLn = 1 .0 nM and KD CLiv = 11 nM). In comparison, Fabs 15430 and 15438, raised against LRP1 CLIV, bound with high affinity to CLiv (Fab 15430, KD CLn = 40 nM and KD CLiv = 10 nM; Fab 15438, KD CLn = ND and KDCLiv = 1 nM). To further evaluate the impact of anti-Lrp1 antibodies on RVFV infection, each of these Fabs were tested in the context of a human IgG framework in cell-based neutralization assays of RVFV MP12^GFP . The resulting data revealed >80% neutralization by the CLn-binding IgGs 15408 and 15409, and >50% neutralization by the CLiv-binding IgGs 15430 and 15438, compared with an isotype control (FIG. 5E). As a follow-up, bi-specific IgG 15408 in a dose-response neutralization assay were evaluated, and the data revealed an ECso of 936 ± 78 ng/mL (FIG. 5F). IgG 15408 was selected on the basis that the Fab 15408 bound both Lrp1 CLn and CLiv. Taken together, these results support the specificity of Lrp1 for RVFV infection and suggest that antibodies targeting Lrp1 clusters, CLn in particular, have the potential to block access to RVFV entry and therefore present a potential therapeutic avenue to prevent RVFV infection.

[00229] mRAP binds Lrp1 and inhibits RVFV infection in cells derived from taxonomically diverse hosts: RAP binding to LRP1 was demonstrated biochemically in multiple previous studies. When recombinantly expressed RAP is exogenously introduced in cell culture, RAP is known to bind to the LRP1 clusters and inhibit interactions with all known ligands. RAP contains three domains (D1-D3) (FIG. 6A), and RAP D3 binds both LRP1 CLn and CLiv. Our BLI data revealed that both human LRP1 CLII and CLiv bind to mouse RAPD3 (mRAPD3) (FIG 6B and FIG. 6C), consistent with previous studies. To establish if mRAPD3 and Gn have overlapping binding sites on LRP1 , Gn binding with LRP1 CLiv was tested in the absence or presence of mRAPD3. At higher concentrations, it was found that mRAPD3 competed with RVFV Gn for binding to LRP1 CLiv in vitro (FIG. 6D). These results suggest that mRAPD3 can function as an inhibitor of Gn binding and as a probe to assess interactions with LRP1 . inhibitory neutralization assays were performed next using mRAPD3. It was found that mRAPD3 potently inhibited RVFV MP12^GFP infection with an ECso of 0.59 ± 0.2 mg/mL (FIG. 6E and FIG. 6F). Consistent with previous studies, mutant mRAPD3 (FIG. 6A) showed weak interaction with LRP1 CLn (FIG. 6G) and CLiv (FIG. 6H). Upon incubation of mutant mRAPD3 with BV2 cells, RVFV MP12^GFP infection was moderately affected (FIG. 6I).

[00230] To determine the relevance of LRP1 as an essential factor for RVFV infection in cells derived from other organisms (mice, hamsters, cows, monkeys, and humans), cells were treated with 5 mg/mL of mRAPD3 (10³ ECso). In all cell lines tested, a substantial inhibition of infection by RVFV MP12^GFP was observed (FIG. 6J) and by the pathogenic RVFV ZH501 (FIG. 6K). Importantly, a dose-dependent reduction in RVFV infection was observed across all cell lines, further supporting the observations.

[00231 ] Because mRAPos can prevent infection in multiple cell types, it was assessed whether mRAP inhibition of RVFV infection occurred at the level of virus binding or post-binding event. Pre-incubation of BV2 cells with mRAPos resulted in significant protection from RVFV MP12^GFP infection, whereas post-infection treatment with mRAPD3 resulted in infection levels similar to control cells lacking mRAPos treatment. These results support a model for mRAPos blocking RVFV Gn interaction with Lrp1 receptor as a pre-infection event. To assess the integrity of mutant mRAPos, size exclusion chromatography was used to evaluate the proteins, which show similar elution profiles for mutant mRAPos and mRAPos, suggesting that the physical properties, including hydrodynamic behavior of both proteins are similar.

[00232] mRAP, an Lrp1 ligand, protects mice from lethal infection with RVFV ZH501: To support Lrp1 as a critical factor for RVFV infection, the effect of mRAPD3 treatment was evaluated in vivo using a mouse model. Mice (C57BL/6) are extremely susceptible to RVFV infection, with an LDso of <1 plaque-forming units (PFU) or TCIDso after footpad injection. Because mRAPos is highly effective at preventing RVFV ZH501 infection of neurons in cell culture (FIG. 6K), it was sought to determine whether mRAPos treatment can prevent RVFV infection of the brain using IC injection as an initial proof-of-concept experiment. Similar to footpad injection, the LDso of RVFV ZH501 by IC injection is <1 PFU with an average survival time (AST) of 3.5 days. The effectiveness of administering 215 mg of mRAPos IC was evaluated simultaneously with 10 PFU (FIG. 7A) or 1 PFU of RVFV ZH501. Most mice lost weight within a day after IC injection but recovered and gained weight thereafter. Groups of infected, untreated mice succumbed to disease in both the 10 PFU group (13 of 13 died; AST 2.5 days) (FIG. 7A) and 1 PFU group (11 of 14 died; AST 4.5 days), respectively. In contrast, coadministration of mRAPos along with IC infection with RVFV ZH501 resulted in a significant increase in survival in both dose groups and an increase in AST of those that succumbed (FIG. 7A and S9B). For the 10 PFU group, 12 of 17 m RAP-treated mice survived with an AST of 5.2 days for the five mice that died (FIG. 7A). For the 1 PFU group, 11 of 17 mRAP-treated mice survived, with an AST 5.8 days for the six mice that succumbed to disease. As controls, groups of mice were given equivalent amounts of either an irrelevant control protein (Ebola virus VP30 protein) or the mutant mRAPos that showed weaker interaction with Lrp1 and reduced neutralization of MP12 (FIG. 6G-6I). Mice in both control groups did not survive co-infection with 10 PFU of RVFV ZH501 and succumbed within an average of 3.5 days (FIG. 7A). These results suggest that mRAP with Lrp1 -binding capability is able to prevent lethal infection with RVFV. [00233] In a follow-up experiment, groups of three mice from each treatment group underwent planned euthanasia at 3 dpi for direct comparison of tissue viral loads and pathology across groups. The liver, spleen, brain, and serum from mRAPD3-treated mice co-infected with 10 PFU of RVFV contained reduced, but not eliminated, levels of both viral RNA and infectious virus (FIG 7B-7C) compared to untreated, mutant m RAP-treated, or control-protein-treated RVFV-infected control mice. Infection levels of the tissues from 3 dpi were confirmed using immunofluorescence with an anti-NP antibody and histopathology. At 3 dpi, mice infected with 10 PFU contained widespread RVFV-antigen-positive cells in both the liver and brain in untreated control, mutant mRAP, and control protein-treated animals (FIG. 7D and FIG. 7E). Hematoxylin and eosin (H&E) staining revealed classic indications of RVFV-mediated hepatic destruction and hemorrhage. Similar results were seen for control mice infected with 1 PFU of RVFV ZH501. In comparison, tissue sections from the mRAPos-treated mice contained undetectable levels of viral antigen staining and no histological damage caused by viral infection (FIG. 7D and FIG. 7E). The mRAPos-treated mice that survived RVFV infection showed anti-RVFV serum titers consistent with infection and survival. Collectively, these in vivo proof-of-concept experiments provide evidence of a significant reduction in viral infection in multiple tissues when mRAPos is co-administered with RVFV at the time of infection. Important controls including a mutant mRAP that shows reduced Lrp1 binding were not able to rescue mice from lethal infection. These results provide further support for a role for Lrp1 as a major cellular factor required for RVFV infection in a rodent model.

[00234] DISCUSSION: Given the broad tropism of mosquito-transmitted zoonotic viruses such as RVFV, host factors that mediate entry are critical in order to fully understand viral emergence, zoonosis, and spread. Previous studies have implicated several cellular factors in RVFV binding and entry. The glycosaminoglycan (GAG) heparan sulfate was identified in a genetic screen as essential for RVFV infection. Although the studies showed that heparan sulfate proteoglycan (HSPG) inhibition resulted in inhibition of RVFV infection in some cell types, the exact role of HSPG in RVFV infection was unclear. Interestingly, the screen also identified an HSPG- related gene, Ext2, a gene involved in the synthesis of GAGs. It was found, however, that deletion of Ext1 or Ext2 did not have a significant impact on RVFV infection in mouse BV2 cells and that treatment with the GAG inhibitor surfen also did not inhibit RVFV infection. These results support a minimal role for HSPGs in BV2 cells despite their importance as a receptor for macromolecular endocytic cargo; instead, they may play a role in augmentation of RVFV infection. Like HSPGs, C-type lectins such as DC- SIGN were identified as mediating RVFV infection of dermal dendritic cells and some other cell types. RVFV has broad tropism and infects a wide range of tissues. Because DC-SIGN was not expressed in many cell types, including BV2 cells in which these assays were conducted, these results do not directly address the significance of the previous findings for DC-SIGN. It is important to note that DC-SIGN interaction with RVFV glycoprotein was glycosylation-dependent, which suggests that DC-SIGN is unlikely to be a proteinaceous receptor for RVFV. In contrast, the results here, including biochemical studies using recombinant bacterially expressed non-glycosylated RVFV Gn and deglycosylated LRP1 , show that the interaction between Gn and Lrp1 does not depend on the glycosylation state of LRP1 or RVFV Gn.

[00235] Given the limitations of previous findings and to address the need to better understand tropism and entry of RVFV, a pooled genome-scale screen using the CRISPR/Cas9 system was conducted. The present example identified an LDL receptor family protein, Lrp1 , as an essential host factor capable of mediating RVFV infection across cell lines from multiple species. A combination of Lrp1 KO cells and cells lacking key chaperones for Lrp1 processing and surface presentation, including RAP and Grp94, provide support for Lrp1 as a proteinaceous entry factor. In cells lacking RAP or Grp94, it was observed reduced Lrp1 expression and concomitantly demonstrated reduced binding by two strains of RVFV. Cells lacking Lrp1 expression also showed reduced binding by a chimeric VSV expressing the RVFV glycoproteins, demonstrating that interaction between Lrp1 and RVFV is at the level of glycoprotein binding and entry. Lrp1 is also important for RVFV infection of primary cells, as primary MEFs from Lrp1^F/F mice transduced with Ad^Cre showed reduced infectivity by RVFV. The biochemical analysis revealed a direct interaction between RVFV Gn with some but not all complement-like repeat clusters in the Lrp1 ectodomain. Notably, Lrp1 CLiv has emerged as an important site of interaction. Exogenous addition of Lrp1 CLiv-Fc resulted in potent neutralization of RVFV infection in vitro.

[00236] The D3 domain from mouse RAP is a known Lrp1 -interacting protein, and when added exogenously, it serves as an inhibitor of all known Lrp1 ligands. The biochemical studies showed that mouse RAPDS, like RVFV Gn, bound to Lrp 1 domain CLiv with higher affinity than CLH. It was also shown that RAPDS competed with Gn for binding to Lrp1 . Blocking the LRP1 receptor with RAPDS inhibited RVFV infection in cells derived from a variety of species including rodents, ruminants, and primates. Notably, RAPDS was also effective in human SH-SY5Y neuronal cells, where infection with ZH501 was rendered undetectable, further supporting the broad importance of Lrp1 in RVFV infection with implications for understanding neuropathogenesis. As an important control, it was shown that m RAPDS containing two point mutations that reduce binding to CLiv and CLn can no longer effectively block RVFV infection, thus supporting the model that Gn binding to CLiv as an important interaction. Finally, it was shown that human antibodies that target Lrp 1 are also potent inhibitors of RVFV infection.

[00237] Similar to RAP, Grp94 impacts Lrp1 cell surface levels and Lrp1 recycling via an indirect mechanism. Pcsk9 is expressed as a pro-protease and is eventually secreted where it binds LRP1 to enhance LRP1 endocytosis. In the endoplasmic reticulum, Grp94 binds to Pcsk9 and prevents its release from the cell. As discussed above, the screen identified Lrp1 , Grp94, and RAP as pro-viral factors. The factors were identified as hits in the screen based on Iog2-fold enrichment relative to an untreated pooled cell population. Importantly, the relative levels of sgRNA relative to an untreated pooled cell population indicates sgRNA targeting Pcsk9 is inversely correlated with RVFV infectivity, suggesting loss of Pcsk9 gene product results in higher levels of Lrp1 and consequent higher levels of infection. Taken together, these observations provide further evidence that a pathway regulating Lrp 1 biosynthesis and surface presentation is essential for RVFV infection.

[00238] In this Example, it was established that RVFV glycoprotein Gn interacts directly with host factor, Lrp 1 , and this interaction is largely driven through direct binding to Lrp1 CLiv, with a weaker interaction with Lrp1 CLn. The data showed that bacterially expressed Gn, lacking glycosylation, binds directly to Lrp1 and also competes with virus and inhibits infection of cells. Similarly, deglycosylated LRP1 also interacted with RVFV Gn lacking glycosylation. Thus, the results point to a model where RVFV Gn interaction with Lrp1 functions as a proteinaceous entry factor for RVFV infection. The exact mechanism by which Lrp1 functions in RVFV entry is under further investigation. Since Lrp1 expression is ubiquitous and the RVFV receptor is conserved across taxonomically diverse species, the results support Lrp1 as a potential host factor that can promote infection in multiple cell types and would explain the broad tropism of RVFV across species. The discovery of Lrp1 as a major cellular factor for RVFV provides a framework to better understand the molecular basis for RVFV attachment and internalization.

[00239] In addition to biochemical and in vitro evidence presented above, provided is compelling data on the in vivo relevance of Lrp1 as a critical factor for RVFV infection through proof-of-concept mouse experiments. Simultaneous intracranial administration of mRAPos and RVFV ZH501 significantly enhanced survival from this otherwise highly lethal infection. In contrast, a mutant mRAPos, which binds weakly to Lrp1 , failed to protect mice, supporting the specificity for the role of Lrp1 in viral entry, ly is suggested that mRAPos was able to block infection of cells in the brain, similar to our results from the exogenously treated cell lines, and thereby reduce dissemination from the brain to the liver and spleen resulting in enhanced survival of the mice. These experiments pave the way for further exploration of the role of Lrp1 in dissemination and tropism in vivo.

[00240] In summary, Lrp1 was identified as a novel proteinaceous host factor important for RVFV entry with potential to support infection. Conservation of Lrp1 across cell types and species, including mosquitos, a vector host for RVFV, highlight the significance of these finding in the context of broad tropism observed for RVFV. Although the exact mechanism by which Lrp1 mediates RVFV entry require further study, the findings provide a foundation for answering many open questions related to RVFV, including mechanisms associated with zoonotic transmission, tropism, spread, and pathogenesis. Knowledge gained from these studies positions us to explore Lrp1 , a conserved cell-surface protein, as a target for prophylactic and therapeutic development of RVFV infections.

[00241 ] Cells. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Cat. 11965084) supplemented with 10% fetal bovine serum (FBS) (Sigma Millipore, Cat. F2442) in a humidified incubator at 37°C and 5% CO2. For the murine microglial BV2 cell lines, the media was supplemented with 10 mM HEPES (Corning, Cat 25-060-CI) and 1 mM sodium pyruvate (Corning, Cat. 25-000-CI). HEK293T (CRL-3216), VeroE6 (CRL-1586), HepG2 (HB-8065), SH-SY5Y (CRL-2266), and COS1 (CRL-1650) cells were obtained from American Type Culture Collection (ATCC). BCE C/D-1 b (ATCC 2048), BHK-21 (ATCC CCL-10), and BV2 cells were provided by M. Diamond (WUSM), S. Whelan (WUSM), and H. Virgin (WUSM), respectively.

[00242] Preparation of primary mouse embryonic fibroblasts. Lrp 1 -flox m ice were purchased from the Jackson Laboratory (B6; 129S7-Lrp1 ^tm2Her /J, Stock# 012604). E14.5 embryos were obtained by timed mating of Lrp1 ^F/+ mice, were genotyped by polymerase chain reaction using genomic DNA from tissue digested with 0.5 mg/ml proteinase K in DirectPCR Lysis Reagent (Viagen, 101 -T) for 30 minutes at 55°C. Genotyped embryos were minced into small pieces and digested with 0.25% Trypsin/0.02% EDTA (Millipore Sigma T4049) for 25 minutes, followed by culture in DMEM supplemented with 10% FBS and cryostock after two days of culture.

[00243] Viruses. RVFV ZH501 (provided by S. Nichol, CDC) was generated from reverse genetics plasmids containing the WT ZH501 sequence, which was confirmed by sequencing. RVFV ZH501 is a select agent and is handled at BSL-3 in the Pitt RBL. Virus was amplified in VeroE6 cells and p2 stock was used for this study (titer 1x10⁷ pfu/mL). A standard viral plaque assay (VPA) was used to measure infectious titers; VPAs used an agarose overlay (1x minimum essential medium, 2% FBS, 1 % penicillin/streptomycin, HEPES buffer, and 0.8% SeaKem agarose) and were incubated for 3 days at 37°C, followed by visualization using crystal violet. RVFV MP12^GFP (provided by M. Diamond, WUSM) was amplified in VeroE6 cells. The virus was collected 5 dpi and then filtered through 0.45 pm, aliquoted, and frozen at -80°C. The titer of the virus stock was calculated (~ 6.5 x 10⁷ lU/mL) and all experiments in this study were performed using the same stock of the virus. Adenoviruses Ad-mCherry (Cat 241 #1767) and Ad-mCherry Cre recombinase (Cat #1773) were purchased from Vector Biolabs. Adenoviruses were used for infection of mouse embryonic fibroblasts (MEFs). Lentiviruses were used to transduce the sgRNA to generate BV2 library cells. Influenza A virus, strain PR8 (IAV PR8) was provided by J. Boon (WUSM). Respiratory syncytial virus, RSV GFP5 (Cat# R125) was purchased from Viratree.

[00244] Antibodies. The following antibodies were used in the study: rabbit anti-LRP1 (Cell Signaling, cat. 64099), rabbit anti-His antibody (Cell Signaling, Cat. 2365), anti-p tubulin (Sigma Aldrich, Cat. T8328-200UL), anti-RVFV clone 4-39-CC (BEI Resources; NR-43195).

[00245] CRISPR Cas9 Screen. BV2 Cas9 library cells were generated as described previously. Briefly, Cas9 activity was evaluated in BV2-Cas9 cells by transducing pXPR 011 plasmid (Addgene 59702) expressing eGFP and sgRNA targeting eGFP. Further, the BV2-Cas9 cells were transduced with the Brie library (Addgene #73633) targeting 19,674 mouse genes with 78,637 gRNAs (~4 gRNAs for each gene). 160 x 10⁶ cells were transduced with the library at 0.25 infectivity rate to achieve a coverage of 500x and two days’ post-transduction, puromycin (Sigma Aldrich, Cat. P833) was added and the cells were selected in puromycin for 5 days. Library cells were expanded and coverage of 500 per sgRNA (40x10⁶ transduced cells). Two vials of library cells each containing 25 x 10⁶ cells were seeded in 150 mm flasks and infected with RVFV ZH501 at MOIs of 0.1 and 0.01 in the University of Pittsburgh RBL BSL-3 facility. Infections were carried out using DMEM with 2% FBS. Cells were observed daily for cytopathic effect (CPE). Dead floating cells were removed and replaced with fresh DMEM/2%FBS. By 4 dpi, the majority of the dead cells were removed. Surviving cells were cultured in DMEM with 10% FBS for an additional 14 days, during which time colonies developed. At 18 dpi, all remaining cells were then trypsin ized ; half of the cells were treated with TRIzol for genomic DNA extraction and the other half were re-infected with RVFV ZH501 at MOIs of 0.1 and 0.01. Three days after re-infection, remaining live cells were treated with TRIzol for DNA extraction.

[00246] Genomic DNA Extraction, Next-Generation Sequencing, and Analysis. Genomic DNA was extracted from TRIzol treated samples as previously. Briefly, 20 pL of chloroform was added to each TRIzol treated sample (~1 mL), incubated at 25 °C for 2-3 mins, and then centrifuged at 12,000 x g for 15 mins at 4 °C. The upper aqueous phase containing RNA was discarded. 300 pL of ethanol was added to each sample and mixed by inverting several times. Samples were incubated for 2-3 mins and then centrifuged at 2000 x g for 5 mins at 4 °C to pellet the DNA. The pellet was resuspended in 1 mL of 100 mM sodium citrate (pH 8.5) in 10% ethanol, incubated for 30 mins, and centrifuged at 2,000 x g for 5 mins at 4 °C. The supernatant was discarded and the process was repeated twice. The pellet was washed with 75% ethanol and gDNA pellets were air-dried and solubilized in 500 pL of 8 mM NaOH. After centrifugation at 12,000 x g at 4 °C for 10 mins, the supernatant was transferred to a new tube, and the pH was adjusted to 7.5 with HEPES. The DNA purity and concentration were determined using the NanoDrop 2000c spectrophotometer (Thermo Scientific).

[00247] Illumina sequencing was performed at the Broad Institute at the Massachusetts Institute of Technology, similar to previous studies. Briefly, gDNA was PCR amplified in a 96-well plate, each well containing up to 10 pg of the DNA, using primers amplifying barcodes associated with each sgRNA in the integrated vector. PCR products were purified and sequenced on Illumina HiSeq 2000. Barcodes were deconvoluted and mapped to the reference file. An array of read counts were generated and normalized to 10⁷ total reads per sample as scores files. The data was then Iog2- transformed to generate Iog2-norm files. The abundance of perturbations was calculated as Iog2 fold change (LFC) by subtracting the average of Iog2 normalized values of each infection condition with the uninfected Iog2-normalized values. Volcano plots were generated to display the primary screening data where the x-axis represents average Iog2 fold change of all perturbations of a gene and the y-axis represents average p-values on the logic scale (github.com/mhegde/volcano_plots).

[00248] Generation of BV2 Knockout cell lines. All knockout cell lines were generated at Genome Engineering and iPSC center at Washington University. Briefly, BV2 cells were nucleofected with Cas9 and gene-specific gRNAs. For Lrp1 KO cells, a dual gRNA targeting approach was employed to delete a 9.4kb coding fragment of the Lrp1 gene. The cells were subjected to single-cell sorting and DNA was extracted from each clone and sequenced to confirm the deletions. For RAP and Grp94 knock cells, a single gRNA was used to target each gene. After cell sorting, each clone was sequenced for indels.

[00249] Neutralization assays with mRAPos and soluble LRP1 CLn, CLm, and CLiv domains. Cell lines (e.g. BV2, HEK-293T, BHK-21 , BCE, HepG2, SH-SY5Y, VeroE6) from different species were seeded in 24-well plates and cultured overnight. The next day, media was removed and cells were incubated with recombinant mRAPD3 protein at concentrations as defined in the figure in DMEM media supplemented with 2% FBS. After 45 mins of mRAP D3 treatment, the cells were infected with either RVFV MP12^GFP or RVFV ZH501 . 15-24 hpi, cells were assessed for virus infection through GFP expression by flow cytometry, intracellular Gn expression by flow cytometry, or viral RNA synthesis by q-RT-PCR analysis. In LRP1 neutralization assays, Fc and Fc- fused LRP1 CLn, CLm, and CLiv domains were pre-incubated with the RVFV MP12GFP virus in serum-free media at increasing concentrations as described in the figure. After 1 hr of incubation at 37°C, the preparations were used to infect the BV2 cells. Virus infection was examined 15 hpi by flow cytometry.

[00250] Plasmids. RVFV glycoprotein Gn ectodomain (amino acid 1 - 316; accession number DQ380200) (PMID: 28827346) derived from ZH501 and mRAP D3 (amino acid 243-360; NM_013587.2) were cloned into a pET28 vector (Novagen). Mouse Lrp1 CLi (residues 26-114), CLn (residues 804-1184), CLm (residues 2482- 2943), and CLiv (residues 3294-3784) domains were cloned into a modified pLVX-EF1 a-vector (Takara) containing the Lrp1 transmembrane (TM) and cytoplasmic tail (CT) domains (residues 3785-4545).

[00251 ] Protein expression and purification. Gn316 expression plasm ids were transformed in BL21 (DE3) E. coli cells (Novagen). Colonies were cultured in Luria Broth media at 37°C to an OD600 of 0.6 and induced with 0.5 mM isopropyl-|3-D- thiogalactoside (IPTG) for 12 hr at 18 °C. Cells were harvested and resuspended in lysis buffer containing 20 mM Tris-HCI pH 8.0, 500 mM NaCI, 5 mM 2-mercaptoethanol. Cells were lysed using an EmulsiFlex-C5 homogenizer (Avestin). The pellet was resuspended in 30 mL cold 2 M urea, 20 mM Tris-HCI pH8.0, 500 mM NaCI, 2% Triton™ X-100 prior to centrifugation at 47,000 x g at 4 °C for 10 min. Inclusion bodies were isolated after repeated rounds of resuspension in urea and centrifugation. The final pellet was resuspended in 20 mM Tris-HCI (pH 8.0), 500 mM NaCI, 5 mM imidazole, 8 M urea, and 1 mM 2-mercaptoethanol. Gn316 was refolded on a NiFF (GE Healthcare) column using a reverse linear urea gradient and eluted with imidazole. Gn316 was further purified using a size exclusion column (SD200 10/300L, GE Healthcare). mRAPD3 was expressed similarly as above, harvested, and resuspended in Tris buffer. mRAPD3 was purified using a series of chromatographic columns, including a size exclusion column as the final step. Protein purity was assessed by Coomassie staining of SDS-PAGE.

[00252] Biolayer Interferometry. BLI assays were conducted at 30 °C at 1 ,000 rpm (Octet Red, ForteBio). Anti-Human IgG Fc Capture biosensors were hydrated in kinetics buffer (Phosphate Buffer Saline (PBS) containing 0.02% Tween-20, 1 mg/mL BSA) for 15 min. Recombinant human LRP1 CLiv Fc Chimera (R&D SYSTEMS, #5395- L4-050), recombinant human LRP1 CLII Fc chimera (R&D SYSTEMS, #2368-L2-050), or recombinant human IgG 1 Fc (R&D SYSTEMS, #110-HG-100) were loaded at 200 nM in buffer for 600s prior to baseline equilibration for 300 s. Association of RVFV_Gn or mRAP D3 at various concentrations (0.5, 1 , 4, 8, and 12 pg/mL) was carried out for 900 s prior to dissociation for 900 s. Data were baseline subtracted to the buffer only controls. Experiments were done in triplicate.

[00253] Competition and pull down assays. Competition assay was performed using rProtein A Sepharose® Fast Flow resin (GE Healthcare, #17-1279-03). Human LRP1 CLiv Fc Chimera (hLRP1_D4, R&D SYSTEMS, #5395-L4-050) was immobilized on resin prior to incubation with RVFV Gn, mRAP DS, or fixed concentration of RVFV Gn in the presence of increasing concentrations of mRAPD3 (1 -10 pg/mL). After a 1 -h incubation at 25 °C, beads were washed six times with PBS-T buffer prior to elution of bound proteins in 2X-laemmli sample buffer. Samples were run on SDS- PAGE and analyzed by western blotting using an anti-H is-tag antibody or anti-human Fc antibody. Similarly, pulldown assay was performed by incubating RVFV Gn with human IgG 1 Fc and recombinant human LRP1 CLn, CLIH and CLiv Fc chimera using rProtein A beads. After washings, the elution were analyzed on western blotting by using anti-His and anti-human Fc antibodies. [00254] Flow Cytometry. RVFV MP12^GFP and RSV^GFP infected cells were analyzed by flow cytometry (BD LSR Fortessa™ X-20 and BD LSR Fortessa™) and the data were analyzed using BD FACS Diva software, as described previously. All flow experiments were done at the Flow Cytometry Facility, Department of Pathology and Immunology, WUSM. For flow experiments with RVFV ZH501 , infected cells were harvested at the indicated time points, stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit for UV excitation (Invitrogen L34961 ), permeabilized with BD fix/perm, then stained with RVFV anti-Gn monoclonal antibody (BEI NR-43195) followed by a FITC-conjugated anti-mouse secondary antibody. Samples were acquired using BDLSRII flow cytometer and analyzed with FlowJo at the University of Pittsburgh Flow core facility. Uninfected cells were run in parallel for subtraction of background.

[00255] Reverse transcription- quantitative PCR. cDNA was synthesized using SuperScriptTM III (Invitrogen) by following the manufacturer’s instructions.

[00256] Virus binding and internalization assays: BV2-WT and BV2-Lrp1 KO (R4) cells (5 x 10⁵) were seeded in 12-well plates and incubated with GAG antagonist surfen (10 mM) for 30 mins. Next, the cells were moved to 4 C for 30 mins and then incubated with RVFV-MP12^GFP virus (MOI 0.5) for 1 hr at 4 C. The cells were washed 5 times with PBS supplemented with 3% bovine serum albumin and 0.02% tween-20. For virus binding assay, the cells were collected and lysed in RLT buffer (QIAGEN) for RNA extraction using RNeasy Mini Kit (QIAGEN). For internalization assay, the cells were incubated at 37 C for 1 more hour. The cells were again washed and collected for RNA extraction using RNeasy kit (QIAGEN). The RT-qPCR was performed using Power SYBR Green Master Mix (Thermo Scientific) with mouse hprt as a control.

[00257] Virus Particle Binding Assay: Gradient purified VSV-RVFV and VSV particles were labeled with AlexaFluor 594 and 647, respectively, as previously described. Both viruses were added to indicated cells and incubated at either 37 C for 15 min or 4 C for 1 h. Three minutes before the end of the incubation, 1 ug/mL Alexa 488 labeled wheat germ agglutinin was added to the media. Cells were then washed two times with ice cold PBS and fixed with 2% PFA for 10 minutes at room temperature. Samples were imaged using a Nikon Ti2 inverted microscope outfitted with a spinning disc head (Yokogawa), Andor Zyla 4.2 Plus sCMOS monochrome camera, and piezo Z stage (Physik Instrument). Images were acquired using Nikon Elements Acquisition Software AR 5.02. Image analysis was performed using Arivis Vision4D. Briefly, cells were masked, and the volume was determined using membrane-based segmentation. Bound viral particles were counted for each image and particle binding per area was calculated by dividing particle counted by the determine cellular volume. At least 3 images were acquired and analyzed for each sample.

[00258] Antibody selections by phage display: A synthetic phage-displayed Fab library was used for binding selections with immobilized Fc-tagged LRP1 -Cu (R&D SYSTEMS, #2368-L2-050) or LRP1-Civ (R&D SYSTEMS, #5395-L4-050), as described. Following 4 rounds of selections, individual clones were characterized for binding to target and control proteins by phage ELISA. Phagemid DNA from binding clones was amplified by the PCR and sequenced to decode the antibody variable region sequences.

[00259] IgG production: DNA encoding the variable regions of phage- derived antibodies was amplified from phagemid DNA by the PCR and sub-cloned in to separate light and heavy chain expression vectors. Equal amounts of DNA from heavy and light chain expression vectors were mixed, diluted in Opti-MEM medium (GIBCO), and complexed with FectoPro transfection reagent (Polyplus Transfection) for 10 minutes. Complexed DNA was transfected in to Expi-293F cells in Expi293 medium and the cultures were incubated for 5 days at 37 C in a humidified, 8% CO2 environment with shaking. Secreted IgG protein was purified from supernatants with Protein A Sepharose (GE Healthcare), eluted in IgG elution buffer (Thermo), neutralized with 1 M Tris buffer pH 8.0 (Invitrogen), and exchanged in to PBS using centrifugal concentrators.

[00260] Enzyme-linked immunosorbent assays: Binding of Fab-phage or IgGs to antigen was measured by ELISA. Wells of microplates (Nunc) were coated overnight at 4 C with a 2 mg/mL antigen solution in PBS pH 7.4 and blocked with PBS, 0.2% BSA for 1 hour at room temperature. Blocking solution was removed, plates were washed 4 times with PBS, 0.05% Tween, and phage or IgG was added and incubated for 30 minutes. Plates were washed, incubated for 30 minutes with an appropriate secondary antibody, and developed with TMB substrate (KPL Laboratories).

Example 2: Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1

[00261 ] Emerging zoonotic viruses are at the forefront due to the ongoing COVID-19 pandemic. Bunyaviruses are a large group of diverse, arthropod-borne viruses that present concern due to reassortment and evolutionary capacity of their segmented RNA genomes. The present Example demonstrated that the conserved host cell surface receptor low-density lipoprotein receptor-related protein (Lrp1 ) facilitates efficient cellular infection by the South American bunyavirus Oropouche virus (OROV). Therefore, Lrp1 is a host factor for multiple bunyaviruses, including Rift Valley fever virus (RVFV), and plays a broader role in bunyavirus infection than has been previously known. This Example identifies a pan-bunyaviral host factor with significant implications for therapeutic targets.

[00262] Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, it was shown that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1 ) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, the biological relevance of the OROV-Lrp1 interaction was demonstrated in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results, along with the recent finding of Lrp 1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp 1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

[00263] Bunyaviruses are a large group of related viruses with singlestranded, segmented, negative, or ambisense RNA genomes. Within the order Bunyavirales, the Peribunyaviridae family contains viruses that infect humans and animals with confirmed or potential zoonotic transmission. Oropouche virus (OROV; Orthobunyavirus genus; Simbu serogroup) is found primarily in the South American regions of Brazil, Trinidad, Peru, Panama, and Tobago. OROV has caused more than 30 epidemics, resulting in excess of 500,000 total cases of human febrile illness, making it the second most common arboviral disease in Brazil, behind Dengue fever. The true case number is likely higher as clinical testing for OROV is lacking and patients are often misdiagnosed as having Chikungunya or Dengue fevers. The arthropod vectors for OROV include Culicoides midges and Culex mosquitoes. In humans, OROV causes a febrile illness that manifests as fever, intense headache, myalgia, joint pain, retro-orbital pain, and photophobia, which can further develop into encephalitis or meningitis. Systemic infection manifests as rash, nausea, vomiting, and diarrhea. Viremia and leukopenia are common features, and virus can be detected in the cerebrospinal fluid. In mice, the virus replicates in the liver and spleen after either subcutaneous or intracerebral infection.

[00264] Due to the broad cellular tropism and ability to infect a variety of species, bunyaviruses are thought to use multiple receptors or attachment factors for entry and/or a protein that is widely expressed across different tissues and conserved across species. Recently, using a CRISPR-Cas9 screen, the conserved host protein low-density lipoprotein receptor (LDLR)-related protein-1 (Lrp1 ) was reported to mediate cellular infection with Rift Valley fever virus (RVFV), a phlebovirus within the Bunyavirales order. Lrp1 (also known as alpha-2-macroglobulin receptor or CD91 ) is a highly conserved multifunctional member of the LDLD family of transmembrane surface proteins. Lrp1 is important for ligand endocytosis, cell signaling, lipoprotein metabolism, blood-brain barrier maintenance, and angiogenesis. Homozygous deletion of Lrp1 is embryonically lethal in mice, further supporting the critical nature of Lrp1 in homeostatic functions. [00265] The M segment of Bunyavirales encodes the surface glycoproteins Gn and Gc, which form heterodimers and multimerize on the surface of the virion. Few studies have been conducted on the binding and entry mechanisms facilitated by OROV Gn/Gc. Given the conserved nature of Lrp1 across taxonomically distinct species and its expression in different tissues, whether OROV, a bunyavirus distantly related to RVFV, also requires Lrp1 for efficient infection of host cells was investigated. Despite having a similar genome organization among members of the Bunyavirales, many of the virally encoded sequences show little sequence homology. Therefore, studies to define similar host protein usage by these two distantly related viruses would have significant implications for pan-bunyavirus therapeutic and diagnostic development.

[00266] Lrp 1 knockout (KO) cell lines were used to show that OROV infection is decreased compared to parental cells expressing Lrp1. Pretreatment of cells with varying concentrations of the high-affinity Lrp1 -binding protein receptor-associated protein (RAP) significantly reduced OROV infection. Zika virus (ZIKV), an arbovirus outside the Bunyavirales order, was unaffected by the loss of Lrp1 or by treatment with Lrp1 -binding RAP protein. Chimeric virions expressing OROV glycoproteins bound to the Lrp1 ectodomain. Finally, the role of Lrp1 in OROV infection was validated in vivo, whereby RAP treatment was able to reduce viral tissue titers and rescue mice from lethal intracerebral infection with OROV. Based on our findings, Lrp1 is a host factor for multiple bunyaviruses, presenting a potential therapeutic approach to address this important group of emerging arboviruses. This work also paves the way for future studies to understand the mechanism of OROV binding to Lrp 1 .

[00267] OROV Infection Is Reduced in Lrp1 KO Cell Lines: OROV strain BeAn19991 was grown in mouse microglial BV2 cells at a multiplicity of infection (MOI) of 0.1 and 0.01 along with RVFV strain ZH501 and ZIKV strain PRVABC59 for comparison. While ZIKV did not replicate well in BV2 cells, OROV and RVFV reached 10 ⁶ PFU/mL by 24 h postinfection (hpi) at MOI 0.1 , and these parameters were used for the remaining cellular infection studies. Clonal BV2 KO cell lines that are deleted for either Lrp1 or the Lrp 1 chaperone protein RAP express significantly reduced levels of Lrp1 , and this was visualized and quantified using immunofluorescence and western blot. Infection of both Lrp 1 and RAP clonal KO cell lines with OROV resulted in significantly less infectious virus produced by 24 hpi when compared to the infection of BV2 wild-type (WT) cells (FIG. 8A), with reductions of 2 to 3 log in titer for both OROV and RVFV as a comparator. Thus, OROV requires Lrp1 or related proteins for efficient cellular infection and production of infectious virus from BV2 cells.

[00268] Additional clonal Lrp1 KO cell lines were established in human HEK293T, A549, and murine N2a cell lines, with the loss of Lrp1 verified by western blot. As with BV2 cells, Lrp 1 KO resulted in significantly reduced OROV infection across all cell lines. While similar reductions were seen with RVFV in Lrp1 KO cells, no significant difference in virus infection or production was observed in cells infected with ZIKV, a flavivirus used as a control (FIG. 8B-8D). The titers for both OROV and RVFV infection of KO cell lines were 10- to 100-fold lower than WT cell lines. By immunofluorescence, Lrp1 was detectable in WT parental A549 cells but was absent from Lrp1 KO lines (FIG. 8E and FIG. 8F). The number of OROV-infected cells at 24 hpi was reduced at least 10-fold in the KO line (FIG. 8E), which corresponds to the observed reduction in titers.

[00269] The Lrp1 -Binding Chaperone Protein RAP Inhibits OROV Infection of Cell Lines from Taxonomically Distinct Species: RAP (or Lrpapl ) is a high-affinity Lrp1 ligand and critical chaperone of Lrp1 and other LDLR family members. Domain 3 of RAP (RAPDS) (FIG. 9A) specifically binds to two extracellular cluster domains of Lrp 1 (CLn and CLiv) and competes for attachment with other compatible ligands while chaperoning the protein through the endoplasmic reticulum (ER) to the cell surface. The ability of exogenous mouse RAPDS (m RAPDS) to block OROV infection was tested by adding it before infection, at the time of infection, or following infection. After determining that pretreatment most effectively blocked infection, m RAPDS was added to murine BV2 microglial cells, monkey Vero E6 kidney cells, and human SH-SY5Y neuroblastoma cells at various concentrations 1 h before infection with MOI 0.1 of OROV, RVFV, or ZIKV as comparators (FIG. 9B and FIG. 9C). At 24 hpi, samples were tested for infectious virus by plaque assay. As a control, mutant mRAPos in which two lysines were changed (FIG. 9A) was tested in parallel, as these mutations have been shown previously to reduce the binding of RAP to CLn and CLiv of Lrp 1 . In all of the cell lines, treatment with mRAPD3 significantly reduced OROV infection compared to untreated cells at all concentrations, and the reduction in infection was comparable to RVFV (FIG. 9B and FIG. 9C). The mutant mRAPos was less effective at inhibiting both viruses at lower concentrations, with significant inhibition of both OROV and RVFV at the highest concentrations tested (10 pg/mL). ZIKV does not efficiently infect BV2 cells, but it does infect SH-SY5Y and Vero cells. Neither mRAPos nor mutant mRAPos inhibited the ZIKV infection of SH-SY5Y or Vero cells (FIG. 9B and FIG. 9C).

[00270] ORO V Interaction with Lrp1 Is Dependent on Viral Glycoproteins: To determine whether the restriction in OROV infection in Lrp1 -deficient cells is at the level of the surface glycoproteins, a chimeric vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and the OROV glycoproteins Gn and Gc (VSV-OROV) was used. Purified VSV-OROV or VSV control virions were used to infect BV2 WT cells or BV2 Lrp 1 KO cells. Samples were collected at 6 to 8 hpi and analyzed for GFP expression by flow cytometry (FIG. 10A) or imaging by fluorescent microscopy (FIG. 10B) . VSV-OROV infection was significantly reduced in BV2 Lrp1 KO cells. VSV infection was also significantly reduced but to a lesser degree, likely due to its utilization of other LDLR family members for viral entry. The reduction in VSV-OROV infection was confirmed by immunofluorescent microscopy (FIG. 10B). Furthermore, because mRAPos is known to bind to Lrp1 CLi and is able to block OROV infection (FIG. 9) biolayer interferometry was used to determine whether chimeric VSV-OROV binds to Lrp1 CLiv. To do this a recombinant Fc-fusion of the Lrp1 CLiv domain was used which was previously shown to block RVFV infection. It was found that VSV-OROV virions cound to immobilized Fc-Lrp1 CLiv but not Fc control (FIG. 10D).

[00271 ] Lrp 1 Cluster Domains CLn and CLiv Inhibit ORO Infection: Many ligands of Lrp1 bind to the CLn and CLiv extracellular domains, including mRAPos. Given the results showing VSV-OROV binding to IV (FIG. 10D), Vero E6 cells were treated with soluble Fc-fused CLn and CLiv proteins (FIG. 10C) and compared the relative infection to untreated cells and Fc-control treated cells. It was observed that Fc-fused CLn and CLiv treatment significantly reduced OROV infection compared to the Fc- control treated cells (FIG. 11 A). These results are comparable to those of treated cells infected with RVFV at the same MOI (FIG. 11 B). In addition, ZIKV infection of Vero E6 cells was unaffected by treatment with any Fc-bound Lrp1 proteins (FIG. 11C). [00272] OROV Infection Is Inhibited by the Glycoprotein Gn from RVFV: It was previously determined that the RVFV Gn protein binds to CLn and CLivof Lrp1 , and that m APD3 can compete with RVFV Gn for Lrp1 binding, indicating overlapping binding sites. Furthermore, the addition of soluble RVFV Gn was able to block RVFV infection. Since mRAPos was also able to block OROV infection and VSV-OROV bound to CLiv, it was next determined whether addition of RVFV Gn can similarly block OROV infection. BV2 cells were treated with RVFV Gn 1 h before OROV infection and kept it in the media for the duration of the experiment. At 24 hpi, it was evaluated OROV titers by plaque assay. RVFV Gn blocking of RVFV infection were compared as a control. In BV2 cells, it was found that pretreatment of cells with RVFV Gn at concentrations of 2, 5, 10, and 20 pg/mL significantly reduced infectious titers of both OROV and RVFV (FIG. 12A). This experiment was repeated in Vero E6 cells with the addition of ZIKV as a control. RVFV Gn significantly reduced OROV and RVFV infection in Vero E6 cells and showed no significant effect on ZIKV infection (FIG. 12B).

[00273] mRAP_D3 Treatment Rescues Mice from Lethal OROV Infection: Because mRAPos reduced OROV infection in vitro, a proof-of-concept experiment was used to determine the in vivo relevance of this interaction. OROV does not cause lethal disease in adult mice when administered subcutaneously. However, the median lethal dose (LDso) of OROV administered by intracerebral (IC) injection in young adult mice is <5 PFU. Mice succumbed to infection with an average time to death of 4.5 days postinfection (dpi) and viral titers in the brain at the time of death were >10 ⁷ PFU/g tissue. Based on the LD50, a dose of 100 PFU (at least 20x OROV IC LD50) was chosen for the mRAPD3 treatment experiments. OROV was administered IC in conjunction with 215 pg mRAPD3, mutant ITIRAPDS, or a similar-sized, unrelated control protein (Ebola VP30) to C57BL/6J mice in a proof-of-concept experiment (FIG. 13A). All untreated and control protein-treated mice succumbed by 4 to 6 dpi. Of the mice that received WT mRAPD3 treatment, 90% survived, while 60% of mice that received mutant mRAPos also survived.

[00274] A cohort of mice underwent planned euthanasia at 3 dpi to directly compare tissue titers across treatment groups. The untreated and control protein- treated mice had high viral titers in their brains (10⁶ to 10⁸ PFU/g tissue), while the mRAPD3-treated mice had levels of infectious virus that were near or at the limit of detection (FIG. 13B). Fitting with the survival data and in vitro data (FIG. 9C), mutant mRAPD3-treated mice had intermediate amounts of virus in the brain compared to mRAPD3 and control animals (FIG. 13B).

[00275] This can be visualized by immunofluorescence microscopy using an anti-OROV N polyclonal antibody on cryosections of the cerebral cortex from 3 dpi brain tissues. The mRAPos-treated mice have little to no staining for OROV N protein in the brain at 3 dpi (FIG. 13C), whereas the untreated and control protein-treated mice had abundant, diffuse OROV staining in the brain (FIG. 13C). The mutant mRAPos- treated mice had reduced, focal regions of OROV staining in the brain at 3 dpi, which was substantially more than mRAPos-treated mice. Thus, the reduced inhibition of OROV infection displayed by the mutant mRAPos protein (FIG. 9) corresponds to reduced binding affinity to CLn and CLiv, intermediate tissue viral loads, and concomitant intermediate levels of survival in IC-infected mice.

[00276] The brain tissues were also stained with the microglial marker lba-1 to examine immune activation within the tissue. Brains from mice receiving mRAPos treatment alone or ITIRAPDS + OROV expressed low levels of lba-1 + cells, indicative of a normal resting state in the brain. However, OROV infected and untreated, mutant mRAPos-treated, or control protein-treated brain sections had more activated microglia (Iba1 + cells), indicating higher levels of inflammatory activation and recruitment (FIG. 13C).

[00277] Finally, serum samples from the surviving WT mRAPos and mutant mRAPos-treated mice were tested for neutralizing activity against OROV using a plaque-reduction neutralization assay (PRNT50). Titers in the surviving mice were

1 :40, while the uninfected control animals had no detectable neutralizing titer, thus confirming that these exposed yet surviving animals were infected with OROV.

[00278] Discussion: The data show that a highly conserved protein Lrp 1 is a host factor that supports efficient cellular infection by Oropouche orthobunyavirus. It was demonstrated that OROV infection of Lrp1 -deficient cells was significantly decreased. It was also shown that high-affinity Lrp1 -binding RAP protein significantly reduced OROV infection in vitro and in vivo. Direct association between OROV and Lrp 1 was established with binding assays using VSV-OROV. The relevance of our finding is also supported by the in vivo studies, in which intracerebral infection of mice with OROV in the presence of ITIRAPDS rescued the animals from an otherwise lethal infection and significantly reduced viral titers in the brain. Taken together, these data support a role for Lrp1 in OROV infection.

[00279] Lrp1 mediates cellular infection by the phlebovirus RVFV. Lrp1 is needed for efficient in vitro cellular infection and to promote lethal RVFV infection in vivo in a mouse model. While important, the previous study was focused on RVFV and did not implicate Lrp1 as having a broader impact. Here, it was revealed that the orthobunyavirus OROV, while classified in a different family than RVFV, also uses Lrp1 to efficiently infect cells and cause disease in vivo, thus implicating Lrp1 as a much broader host factor for bunyaviral infection. Both viruses bind similar and potentially overlapping regions within Lrp1 extracellular domains CLH and CLiv. This finding implies some structural similarities between the two Gn proteins despite sequence diversity.

[00280] Consistent with the results, recent studies have examined the role of the LDLR family and related proteins in the context of viral infections. These efforts led to several reports, including a role for LDLR in binding and entry of dengue virus, hepatitis C virus, VSV, and rhinovirus. The LDLR-related protein LDLRADS was recently reported to facilitate the entry of Venezuelan equine encephalitis virus. Finally, VLDLR and ApoER2 were recently identified as entry receptors for multiple alphaviruses, including Semliki Forest virus, eastern equine encephalitis virus, and Sindbis virus, despite differences in E2/E1 amino acid sequence homology. These findings of related LDLRs, but not Lrp1 , further support a wide-ranging role for this family of receptors in viral infections. However, the studies define a role for Lrp1 in mediating efficient infection by both OROV and RVFV. While consistent with this broader role of LDLRs in viral infections, this study highlights distinct drivers of specificity in the viral entry of bunyaviruses that are related to Lrp1 .

[00281 ] The strain of OROV used here, BeAn 19991 , was isolated from a sloth in Brazil in 1960 and falls within Lineage I. This is also the strain that served as the basis for the OROV reverse genetics system. While further studies testing OROV strains from Lineage II and III strains would be informative, the conserved nature of the interactions and the studies using heterologous proteins such as RVFV Gn and RAPDS to inhibit OROV infection suggest that Lrp1 may support infection by multiple OROV lineages.

[00282] Lrp1 has a large ectodomain and binds many different ligands. RAP protein is one such ligand, and the mutant mRAPos mutation reduces affinity for Lrp1 , but it does not abolish binding. In the in vitro results presented here, it was observed an intermediate reduction in both OROV and RVFV infection in the presence of the mutant mRAPD3 protein at the highest concentrations tested (10 pg/mL), whereas mRAPos inhibits both viruses at 1 pg/mL. The intermediate in vitro phenotype of the mutant mRAPos correlated well with the in vivo OROV findings, in which intermediate levels of virus were seen in the brain as well as 60% survival of the mice (compared to 953% survival with the WT mRAPos). In contrast to the in vitro data presented here, mutant mRAPos is unable to out compete RVFV in vivo. This observation may be attributable to either differences in the affinity of each of the Gn proteins for Lrp 1 or, alternatively, to differences in inherent pathogenicity between OROV and RVFV, as RVFV is much more pathogenic. Studies to address these key observations and differences are ongoing.

[00283] The work presented here underscores a previously unappreciated role played by Lrp1 in cellular infection by diverse bunyaviruses. The results with OROV and RVFV suggest that it is likely that Lrp1 is used by other members of the Bunyavirales order. Importantly, given the need to identify broadly acting inhibitors of emerging viruses, these results highlight the feasibility of Lrp1 as a pan-bunyaviral target. The dependence on Lrp1 by OROV (Peribunyaviridae) and RVFV (Phenuiviridae) suggest common structural elements that transcend sequence homology. Future studies to characterize the binding and internalization mechanisms of both OROV and RVFV will also identify potentially conserved binding epitopes as targets for therapeutic strategies.

[00284] Cells: The LPR1 KO R4 and RAP KO A7 cells were generated, as previously described (11 ). All BV2 and Vero cells (American Type Culture Collection [ATCC], CRL-1586) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (ATCC, 30-2002) supplemented with 1 % penicillin/streptomycin (Pen/Strep), 1 % I- glutamine (l-Glut), and either 2% (D2), 10% (D10), or 12% (D12) fetal bovine serum (FBS). SH-SY5Y cells were obtained from ATCC (CRL-2266) and cultured in D120/F12 media (ATCC, 30-2006) supplemented with 1 % Pen/Strep and 1 % l-Glut. HEK293T, A549, and N2a clonal KO cells were generated by CRISPR-Cas9 using ribonucleoprotein complexes of Cas9 and Lrp1 -specific guide RNAs, as previously described. The resulting cells were subcloned and subjected to next-generation sequencing analysis and short tandem repeat profiling to confirm the deletion and homogeneity of the clones. HEK293T Lrp1 KO cells were maintained in D10 media, A549 Lrp1 KO cells were maintained in D12/F12 media, and N2a Lrp1 KO cells were maintained in Eagle’s minimum essential medium (ATCC, 30-2003) with 10% FBS.

[00285] Viruses: The BeAnl 9991 strain of OROV was rescued through reverse genetics and was generously provided by Paul Duprex and Natasha Tilston- Lunel (CVRPitt Center for Vaccine ResearchAQ8). RVFV ZH501 was rescued through reverse genetics and provided by Stuart Nichol (Centers for Disease Control and Prevention [CDC]). The PRVABC59 (Human/2015/Puerto Rico) strain of ZIKV was obtained from BEI Resources (NR-50240) from the Arbovirus Reference Collection (CDC, Fort Collins, CO, USA). VSV-OROV virus was generated as described previously. All of the viruses were propagated in Vero E6 cells with standard culture conditions using D2 media supplemented with 1 % Pen/Strep and 1 % l-Glut. A standard viral plaque assay (VPA) was used to determine the titer of the stocks. The agar overlay for the VPA was comprised ofs 1 x minimal essential medium, 2% FBS, 1 % Pen/Strep, 1 % HEPES buffer, and 0.8% SeaKem agarose; the assay incubateds at 37°C for 4 d (OROV) and 3 d (RVFV), followed by visualization of plaques with 0.1 % crystal violet.

[00286] Antibodies: The following antibodies were used: rabbit anti-Lrp1 (Abeam ab92544), mouse anti-Lrp1 (Santa Cruz, sc-57353), rabbit anti-OROV N (Custom Genescript), and mouse anti-RVFV N (BEI Resources, NR-43188) for fluorescence immunostaining, and rabbit anti-Lrp1 (Cell Signaling Technology, 64099S) and rabbit anti-GAPDH (Thermo Fisher, PA1 -987) for western blots.

[00287] Neutralization Assays with mRAPD3, RVFV Glycoprotein Gn, and Lrp1 clusters: Cells were seeded in a 24-well plate at a density of 2.4E-5 cells/mL in D10 the day before infection. The following day, the media was removed and the virus was diluted in D2 (MOI 0.1 ) and added to the cell monolayer in a 200- L volume. Following a 1 -h incubation at 37 °C, the inoculum was removed. The monolayer was washed with 1 x Dulbecco’s phosphate-buffered saline (dPBS), and fresh D2 media was added to each well. At the designated timepoint(s), supernatants were collected and infectious virus titers were determined by VPA. In the treatment assays, mRAPD3, mutant mRAPD3, RVFV Gn, Fc-CLII, IV, or Fc-control (trastuzumab) were diluted in D2 and added to the cell monolayer, followed by a 1 -h incubation at 37 °C. Following the incubation, virus diluted in D2 (MO1 1 , 0.1 ) was added to the media and incubated for 1 h at 37 °C. After the absorption period, the inoculum was removed, the cell monolayer was washed once with 1 x dPBS, and D2 media containing the designated proteins was added in a 500- L volume. Supernatants were collected at 24 hpi (OROV and RVFV) or 48 hpi (ZIKV) and viral titers were determined through VPA. Vial titers for RVFV and ZIKV were also analyzed through qRT-PCR as previously described.

[00288] Immunofluorescence: Cover glass (FisherBrand, 12-546-P) was sterilized in 70% EtOH and coated with BME Cultrex (R&D Systems, 3432-010-01 ) before seeding. Cells were seeded the day before staining at a density of 1 E-5 cells/mL in D10 media. Upon harvest, virus-infected cells were fixed in 4% paraformaldehyde for 20 min, followed by permeabilization with 0.1 % Triton X-100 detergent + 1xPBS for 15 min at room temperature (RT). Cells were blocked using 5% normal goat serum (Sigma, G9023) for 1 h at RT, followed by incubation with the primary antibodies rabbit anti- LRP1 at 1 :200, mouse anti-Lrp1 at 1 : 50, mouse anti-RVFV N at 1 :200, or rabbit anti- OROV N at 1 :200 for 1 h at RT. The secondary antibodies goat anti-mouse Cy3 (Jacksonlmmuno, 115-165-003), goat anti-rabbit 488 (Jacksonlmmuno, 111-545-003), goat anti-mouse 488 (Jacksonlmmuno, 115-545-003), or goat anti-rabbit Cy3 (Jacksonlmmuno, 111 -165-144) were added (1 :500 dilution) for 1 h at RT. The cells were counterstained with Hoescht and mounted using Gelvatol.

Example 3: Engineered viral proteins to limit Tau spreading

[00289] There are roughly 5.8 million people living with Alzheimer’s disease (AD) in the U.S. in 2020, and this number is projected to increase to 12 to 16 million by 2050. In addition to the devastating burden of disease, the cost of caring for these patients will mushroom to levels that will have overwhelming impact on U.S. healthcare and economy unless effective treatments are developed to address the underlying causes of the disease. There is a critical need to identify new molecular targets and understand mechanistically how they contribute to the development of AD. Along these lines, the low-density lipoprotein receptor-related protein 1 (LRP1 ) was recently demonstrated to be a receptor mediating cellular uptake of pathogenic tau into neurons, thus facilitating cellular spread of neurotoxic tau and identifying LRP1 and LRP1 -tau interactions as exciting potential new targets to combat AD.

[00290] The cell-to-cell spread of pathogenic tau is a major contributor to the progression of neurodegeneration in AD. In these events, pathogenic misfolded tau is internalized by healthy neurons, providing a template upon which normal cellular tau assembles and then misfolds, thus propagating pathology across a neural network. Recent investigations indicate that the extracellular receptor LRP1 plays a primary role in this process by mediating cellular internalization of tau, suggesting that blocking this interaction is a potential therapeutic route to inhibit cellular propagation of and progressive neurodegeneration caused by pathogenic tau. Towards this end, using robust biophysical methods, it was demonstrated that LRP1 directly engages tau and identified a novel viral protein, Rift Valley Fever Virus glycoprotein N (RVFV Gn), which binds LRP1 with high affinity. Importantly it was also demonstrated that RVFV Gn potently blocks the ability of LRP1 to engage tau, suggesting that there is substantial overlapping binding surfaces on LRP1 for tau and RVFV Gn. The goal of this Example to comprehensively characterize the engagement of tau by LRP1 and characterize novel inhibitors based on RVFV Gn that limit tau uptake by LRP1 and how Lrp1 deletion in the context of established mouse models contribute to a better understanding of the role of Lrp 1 . Thus providing the framework to develop new RVFV Gn-based therapies targeting the LRP1 -tau interaction as a potential treatment to inhibit pathogenic tau spreading.

[00291 ] The LRP1 receptor has a large ectodomain (600 kDa) that can be subdivided into 4 clusters (CLi, CLn, CLIII, and CLiv). Individual clusters were produced as well as multi-cluster proteins (e.g., LRP1 CLi - CLiv, LRP1 CLII-CLIV, etc.) and characterized tau binding affinity and kinetics using biolayer interferometry (BLI) to identifying LRP1 domains that contribute to tau binding. Complementarily, tau fragments were used to characterize binding to LRP1 using the same methods. These studies define the major determinants of each protein that contribute to interaction, and drive the design of minimal constructs that can be co-crystallized to characterize the binding interface. These experiments define the critical molecular surfaces mediating the interaction between LRP1 and tau that are utilized in pathogenic tau spreading.

[00292] In the viral studies, an Lrp1 ^F/F model was developed to delete Lrp1 in a tissue specific manner. Using CamK2a Cre mouse, Lrp1 was eliminated in adult mouse brain and evaluated the impact of Lrp1 loss in established AD models. RVFV Gn and multimeric Gn proteins were used to prevent tau spreading, avidity-enhanced dimeric and tetrameric RVFV Gn fusion proteins were produced and assayed for their potential to inhibit tau binding and uptake, cellular uptake assays of fluorescent tau were utilized into H4 cells to determine if RVFV Gn proteins can inhibit cellular internalization of tau in a dose-dependent manner. Competition binding studies by BLI were used to quantify the potency (ICso) with which RVFV Gn proteins inhibit the binding of tau to LRP1 . Inhibitory molecules will then be further characterized to define whether their binding regions on LRP1 are independent of or overlapping with the tau-binding site using HDX-MS. Inhibitors that exhibit ICso < 500 nM and overlap the tau binding site (>50% solvent accessible surface area) will be considered as optimal leads for developing therapies.

[00293] This study developed engineered decoy viral inhibitors (DEVi), to prevent tau uptake as a treatment for AD. Based on the widely accepted, yet incomplete, model known as the “amyloid hypothesis”, cells can uptake tau which can lead to neurofibrillary tangle formation and contribute to Alzheimer’s disease (AD) and related dementias. An estimated 5.8 million individuals currently suffer from AD and the soaring cost of caring for these patients will have devastating impacts on the US healthcare system and the economy in the next 25-30 years. There is a critical need to identify new molecular targets, gain mechanistic insights into their pathogenic functions, and develop molecular-level strategies to deter those functions. A hallmark of AD is the propagation of pathogenic tau protein between neurons forming neurofibrillary tangles, blocking neuronal transport, and leading to widespread neuronal death. The lipoprotein receptor-related protein 1 (LRP1 ) was recently identified as a major receptor for tau endocytosis into neurons. Inhibition of LRP1 -mediated tau uptake represents a promising new target to prevent neurodegenerative pathogenesis. Leveraging our recently discovered novel binding partner for LRP1 , Rift Valley Fever Virus glycoprotein N (RVFV Gn), as engineered decoy viral inhibitors (DEVi) of tau uptake.

[00294] Regulation of LRP1 surface expression: LRP1 has been characterized for its role as an LDL receptor and its role in Alzheimer’s disease by regulating tau uptake and spread in the brain. Lrp1 processing and surface presentation are modulated by the molecular chaperone RAP. Lrp1 has been recombinantly expressed and its interaction site on RAP has been previously mapped by biophysical studies, including an engineered truncated RAP (mRAPos) that stably binds Lrp1. Lrp1 surface levels are likely regulated by RAP, which enables chaperoning Lrp1 from the ER to the cell surface. Once on the surface, PCSK9 (proprotein convertase subtilisin/kexin type 9) binds Lrp1 and promotes internalization and degradation. Grp94 also impacts the levels of cell surface Lrp1 (and other LDL receptors) by binding to PCSK9 in the lumen of the ER 23. Loss of RAP or Grp94 leads to lower Lrp1 surface expression.

[00295] RVFV Gn inhibits binding of tau to LRP1 CLiv LRP1 CLiv can co-IP with tau. Since similar results were obtained examining RVFV Gn binding to LRP1 , whether RVFV Gn could block tau engagement of LRP1 was investigated. As a first step, it was examined if LRP1 could directly bind to 2N4R tau using BLI. It was found that both tau produced in E. coli (not post-translational ly modified) and tau produced in HEK293 cells (post-translationally modified) bound to LRP1 with nearly identical binding profiles and high affinities (single nM) (FIG. 14A and FIG. 14B). These results demonstrated, for the first time, direct engagement of tau by LRP1 , and these results also indicate that post-translational modification of tau are not required for the interaction with LRP1. Next, BLI-based sequential binding assays were designed to examine whether RVFV Gn could block tau binding to LRP1 . In a first set of experiments, immobilized Fc-LRP1 CLiv was first bound to RVFV Gn (100 nM) and then, after a short dissociation period, binding to tau (100 nM) was examined. It was found that pre-association of RVFV Gn with LRP1 CLiv almost completely prevented binding of tau to LRP1 CLiv (FIG. 15A and FIG. 15B) and found that pre-association of tau with LRP1 CLiv did not noticeably inhibit binding of RVFV Gn to LRP1 (FIG. 15C- 15D).

[00296] Avidity-enhanced multimeric Gn proteins were engineered and the competitive binding quantified using cellular and biophysical assays and characterized the mechanism of inhibition. As shown in FIG. 16, based on the established SpyTag/SpyCatcher system, an LRP1 inhibitor system has been designed. Using this system, tau uptake inhibition was tested, providing a lead for cellular tau inhibition. For RVFV Gn competition experiments, proteins were added at specific concentrations at the same time FAM-tau. Cellular inhibition constants (ICso) were derived from experiments using increasing concentrations of RVFV Gn proteins versus constant FAM-tau. As controls, the LRP1 chaperone RAP were used as a positive control inhibitor and fluorescent-labeled LDL as a positive control of uptake. Transcomplement experiments were also carried out using BV2 cell lacking mouse Lrp 1 expressing human LRP1 CLIV as independent validation. Altogether, the results define the critical interface required for high affinity interactions between RVFV Gn and LRP1 that preclude tau binding and identify RVFV Gn-based inhibitors of LRP1 -tau binding, which validate RVFV Gn as a potential inhibitor lead.

EQUIVALENTS

[0182] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0183] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0184] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0185] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0186] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

Claims

What is claimed is: A method of reducing or treating a viral infection in a subject, the method comprising: administering to the subject an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and decoy receptor comprising an LRP1 polypeptide or fragment thereof. The method of claim 1 , wherein the viral infection is a bunyaviral infection. The method of claim 1 or claim 2, wherein the viral infection is a Rift Valley Fever virus (RVFV) infection, a oropouche virus (OROV) infection, or La Crosse virus (LACV) infection. The method of any one of claims 1 to 3, wherein the subject is having symptoms of a viral infection or is suspected of having a viral infection. The method of any one of claims 1 to 4, wherein the decoy receptor comprises one or more LRP1 CLiv domains. The method of any one of claims 1 to 5, wherein the decoy receptor comprises one or more LRP1 CLII domains. The method of any one of claims 1 to 4, wherein the decoy receptor comprises a LRP1 polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 1 . The method of any one of claims 1 to 4, wherein the decoy receptor comprises an extracellular domain of a LRP1 polypeptide. The method of any one of claims 1 to 8, wherein the decoy receptor comprises a LRP1 polypeptide as a fusion protein. The method of claim 9, wherein the fusion protein is a LRP1 polypeptide-Fc fusion protein. The method of claim 9, wherein the fusion protein is a LRP1 polypeptide- SpyTag/SpyCatcher fusion. The method of any one of claims 1 to 11 , wherein the method further comprises administering to the subject an additional anti-viral agent. The method of any one of claims 1 to 12, wherein infectivity of the virus for a host cell is reduced. The method of any one of claims 1 to 13, wherein infectivity of the virus is reduced by reducing internalization of a virus into the cell. The method of any one of claims 1 to 14, wherein infectivity of the virus is reduced by reducing replication or internalization of a viral genome into the cell. The method of any one of claims 1 to 15, wherein infectivity of the virus is reduced by disrupting or preventing an interaction between a viral surface protein and a host receptor protein. The method of any one of claims 1 to 16, wherein the viral surface protein is a Gn viral glycoprotein protein and the host receptor protein is LRP1 . A decoy receptor composition comprising a recombinant LRP1 polypeptide. The composition of claim 18, wherein the decoy receptor comprises one or more LRP1 CLiv domains. The composition of claim 18 or 19, wherein the decoy receptor comprises one or more LRP1 CLn domains. The composition of any one of claims 18 to 20, wherein the decoy receptor comprises a LRP1 polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 1. The composition of any one of claims 18 to 21 , wherein the decoy receptor comprises an extracellular domain of a LRP1 polypeptide. The composition of any one of claims 18 to 22, wherein the decoy receptor comprises a LRP1 polypeptide as a fusion protein. The composition of claim 23, wherein the fusion protein is a LRP1 polypeptide-Fc fusion protein. The composition of claim 23, wherein the fusion protein is a LRP1 polypeptide- SpyTag/SpyCatcher fusion. The composition of any one of claims 18 to 25 for use in treating a viral infection. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a decoy receptor of any one of claims 18 to 25.

118 A method of reducing or treating a viral infection in a subject, the method comprising: administering to the subject an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and decoy viral inhibitor comprising a viral Gn polypeptide or fragment thereof. The method of claim 28, wherein the viral infection is a bunyaviral infection. The method of claim 28 or claim 29, wherein the viral infection is a Rift Valley Fever virus (RVFV) infection, a oropouche virus (OROV) infection, or La Crosse virus (LACV) infection. The method of any one of claims 28 to 30, wherein the subject is having symptoms of a viral infection or is suspected of having a viral infection. The method of any one of claims 28 to 31 , wherein the decoy viral inhibitor comprises a Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 2. The method of any one of claims 28 to 32, wherein the decoy viral inhibitor comprises the extracellular domain of a Gn polypeptide. The method of any one of claims 28 to 33, wherein the decoy viral inhibitor comprises amino acids 1 to 316 of SEQ ID NO: 2. The method of any one of claims 28 to 34, wherein the decoy viral inhibitor is a RVFV Gn polypepide.

119 The method of any one of claims 28 to 35, wherein the decoy viral inhibitor comprises a Gn polypeptide as a fusion protein. The method of claim 36, wherein the fusion protein is a Gn polypeptide-Fc fusion protein. The method of claim 36, wherein the fusion protein is a Gn polypeptide- SpyTag/SpyCatcher fusion. The method of any one of claims 28 to 38, wherein the method further comprises administering to the subject an additional anti-viral agent. The method of any one of claims 28 to 39, wherein infectivity of the virus for a host cell is reduced. The method of any one of claims 28 to 40, wherein infectivity of the virus is reduced by reducing internalization of a virus into the cell. The method of any one of claims 28 to 41 , wherein infectivity of the virus is reduced by reducing replication or internalization of a viral genome into the cell. The method of any one of claims 28 to 42, wherein infectivity of the virus is reduced by disrupting or preventing an interaction between a viral surface protein and a host receptor protein. The method of any one of claims 28 to 43, wherein the viral surface protein is a Gn viral glycoprotein protein and the host receptor protein is LRP1 . A decoy viral inhibitor composition comprising a recombinant viral Gn polypeptide.

120 The composition of claim 45, wherein the decoy viral inhibitor comprises a Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 2. The composition of claim 45 or 46, wherein the decoy viral inhibitor comprises the extracellular domain of a Gn polypeptide. The composition of any one of claims 45 to 47, wherein the decoy viral inhibitor comprises amino acids 1 to 316 of SEQ ID NO: 2. The composition of any one of claims 45 to 48, wherein the decoy viral inhibitor is a RVFV Gn polypepide. The composition of any one of claims 45 to 49, wherein the decoy viral inhibitor comprises a Gn polypeptide as a fusion protein. The composition of claim 50, wherein the fusion protein is a Gn polypeptide-Fc fusion protein. The composition of any one of claims 45 to 51 for use in treating a viral infection or tauopathy. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a decoy viral inhibitor of any one of claims 45 to 52. A method of reducing or treating a tauopathy or reducing a tau-related pathology in a subject, the method comprising:

121 administering to the subject an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and decoy viral inhibitor comprising a viral Gn polypeptide or fragment thereof. The method of claim 54, wherein the subject is amyloid negative. The method of claim 54, wherein the subject has no dementia. The method of claim 54, wherein the subject has dementia. The method of claim 54, wherein the subject is amyloid positive. The method of claim 54, wherein the tauopathy is progressive supranuclear palsy (PSP), dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease, Parkinson-dementia complex of Guam, tangle- predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick’s disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Frontotemporal lobar degeneration (FTLD), Alzheimer’s disease (AD), or frontotemporal dementia (FTD). The method of claim 54, wherein the tauopathy is AD. The method of any one of claims 54 to 59, wherein the decoy viral inhibitor comprises a Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full-length amino acid sequence of SEQ ID NO: 2.

122 The method of any one of claims 54 to 60, wherein the decoy viral inhibitor comprises the extracellular domain of a Gn polypeptide. The method of any one of claims 54 to 61 , wherein the decoy viral inhibitor comprises amino acids 1 to 316 of SEQ ID NO: 2. The method of any one of claims 54 to 62, wherein the decoy viral inhibitor is a RVFV Gn polypepide. The method of any one of claims 54 to 63, wherein the decoy viral inhibitor comprises a Gn polypeptide as a fusion protein. The method of claim 64, wherein the fusion protein is a Gn polypeptide-Fc fusion protein. The method of any one of claims 54 to 65, wherein cell-to-cell spread of pathogenic tau is reduced relative to the spread of tau in the absence of the decoy viral inhibitor. The method of any one of claims 54 to 66, wherein cell-to-cell spread of pathogenic tau is reduced by reducing internalization of a pathogenic tau into the cell. The method of any one of claims 54 to 67, wherein cell-to-cell spread of the pathogenic tau is reduced by disrupting or preventing an interaction between the pathogenic tau and a host receptor protein. The method of claim 68, wherein the host receptor protein is LRP1 . A method of increasing the amount of an imaging agent or therapeutic agent in the central nervous system of a subject, the method comprising;

123 administering to the subject a composition comprising the imaging agent or therapeutic agent conjugated to a viral Gn poly peptide, thereby improving transfer of a therapeutic or imaging agent transfer across the blood-brain-barrier. The method of claim 70, wherein the Gn polypeptide having an amino acid sequence that has greater than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the full- length amino acid sequence of SEQ ID NO: 2. The method of claim 70 or 71 , wherein the viral Gn polypeptide comprises the extracellular domain of a Gn polypeptide. The method of any one of claims 70 to 72, wherein the Gn polypeptide comprises amino acids 1 to 316 of SEQ ID NO: 2. The method of any one of claims 70 to 73, wherein the viral Gn polypeptide is a RVFV Gn polypeptide. The method of any one of claims 70 to 74, wherein the imaging agent or therapeutic agent is directly or indirectly conjugated to the viral Gn polypeptide.

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